orthodontic tooth movement is associated with orofacial … · with orofacial mechanical and...
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ORTHODONTIC TOOTH MOVEMENT IS ASSOCIATED
WITH OROFACIAL MECHANICAL AND THERMAL
HYPERSENSITIVITIES AND FACE SENSORIMOTOR
CORTEX NEUROPLASTICITY
by
Mandeep Sood
B.D.S., M.D.S. (Orthodontics)
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Faculty of Dentistry
University of Toronto
© Copyright by Mandeep Sood (2013)
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ORTHODONTIC TOOTH MOVEMENT IS ASSOCIATED
WITH OROFACIAL MECHANICAL AND THERMAL
HYPERSENSITIVITIES AND FACE SENSORIMOTOR
CORTEX NEUROPLASTICITY
Mandeep Sood
Doctor of Philosophy
Graduate Department of Dentistry and Collaborative Program in Neuroscience
University of Toronto
2013
ABSTRACT
This study addressed the General Hypothesis that orthodontic tooth movement (OTM) is
associated with neuroplasticity of the rat’s primary face motor cortex (face-M1) and
somatosensory cortex (face-S1) motor representations and with mechanical and thermal
hypersensitivities in the orofacial region.
Objective 1 was to develop an OTM rat model, and design and manufacture a cephalostat
for standardized radiographic measurements of the amount and the rate of OTM. A constant
force of 10 ± 4 cN applied for 28 days produced a uniform rate of OTM of the right three
maxillary molars and maxillary incisors of 1.75 ± 0.23 mm. The well-defined force
parameters correlated with clinical observations of OTM in humans.
Objective 2 was to use intracortical microstimulation (ICMS) and electromyographic
(EMG) recordings to test if neuroplastic changes occur in the ICMS-defined motor
representations of anterior digastric (LAD, RAD), masseter (LMa, RMa), buccinator (LBu,
RBu), and genioglossus (GG) muscles within the rat’s face-M1 and face-S1 during OTM;
the analyses to include any alterations in the number of ICMS sites representing these
muscles and in the onset latencies of ICMS-evoked responses in the muscles. OTM resulted
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in significant changes in LAD, RAD, and GG motor representations in face-M1 and face-
S1 at days 1, 7, and 28; there were no significant difference in the onset latency for these
muscles across the groups, and between face-M1 and face S1 within each group.
Objective 3 was to test if orofacial mechanical and thermal hypersensitivities occur in rats
during OTM. The mechanical and thermal sensitivities at ipsilateral and contralateral
orofacial sites were significantly increased in the early postoperative period (1 – 5 days),
with peaks reached on day 1, and then returned to preoperative levels until postoperative
day 28. These findings suggest that the cortical neuroplastic changes are unlikely related to
maintained OTM-induced pain, although pain may have contributed to the changes in the
early postoperative period.
Thus, this new OTM rat model was successfully used for neurophysiological and
behavioural studies that revealed the relevance of cortical neuroplasticity to orthodontic
therapy and concepts of orofacial growth and development.
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ACKNOWLEDGEMENTS
This doctoral work was carried out under the supervision of Dr. Barry J. Sessle in
the Graduate Department of Dentistry & the Collaborative Program in Neuroscience,
University of Toronto, Canada, spanning the years 2005-2013.
I express my most humble and sincere thanks to Dr. Barry Sessle for his proficient
navigation through this incredible journey. I am indebted to him for his expert guidance,
meaningful criticisms, unrelenting support and wisdom that he has imparted to me and has
helped me to develop into a confident and knowledgeable clinician scientist.
I am fortunate to have been advised by renowned experts in motor control and pain
neuroscience research and in orthodontics, who are also exceptional individuals. I sincerely
thank Drs. Karen Davis, Angelos Metaxas, and Ze’ev Seltzer who have provided their
expertise and have been very supportive throughout the years of being on my supervisory
committee.
I owe many thanks to Drs. Richard Ellen and Christopher McCulloch of the Cell
Signals CIHR Strategic Training Program and Dr. Michael Salter of M2C (Molecules to
Community) Strategic Training Program. These programs provided me with multiple
opportunities to collaborate with a multidisciplinary group of international scientists and
clinicians and have been crucial to my development as a clinician scientist.
I would like to thank all those responsible for my training as a specialist in the
Department of Orthodontics and Dentofacial Orthopedics, University of Toronto. In
particular, Drs. Bryan Tompson, William Wilson, James Posluns, Norm Riekenbrauck, Jim
Marco, and John Daskalogiannakis - thank you for your support and providing me with the
very best clinical and research training experience possible. A special thank you is due to
my co-residents Drs. Michael O’Toole, Mathew McLeod, and Joanie Roy.
I would like to profoundly thank my mentor Dr. Angelos Metaxas from the
orthodontic department for his confidence in me and for his constant support and
encouragement during the last 13 years. His guidance has played a crucial part in the career
path choices I have made in Canada.
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I acknowledge the work of Poolak Bhatt, summer/winter student in the laboratory,
for conducting the data analyses. I also acknowledge the support of my colleagues in the
laboratory Drs. Kajunori Adachi, Pavel Cherkas, Vidya Varathan, Hua Wang and Dong
Yao. A special token of appreciation is due to Dr. Jye-Chang Lee for his guidance on the
electrophysiological procedures and the development of the software programs. Drs. Limor
Avivi-Arber and James Hu - your help and support are highly appreciated.
I am also grateful to Ms. Susan Carter for providing support and technical expertise
at the Faculty of Dentistry, University of Toronto. The contributions of statisticians Xiuyan
Zhao and Derek Stephens from Hospital of Sick Kids are highly appreciated.
I would like to thank my family, especially my mother Kanta Sood and father
Brahm Prakash Sood for ingraining the value and love for academics in me and
wholeheartedly supporting and encouraging me through the many “adventurous”
endeavours that I have undertaken. You are the best parents one could hope for and aspire
to emulate.
I owe a great deal of loving thanks to my son Rohan Sood and my daughter Riya
Sood whose patience can be best described as amazing. Their acceptance of my
commitments to my program made this journey possible. Rohan, hereafter I will be there
for any number of T.T. games that you desire to play; Riya, I will be cheering for you from
the stands for every gymnastic sequence you participate in.
Lastly, I express my heartfelt thanks to my extraordinary wife Rohini Sood for her
constant unconditional love, understanding, support and encouragement during the many
challenging times we faced together. Rohini, you indeed are the wind beneath my wings – I
could not have completed this journey for the last 8 years without you by my side – to you I
owe my deepest gratitude.
I dedicate this thesis to my wife and my children as it would not have seen the light
of the day had it not been for their countless sacrifices.
This work was supported by CIHR grant MOP-4918 to Dr. Barry Sessle, Cell
Signals CIHR Training program, and Harron Scholar funding.
Toronto, July, 2013 Mandeep Sood.
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TABLE OF CONTENTS
1.2.1. Peripheral sensory mechanisms ................................................................................ 4
1.2.1.1. Peripheral receptors and their afferent processes .............................................. 4
1.2.2. Central sensory mechanisms ..................................................................................... 7
1.2.2.1. Primary afferent projections to the brainstem .................................................... 7
1.2.2.2. Role of brainstem networks in somatosensory and other functions .................. 9
1.2.3. Ascending projection systems from the trigeminal brainstem sensory nuclear
complex ............................................................................................................................... 9
1.2.4. Organization of the primary somatosensory cortex (S1) ........................................ 11
1.2.5. Role of face-S1 in the control of orofacial movements .......................................... 17
1.3.1. Cytoarchitecture of M1 ........................................................................................... 20
1.3.2. Somatosensory, intra-cortical and extra-cortical inputs to M1 ............................... 21
1.3.3. Motor outputs from M1 .......................................................................................... 22
ABSTRACT .......................................................................................................................... ii
ACKNOWLEDGEMENTS ................................................................................................ iv
LIST OF FIGURES .......................................................................................................... xiii
LIST OF TABLES .............................................................................................................. xv
LIST OF ABBREVIATIONS ........................................................................................... xvi
APPENDIX ...................................................................................................................... xviii
CHAPTER 1: LITERATURE REVIEW ........................................................................... 1
1.1. Introduction .................................................................................................................... 2
1.2. Sensory pathways and related mechanisms ................................................................ 4
1.3. Motor pathways and related mechanisms ................................................................. 19
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1.3.4. Organization of M1 ................................................................................................. 24
1.3.4.1. Motor map as a motor engram ......................................................................... 24
1.3.5. Role of face-M1 in the control of elemental and semiautomatic orofacial motor
functions ............................................................................................................................ 26
1.3.5.1. The role of face-M1 defined from ICMS studies ............................................ 27
1.3.5.2. The role of face-M1 defined from movement-related neuronal activity ......... 34
1.3.5.3. The role of face-M1 defined from cold block and ablation studies ................. 35
1.3.5.4. The role of face-M1 defined from functional organization of sensory inputs . 36
1.3.5.5. The role of face-M1 defined from functional overlapping of somatosensory
inputs and motor outputs in sensorimotor cortex .......................................................... 37
1.4.1. Neuroplasticity of face-M1 associated with altered somatosensory and motor
experience ......................................................................................................................... 39
1.4.2. Neuroplastic changes in face-S1, other cortical and subcortical areas ................... 43
1.4.3. Mechanisms underlying cortical neuroplasticity .................................................... 45
1.4.4. Potential applications of neuroplastic mechanisms in a clinical setting ................. 48
1.4.4.1. Stroke, trauma and spinal cord injury .............................................................. 49
1.4.4.2. Neuroplasticity-based interventions ................................................................ 50
1.5.1. Periodontal mechanoreceptors (PMRs) and their role in orofacial motor control .. 52
1.5.2. Receptor function during and after OTM ............................................................... 54
1.5.3. OTM-induced pain and motor functions ................................................................ 56
1.5.4. Animal models used to study OTM ........................................................................ 60
1.4. Neuroplasticity and its relationship to the role of face-M1 and face-S1 in adaptive
and learning processes ........................................................................................... 38
1.5. Orthodontic tooth movement (OTM) –- its neural regulation ................................ 51
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1.7.1. Statement of the problem ........................................................................................ 62
1.7.2. General hypothesis .................................................................................................. 64
1.7.3. Objectives ............................................................................................................... 64
2.3.1. Animal preparation ................................................................................................. 69
2.3.2. Study groups and dental procedures ....................................................................... 69
2.3.3. Cephalostat and radiographic measurement of OTM ............................................. 71
2.3.4. Statistical Analyses ................................................................................................. 72
2.4.1. Weight gain during the course of the experiment ................................................... 72
2.4.2. Force characteristics ............................................................................................... 73
2.4.3. OTM ........................................................................................................................ 73
2.4.3.1. Amount and rate of OTM ................................................................................ 73
2.4.3.2. Amount of DD of the maxillary molars ........................................................... 74
2.5.1. Force system ........................................................................................................... 75
2.5.1.1. Force delivery method ..................................................................................... 75
1.6. Models used to study orofacial nociceptive behaviour ............................................. 60
1.7. Statement of the problem, hypothesis, and objectives .............................................. 62
CHAPTER 2: DEVELOPMENT OF A RAT MODEL FOR STUDYING
NEUROPHYSIOLOGICAL CHANGES DUE TO ORTHODONTIC TOOTH
MOVEMENT .......................................................................................................... 65
2.1. Abstract ......................................................................................................................... 66
2.2. Introduction .................................................................................................................. 67
2.3. Material and Methods ................................................................................................. 69
2.4. Results ........................................................................................................................... 72
2.5. Discussion ..................................................................................................................... 74
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2.5.1.2. Force parameters .............................................................................................. 76
2.5.2. Amount and rate of OTM ....................................................................................... 77
3.3.1. Animal preparation ................................................................................................. 94
3.3.2. Study groups and dental procedures ....................................................................... 95
3.3.3. ICMS and EMG recordings .................................................................................... 96
3.3.4. Data acquisition and analysis .................................................................................. 99
3.3.5. Statistical Analyses ............................................................................................... 100
3.4.1. General features of face-M1 and face-S1 motor representations.......................... 101
3.4.2. Effects of OTM ..................................................................................................... 102
3.4.2.1. LAD, RAD and GG motor representations in face-M1 ................................. 102
3.4.2.2. LAD, RAD and GG motor representations in face-S1 .................................. 105
3.4.2.3. Distribution of positive ICMS penetrations in face-M1 and face-S1 ............ 106
3.4.2.4. CoG of positive ICMS sites in face-M1 and face-S1 .................................... 107
3.5.1. Features of jaw and tongue motor representations in the face-M1 ....................... 108
3.5.2. Features of jaw and tongue motor representations in the face-S1 ........................ 109
2.6. Conclusions ................................................................................................................... 81
2.7. Figures and Graphs ..................................................................................................... 82
CHAPTER 3: NEUROPLASTIC CHANGES IN THE FACE-M1 AND FACE-S1
ASSOCIATED WITH ORTHODONTIC TOOTH MOVEMENT ................... 89
3.1. Abstract ......................................................................................................................... 90
3.2. Introduction .................................................................................................................. 91
3.3. Materials and Methods ................................................................................................ 94
3.4. Results ......................................................................................................................... 101
3.5. Discussion ................................................................................................................... 108
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3.5.3. Neuroplasticity associated with altered jaw and tongue motor representations
induced by OTM ............................................................................................................. 110
4.3.1. Animal preparation ............................................................................................... 138
4.3.2. Study groups and mechanical and thermal testing procedures ............................. 138
4.3.2.1. Testing procedure .......................................................................................... 139
4.3.2.2. Nature of stimuli and the stimulation sites .................................................... 139
4.3.2.3. Response scoring ........................................................................................... 141
4.3.3. Statistical analyses ................................................................................................ 141
4.4.1. Response threshold related to mechanical stimulation ......................................... 142
4.4.2. Responses related to thermal stimulation ............................................................. 143
4.5.1. OTM pain - Peripheral mechanisms ..................................................................... 145
4.5.2. OTM pain - Central mechanisms .......................................................................... 146
4.5.3. Mechanical and thermal hypersensitivities of the orofacial region as an index of
OTM-induced pain .......................................................................................................... 147
3.6. Conclusions and clinical implications ...................................................................... 115
3.7. Figures and Tables ..................................................................................................... 116
CHAPTER 4: MECHANICAL AND THERMAL HYPERSENSITIVITIES
ASSOCIATED WITH ORTHODONTIC TOOTH MOVEMENT (OTM): A
BEHAVIOURAL RAT MODEL FOR OTM-INDUCED PAIN ...................... 134
4.1. Abstract ....................................................................................................................... 135
4.2. Introduction ................................................................................................................ 136
4.3. Materials and Methods .............................................................................................. 138
4.4. Results ......................................................................................................................... 142
4.5. Discussion ................................................................................................................... 144
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4.5.4. Orofacial mechanical and thermal hypersensitivities contralateral to the side of
orthodontic spring application ........................................................................................ 148
4.5.5. Strengths, limitations and future directions .......................................................... 148
5.1.1. OTM rat model ..................................................................................................... 155
5.1.2. A behavioural rat model that measures mechanical and thermal hypersensitivities
associated with OTM ...................................................................................................... 157
5.1.3. Neuroplasticity in the face-M1 and face-S1 associated with OTM ...................... 159
5.1.3.1 Features of jaw and tongue motor representations in the face-M1and face-S1
.................................................................................................................................... 159
5.1.3.2. Effects of OTM on face-M1 and face-S1 ...................................................... 160
5.4.1. The classic concepts of growth and development of the face vis-à-vis new insights
in neuroscience ............................................................................................................... 167
5.4.1.1. Functional matrix theory ................................................................................ 167
5.4.1.2. Petrovic’s servosystem theory ....................................................................... 168
5.4.2. Equilibrium theory revisited yet again .................................................................. 168
5.4.3. Retention mechanisms and other orthodontic implications .................................. 170
4.6. Conclusions ................................................................................................................. 150
4.7. Figures ......................................................................................................................... 151
CHAPTER 5: GENERAL DISCUSSION AND CONCLUSIONS .............................. 153
5.1. Findings related to specific objectives of the study ................................................. 155
5.2. Possible mechanisms of OTM-induced neuroplasticity in the sensorimotor cortex
................................................................................................................................ 162
5.3. Are OTM-induced neuroplastic changes adaptive or maladaptive in nature? .... 165
5.4. Targeting neuroplasticity mechanisms in orthodontics ......................................... 166
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5.5.1. Validity of the use of ICMS technique to study sensorimotor cortical changes ... 171
5.5.2. Time course and mechanisms involved in OTM-induced sensorimotor cortical
neuroplasticity ................................................................................................................. 173
5.5.3. Sex differences in sensorimotor cortex neuroplasticity, mechanical and thermal
hypersensitivity states, and amount and rate of OTM .................................................... 173
5.5.4. Translation of experimental animal models to human OTM sensorimotor
neuroplasticity and pain states ........................................................................................ 174
5.5. Study limitations and future directions ................................................................... 171
REFERENCES .................................................................................................................. 178
APPENDIX ........................................................................................................................ 240
xiii
LIST OF FIGURES
CHAPTER 2
Fig. 2-1. NiTi closed-coil orthodontic spring activated and stretched between the right three
maxillary molars and the around the maxillary incisors.
Fig. 2-2. A, B The cephalostat assembly to position the head of the rat for taking the
radiographs.
Fig. 2-3. The mean ± SEM change in weight gain for the duration of the experiment in the
E and the S groups.
Fig. 2-4. Force deflection curve of the NiTi closed-coil orthodontic spring.
Fig. 2-5. The mean ± SEM change in the I-M distance during the course of 28 days is
shown in the orthodontic spring side and the control side in the E and the S groups.
Fig. 2-6. The mean ± SEM rate of OTM per week on the orthodontic spring side in the E
group for the 28 days duration of the experiment.
CHAPTER 3
Fig. 3-1. Experiment timeline.
Fig. 3-2. A. Illustration of LAD, RAD, GG, Ma, and Bu muscles where EMG electrodes
were inserted.
Fig. 3-2. B. The intracortical microstimulation (ICMS) mapping area at AP2.5, AP3.0,
AP3.5, and AP4.0 anterior to Bregma.
Fig. 3-2. C. Example of a set of 5 ICMS-evoked EMG responses recorded from LAD of a
single rat.
Fig. 3-2. D. Example of data from C after being rectified and averaged by a 4ms-moving
average window.
Fig. 3-3. A, B, C, D. Nissl-stained coronal sections (100 μm) from a representative rat
sensorimotor cortex at AP planes 4.0 (A), 3.5 (B), 3.0 (C), and 2.5 (D) mm anterior to
bregma.
Fig. 3-4 A, B, C. Representative motor maps of LAD, RAD, and GG in a rat face-M1 and
S1.
xiv
Fig. 3-5. A, B, C, D. The number of positive ICMS sites in left and right face-M1 at ICMS
intensity of 60 μA for LAD (A), RAD (B), and GG (C) in all the groups, and for LAD,
RAD, and GG in the E7 group (D).
Fig. 3-6. A, B, C, D. The number of positive ICMS sites in left and right face-M1 at ICMS
intensity of 60 μA for LAD + GG (A), RAD + GG (B), RAD + LAD (C) and RAD + LAD
+ GG (D).
Fig. 3-7. A, B, C. The number of positive ICMS sites in left and right face-S1 at ICMS
intensity of 60 μA for LAD (A), RAD (B), and GG (C).
Fig. 3-8. A, B, C. The number of positive ICMS sites in left and right face-S1 at ICMS
intensity of 60 μA for LAD, RAD, and GG in the E1 (A), E7 (B), and E28 (C) groups.
Fig. 3-9. AP distribution of the positive ICMS penetrations in the left (-ve coordinates on x-
axis) and the right (+ve coordinates on x-axis) face-M1 and face-S1 irrespective of the
muscle and the ML position.
Fig. 3-10. ML distribution of the positive penetrations in the left (-ve coordinates on x-axis)
and the right (+ve coordinates on x-axis) face-M1 and face-S1 irrespective of the muscle
and the AP position.
Fig. 3-11. A, B, C, D, E, F. CoG of the positive ICMS sites in face-M1 and face-S1 for
LAD, RAD, and GG.
CHAPTER 4
Fig. 4-1. A, B, C. Response threshold evoked by mechanical stimulation at the bilateral
cheek (A), upper lip (B), and maxillary incisor gingival (C) sites.
Fig. 4-2. A, B, C. Responses evoked by noxious thermal stimulation on the cheek site
tested using the total response duration (TRD) (A), response score (B), and response
percentile rate (C).
CHAPTER 5
Fig. 5-1. Petrovic’s servosystem theory on maxillomandibular growth that suggests that
CNS mechanisms play a critical role in the regulation of the growth of the mandible.
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LIST OF TABLES
CHAPTER 3
Table 3-1. Onset latencies (ms) of ICMS (60 μA) –evoked EMG activites in LAD, RAD,
and GG in face-M1 and face-S1 (mean ± SEM).
Table 3-2. Number of positive ICMS sites in the left and right face-M1 and face-S1 for
LMa, RMa, LBu, RBu, and at ICMS intensity of 20 μA for LAD, RAD, and GG.
Table 3-3. Mixed model repeated measures ANOVA, followed by post-hoc Sidak-adjusted
pairwise comparisons to determine whether study group, cortical side, or any interactions
of these effects significantly affected the number of positive ICMS sites for each muscle
and each combination of muscles in face-M1.
Table 3-4. Mixed model repeated measures ANOVA, followed by post-hoc Sidak-adjusted
pairwise comparisons to determine whether study group, cortical side, or any interactions
of these effects significantly affected the number of positive ICMS sites for each muscle in
face-S1.
CHAPTER 5
Table 5-1. Requirements of an ideal OTM Animal Model. Parameters investigated in the
present study.
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LIST OF ABBREVIATIONS
AD anterior digastric
A-OTM adjusted OTM
AP anteroposterior
Bu buccinator
BMI brain-machine interface
CL central lateral nucleus of thalamus
CMA cortical masticatory area
CNS central nervous system
CNX cervical nerve transection
CoG centre of gravity
CPG central pattern generator
DD distal drift
EMG electromyographic
fMRI functional magnetic resonance imaging
GABA γ-amino butyric acid
GG genioglossus
IANX inferior alveolar nerve injury
IASP international association for the study of pain
ICMS intracortical microstimulation
ION-CCI infraorbital nerve-chronic constriction injury
LAD left anterior digastric
LTD long-term depression
LTP long-term potentiation
M1 primary motor cortex
Ma masseter
MEG magnetoencephalograph
MMRM ANOVA mixed model repeated measures ANOVA
ML mediolateral
M-OTM measured OTM
Ni-Ti nickel titanium
NMDA N-methyl-D-aspartate
OTM orthodontic tooth movement
PDL periodontal ligament
PET positron emission tomography
PF parafascicular nucleus of thalamus
PGE prostaglandin
PMA premotor cortex
PMR periodontal mechanoreceptor
PO thalamic posterior nucleus
PPT pressure pain threshold
PVparietoventral
RAD right anterior digastric
RF receptive field
Rt reticular thalamic nucleus
S1 primary somatosensory cortex
xvii
S2 secondary somatosensory cortex
SD standard deviation
SEM standard error of the means
SMA supplementary motor area
TES transcranial electrical stimulation
TMJ temporomandibular joint
TMS transcranial magnetic stimulation
TRD threshold response duration
VB thalamic ventrobasal nucleus
VBSNC trigeminal brainstem sensory nuclear complex
VPLventroposterolateral nucleus of the thalamus
VPM ventroposteromedial nucleus of the thalamus
V - trigeminal
Vm trigeminal motor nucleus
VII facial nucleus
xviii
APPENDIX
Appendix 1. Data from individual animals
CHAPTER 1
LITERATURE REVIEW
Chapter 1
2
1.1. Introduction
The brain has the potential to differentiate and adapt to events in both the external
and the internal worlds. The continuous rapid flow of information from the sensory
receptors is organized in the brain and may result in perceptions that are influenced by the
memory of past experiences, motivations, and emotions to formulate an appropriate
response, such as a well-executed movement. This feed-forward capability of the brain to
coordinate precise muscle activities for a specific function is an integral element of fine
motor skill performance by humans.
Mechanisms of motor control impact the regulation of jaw and tongue function, and
may be influenced for example by a change in the occlusion of teeth through intraoral
alterations such as orthodontic tooth movement (OTM), which is a response to applied
mechanical force that is associated with remodelling of the alveolar bone. It is also known
that the orofacial region (face-M1) of the primary motor cortex (M1) serves a crucial role in
the generation and control of orofacial motor functions, such as jaw, facial and tongue
movements during mastication, speech, and facial expressions. In addition, it is clear that
the orofacial region (face-S1) of the primary somatosensory cortex (S1) uses sensory inputs
ascending the brain and derived from the peripheral receptors to help adapt the orofacial
motor function to the specific task at hand. Further, it has been recently recognized through
studies on rodents, monkeys and humans that the face-M1 also plays a role in the learning
and the adaptive processes associated with alterations of orofacial sensorimotor functions.
This role is effectively performed by the face-M1, in association with the face-S1, through
the property of “neuroplasticity” which refers to the ability of the nervous system to change
its function and structure throughout life in response to learning or alterations in the
external or internal environment.
The goal of the research project underlying this thesis was to establish a rat model
of OTM and to use this model to study the neuroplastic changes of the jaw, cheek, and
tongue motor representations in the face-M1 and face-S1 of adult rats following OTM.
OTM was induced by an activated closed-coil spring that extended from the right maxillary
Chapter 1
3
molar teeth to the maxillary incisors and that produced mesial movement of the right
maxillary molars. Possible neuroplastic changes related to this alteration in the oral cavity
were electrophysiologically studied in the face-M1 and face-S1 by investigating the
organization of motor representations. Motor representations can be evaluated by recording
muscle activities triggered through intracortical microstimulation (ICMS)-activated
projections descending from the face-M1 and face-S1 to excite brainstem motoneurons that
supply the orofacial muscles.
The activation of motoneurons is governed by a complex plethora of networks in
ascending and descending pathways, and the response of motoneurons may be influenced
by alterations in the sensory inputs. Therefore, the following literature review outlines the
anatomical substrate and the physiology of the peripheral mechanisms and ascending
pathways that may be related to OTM. Further, the role of face-M1 and face-S1 and their
mechanisms in influencing jaw, facial and tongue function are discussed. Emphasis in the
review is given to the features of both the rat and the primate sensorimotor cortex because
of the history of primate studies in this field and because this thesis used a rat animal
model. This section also includes a description of the ICMS technique used in this study.
This is followed by a review of the concept of neuroplasticity, along with the studies that
have been performed of face-M1 and face-S1 neuroplasticity and the various proposed
underlying mechanisms. Another segment covers the importance of periodontal
mechanoreceptor (PMR) outputs in control of jaw movements and the role of PMRs in
OTM. Since it is conceivable that OTM-induced pain may influence face-M1 and face-S1
neuroplasticity, a section on OTM-induced pain studies is presented along with the
electromyographic (EMG) studies of muscle function during and after OTM. Further, since
a new OTM rat model has been developed for this study, existing animal OTM models are
also described in the section on various animal models used for OTM studies. Since facial
mechanical and thermal sensitivities were studied during various time periods of OTM to
determine if changes in sensitivities are associated with the changes in face-M1 and face-
S1, animal models that have been used to study orofacial nociceptive behaviour are
described.
Chapter 1
4
1.2. Sensory pathways and related mechanisms
1.2.1. Peripheral sensory mechanisms
1.2.1.1. Peripheral receptors and their afferent processes
Most primary afferents from the orofacial region project to the central nervous
system (CNS) through branches of the trigeminal (V) nerve. The maxillary division
innervates the maxillae and the maxillary teeth, the mandibular division innervates the
mandible, mandibular teeth and anterior 2/3rd of the tongue, while the ophthalmic branch
supplies the eyeball, lachrymal gland, mucous lining of the eye and nose, and muscles of
the eyebrow and forehead. Primary afferents from the posterior tongue, larynx and pharynx
project through the glossopharyngeal (IX) and the vagus (X) nerves. Ear and jaw angle
afferents project through the facial (VII), the glossopharyngeal (IX) and the vagus (X) or
cervical nerves (Byers and Holland, 1977; Hildebrand et al., 1995; Tal and Devor, 2008).
The orofacial region manifests many complex functions that include speech,
mastication, and facial expressions that would not be efficiently performed without the fine
coordination of various muscles of the face and jaws. To accomplish these tasks effectively,
the brain must gather and process extensive sensory information from the lips, cheeks,
tongue, teeth, and the jaws. It is the presence of specialized and non-specialized sensory
receptors in the oral cavity and the face that make these functions possible. The different
types of receptors present are mechanoreceptors, thermoreceptors, chemoreceptors, and
nociceptors (see below). The orofacial tissues have sensory, sympathetic, and
parasympathetic innervations, although the branches of the V sensory nerve axons that are
grouped together into bundles or fascicles are the most predominant (Sessle, 2000).
The skin of the face and the oral mucosa has a rich plexus of free nerve endings and
specialized mechanoreceptors that function as exteroceptors, and these include low-
threshold mechanoreceptors as well as proprioceptors that respond to deformations of
underlying muscles and jaw opening and closing during orofacial function. The facial
muscles that are innervated by the facial nerve lack specialized proprioceptors as these
muscles do not have muscle spindles, Golgi tendon organs, or joint receptors (Trulsson and
Chapter 1
5
Essick, 2004), but since these muscles directly attach to the skin, the activity of these
muscles can stretch the skin and thereby activate skin receptors that transmit sensory
information to the CNS. The mechanoreceptors are also important for the control of the
jaw-opening muscles that have few or no muscle spindles, while the jaw-closing muscles
are endowed with muscle spindles. Muscle spindles are receptors that are extremely
sensitive to stretch of these muscles and aid in guiding the contractile activity during
mastication and other jaw-closing activities (Lennartsson, 1980; Rokx et al., 1984; Miles,
2004). In the tongue, the fast-adapting mechanoreceptors at the tip may respond each time
the tip touches the teeth or other soft tissues, whereas the mechanoreceptors situated deeper
in the tongue muscles may encode tongue movements (Trulsson and Essick, 1997). The
orofacial tissues have the highest tactile sensitivity in the body made possible through their
high innervation density of mechanoreceptors and a highly permissible elastic
deformability of the tissues (Sessle, 2000).
A ‘nociceptor’ is a high-threshold sensory receptor of the peripheral somatosensory
nervous system that is capable of transducing and encoding noxious stimuli (IASP
taxonomy, 2013). Free nerve endings in the tissues function as nociceptors and are
numerous in the orofacial region (Svensson and Sessle, 2004). These receptors are
activated by application of mechanical pressure and also by chemical mediators (e.g. K⁺ ,
prostaglandins, and bradykinins) that are released from cells and vessels damaged by a
peripheral noxious stimulus. Further, the excitability of the nociceptors can be altered
(peripheral sensitization) by multiple factors such as peripheral tissue trauma-induced
inflammation that involves products released from blood vessels or from the cells of the
immune system, release of neurochemicals that are synthesized in the primary afferent cell
bodies [substance P, calcitonin gene-related peptide (CGRP), glutamate, and neurotrophins
such as nerve growth factor], chemicals such as noradrenaline released from the
sympathetic efferents that innervate peripheral tissues, and nerve sprouting or abnormal
nerve changes subsequent to peripheral tissue damage that results in ectopic or aberrant
neural discharges. Neurogenic inflammation involves the release of neuropeptides (e.g.
substance P, CGRP, and neurotrophins) that act on platelets, macrophages, mast cells and
other cells of the immune system to release inflammatory mediators such as histamine,
Chapter 1
6
serotonin (5-HT), bradykinins and cytokines. Since neurogenic inflammation of the
periodontal ligament (PDL) is associated with OTM (Vandevska-Radunovic, 1999; Meikle,
2006) it is discussed in detail in the section on OTM (Section 1.5). The inflammatory
mediators may diffuse through tissues and influence the activity of adjacent nociceptive
endings thereby contributing to the process of “pain spread”. In addition, the increased
peripheral nociceptive activity may lead to an increase in afferent inputs into the CNS
where concomitant functional changes can lead to chronic pain through processes such as
central sensitization (for review, see Sessle, 2000, 2011a; Woolf, 2011).
The PDL also has specialized mechanoreceptors (PMRs); these are distributed
along the dental roots and form a dental plexus. These low-threshold somatosensory
receptors and their afferents to the CNS are responsible for perceptual as well as reflex
responses to intraoral stimuli during functioning of the orofacial tissues. These receptors
provide tactile information and respond to stretch of the PDL collagen fibres, and are found
between the fulcrum and the apex of the tooth (Linden, 1990). Ruffini-like receptors in
close proximity of collagen fibres of the PDL in both animals and humans have been
reported (Byers, 1985; Maeda et al., 1999; Loescher and Holland, 1991) that transmit
inputs via fast-conducting, large-diameter, myelinated A-beta axons. The PDL also
contains nociceptors that are high threshold and are stimulated by a heavy bite or
orthodontic forces, tissue injury, and inflammatory mediators and are associated with
unmyelinated C fibres or small myelinated A-delta fibres (Jyvasjarvi et al., 1988; Sato et
al., 1992; Toda et al., 2004). Since the functions of PMRs are related to the process of
OTM, their morphology and functional properties are covered in detail in the subsequent
section on OTM (Section 1.5).
The dental pulp has a rich innervation and is sensitive to mechanical, thermal and
chemical stimuli. The extensive axon branching of the dental nerve fibres in the pulp is
related to their unique physiological properties and provides a substrate for relatively a
small number of dental afferent fibres in the pulp to be able to innervate an extensive
number of dentinal tubules (Hu, 2004). In contrast to humans, the continuously erupting
Chapter 1
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incisor teeth in rats have a relatively diminutive pulpal innervation and a majority of the
nerve fibres innervate the PDL (Naftel et al., 1999).
Sensory innervation of the temporomandibular joint (TMJ) is mainly derived from
the auriculotemporal (posterior and lateral aspect) , masseteric and temporalis (anterior
portion of the capsule) branches of the mandibular branch of the trigeminal nerve; there is
no innervation within the central portion of the articular disc (Thilander, 1964; Turker,
2002; Suzuki et al., 2012).
The extensive innervation of the orofacial region is responsible for the elaborate
peripheral feedback and feed-forward information that is essential for the coordination of
masticatory muscles in a predictive manner based on learned relationships between patterns
of receptor signals and appropriate efferent signals (for review, see Trulsson and Essick,
2004; Trulsson, 2007). Hence, any alteration in the orofacial environment, either from
changes in the consistency of the diet or dental manipulations may affect the exteroceptive,
proprioceptive and perhaps also the nociceptive inputs into the CNS and thereby may alter
the patterns of jaw and tongue movements during mastication (Murray, 2004; Sessle,
2006).
1.2.2. Central sensory mechanisms
1.2.2.1. Primary afferent projections to the brainstem
The primary afferents of the V system supplying the superficial and the deep tissues
have their primary afferent cell bodies in the V (Gasserian or semilunar) ganglion, except
for the afferents originating from the jaw muscle spindles (e.g. jaw-closing muscles) and
some PMRs that have their cell bodies in the V mesencephalic nucleus within the CNS.
These afferents then project to the V motor nucleus or to the interneuronal sites such as
supratrigeminal nucleus and V subnucleus oralis where they contribute to circuits
underlying craniofacial reflex activity. The central projections from the V ganglion enter
the brainstem and may ascend or descend in the V spinal tract to terminate in one or more
subdivisions of the V brainstem sensory nuclear complex (VBSNC) which consists of the V
main sensory nucleus as well as the V spinal tract nucleus that is subdivided into subnuclei
Chapter 1
8
oralis, interpolaris and caudalis (Capra, 1995; Sessle, 2000; Waite, 2004). Primary afferents
especially of cranial nerves VII, IX, and X form the solitary tract and terminate in the
nucleus of the solitary tract in the brainstem. The low-threshold V mechanosensitive
primary afferents mostly terminate in the more rostral components of the VBSNC and in
laminae III-VI of subnucleus caudalis. A few V nociceptive cutaneous and intraoral
afferents also terminate in some of the rostral components, but a majority of the small-
diameter V nociceptive afferents terminate in subnucleus caudalis, in its laminae I, II, V
and VI (Sessle, 2000).
Many neurons in all the four components of VBSNC contribute to ascending
nociceptive or non-nociceptive pathways that are involved in somatosensory modulation
through their projections to ventrobasal thalamus, periaqueductal gray, pontine parabrachial
area, or reticular formation (see below). Each nucleus/subnucleus has a differential
contribution to each of these projections, e.g. the main sensory nucleus is the principal
direct brainstem relay to ventrobasal thalamus of mechanosensitive afferent input from
most parts of the orofacial region, whereas most subnucleus oralis neurons directly project
to the other brainstem structures. The major projections from subnucleus interpolaris are to
the local circuits and modest projections have been documented to contralateral thalamus,
superior colliculus, reticular formation, periaqueductal grey, inferior olive; ipsilateral
solitary and parabrachial nuclei, and spinal cord (Jacquin et al., 1989a, b).
Electrophysiological investigations and anatomical findings provide evidence that VBSNC
neurons, including nociceptive neurons in subnuclei caudalis and interpolaris, project to the
periaqueductal gray and the parabrachial area (from where there are projections to
amygdala and hypothalamus) as well as to other components of the ipsilateral VBSNC, the
V motor nucleus, contralateral VBSNC and contralateral ventrobasal thalamus (Craig and
Dostrovsky, 1997; Meng et al., 1997; Sessle, 2000, 2009b; Gauriau and Bernard, 2002).
A few of the connections to the reticular formation and other parts of the brainstem
are related to autonomic reflex responses to orofacial stimuli, while other neurons in and
adjacent to the VBSNC (e.g. supratrigeminal nucleus) also serve as interneurons in
craniofacial and cervical muscle reflex pathways. Intrinsic connections between neurons in
Chapter 1
9
the different components of the VBSNC are responsible for the modulatory influences
between rostral and caudal V brainstem neurons (for review, see Capra, 1995; Sessle, 2000;
Waite, 2004).
1.2.2.2. Role of brainstem networks in somatosensory and other functions
Through their projections to the other nuclei in the brainstem or higher brain centres
(see above), the neurons in the VBSNC are involved in perceptual functions (e.g., touch
and pain) as well as in regulating reflex or complex behavioural responses to the peripheral
inputs, e.g. nociceptive orofacial stimuli influences on masticatory muscle activity or
postural activity of the mandible. The subnucleus caudalis, especially its transitional region
with subnucleus interpolaris, appears to be crucial for autonomic responses in cardiac,
adrenal or respiratory function evoked by noxious orofacial stimuli (Meng et al., 1997;
Dubner and Ren, 2004; Ren and Dubner, 2011). The caudalis neurons are also crucial in the
prolonged reflex co-activation of jaw opening and closing muscles that can be evoked by
the noxious stimulation of the craniofacial musculoskeletal tissues (Svensson and Sessle,
2004). The neurons in the rostral components of the VBSNC are involved in reflex circuits,
e.g. the jaw-opening reflex (Bereiter et al., 2000; Sessle, 2000; Svensson and Sessle, 2004).
This demonstrates the close interplay between sensory and motor pathways. Central
NMDA (N-methyl-D-aspartate) and non-NMDA receptors appear to be involved in the
EMG changes reflexly evoked in the rat jaw muscles in response to noxious stimuli. These
observations suggest that nociceptive afferent inputs evoke central NMDA and non-NMDA
receptor activity in the subnucleus caudalis that influences neuromuscular changes,
although these neural changes may be restricted by the involvement of central opioid
mechanisms (Cairns et al., 2001; Svensson and Sessle, 2004).
1.2.3. Ascending projection systems from the trigeminal brainstem
sensory nuclear complex
A majority of the second-order axons arising from the VBSNC cross the midline
and project in a somatotopic manner through the ventral trigeminothalamic tract to the
thalamus. Second-order axons arising from the V main sensory nucleus ascend bilaterally
as the dorsal trigeminothalamic tract and also project in a somatotopic manner to the
Chapter 1
10
thalamus (Asanuma et al., 1980; Chiaia et al., 1991; Diamond et al., 1992; Iyengar et al.,
2007).
The thalamus includes the ventrobasal complex [ventroposterior nucleus (VP) for
primates], the posterior group of nuclei and the medial thalamus (Jones, 1998; Craig and
Dostrovsky, 2001; Sessle, 2005). The ventrobasal complex is composed of two nuclear
subdivisions: the ventral posterior medial subnucleus (VPM) and the ventral posterior
lateral subnucleus (VPL). In primates, VPM comprises the dorsomedial portion of VP, with
VPL nestled around it in the ventrolateral portion of VP. The reticular thalamic nucleus
(Rt) is ventrolateral to VPL. Most of the ventrobasal thalamic neurons are low-threshold
mechanosensitive neurons (Tabata et al., 2002; Henry and Catania, 2006; Iyengar et al.,
2007). These neurons relay detailed thalamic sensory information to the overlying
somatosensory areas of the cerebral cortex in contrast to the neurons present in the posterior
and medial thalamus that have more divergent projections, are less somatotopically
organized and relay less specific information (Asanuma et al., 1980; Bushnell and Duncan,
1987; Chiaia et al., 1991; Diamond et al., 1992; Henry and Catania, 2006; Iyengar et al.,
2007). The posterior nucleus (PO) is a heterogeneous nucleus and is divided into lateral,
intermediate, and medial subdivisions (Jones, 2009). PO serves as a specific paralemniscal
relay station for the somatosensory information in coordination with the lemniscal relay of
the VPM and VPL. Some PO neurons receive information from the spinal trigeminal
nucleus and project to the somatosensory cortex (for review, see Diamond et al., 2003;
Rubio-Garrido et al., 2009; Ohno et al., 2012).
The VPM has pronounced projections to the S1 that are of reciprocal nature. For
the nucleus PO, the ventromedial parts are with major projections to the S1 with reciprocal
inputs and concentrate along the ventral border of PO that neighbours areas of VPM
(Veinante et al., 2000; Henry and Catania, 2006). A collection of intralaminar nuclei are
present dorsomedial to PO, including the central lateral nucleus (CL) and the parafascicular
nucleus (PF). In rats, these intralaminar nuclei have projections to areas in M1 and S1, as
well as to the striatum, thus creating a circuit for the coordination of sensory-motor and
limbic responses (Berendse and Groenewegen, 1990; Fabri and Burton, 1991a, b;
Chapter 1
11
Carmichael and Price, 1995). In primates, the VP projects to Brodmann areas 3b and 1 (see
below) only, whereas area 2 (see below) receive inputs from thalamic nuclei dorsal to VP,
termed the ventroposterior superior nucleus, and area 3a (see below) receives projections
from a separate thalamic nucleus rostrodorsal to VP (Kaas, 1983, Kaas et al., 2006; Iyengar
et al., 2007). The connections to S1 and each nucleus are organized topographically with
discrete representations for the body parts. There are some nociceptive neurons in the
ventrobasal thalamus that have connections with the overlying S1 and are involved in the
sensory-discriminative dimension of pain, whereas nociceptive neurons in the more medial
nuclei of thalamus are involved primarily with the affective or motivational dimension of
pain through their links to the other higher brain areas such as the hypothalamus and
anterior cingulate cortex that also participate in neuroendocrine responses related to the
pain (Svensson and Sessle, 2004).
1.2.4. Organization of the primary somatosensory cortex (S1)
Based on multiple electrophysiological and histological studies, four distinct
architectonic divisions of the primary somatosensory cortex in primates have been
identified, namely, Brodmann areas 3a, 3b, 1, and 2. As the 3b region of the cortex receives
the majority of ascending somatosensory input from the cutaneous tissues it is termed the
S1 proper (Kaas, 1983, Kaas et al., 2006; Iyengar et al., 2007). Cytoarchitecturally, area 3b
is characterized by densely packed cells in layers IV and III and to a lesser extent in layer
VI, and is termed the granular cortex. Layer IV is not as well developed in area 3a, so that
the layer is thin and less granular. In addition, the pyramidal cells in layer V are more dense
and larger than those in area 3b, but not as large or dense as those in the M1 (area 4), where
layer IV all but disappears. In area 1, layer IV, layer III, and layer VI are less densely
packed with cells than in area 3b, thus providing less of a contrast with layer V. In area 2,
layer IV and layer VI become denser again. The areas 3a and 2 receive inputs from muscle
spindles and joints respectively (Kaas, 1983; Kaas et al., 2006; Iyengar et al., 2007).
Neurons receiving orofacial muscle afferent inputs are concentrated in the depth of the
central sulcus within area 3a and in the rostral bank in area 4, while neurons receiving
afferents from the orofacial cutaneous tissue are concentrated in areas 3b and 1, and to
some extent in the area 3a (Huang et al., 1988; Murray et al., 2001). Area 1 also receives
Chapter 1
12
projections from cutaneous tissues, however, the presence of Pacinian receptor inputs to
area 1 only, distinguishes this area from 3b (Kaas, 1983; Kaas et al., 2006; Iyengar et al.,
2007).
There is evidence in primates of three representations of the teeth and tongue, one in
area 3b, another in area 3a, and a third in presumptive area 1. In 3b area, just ventrolateral
to the hand-face septum representation, three partially separated ovals of densely
myelinated cortex represent different structures of the face numbered as F1, F2, and F3,
with F1 corresponding to the representation of the upper face, F2 to the region of the upper
lip, and F3 to the lower lip, chin, and lower face (Kaas et al., 2006). Rostroventral to the
three face ovals are a series of myelin-dense ovals in owl and squirrel monkeys that
represent receptors from the oral cavity, primarily from the tongue and the teeth. In a
caudorostral sequence, these ovals are O1, O2, O3, and O4. Microelectrode recordings in
these ovals indicate that O1 represents the upper and lower contralateral teeth, O2 responds
to touch on the tongue and to a lesser extent to the hard palate, with a majority portion of
the O2 being devoted to the contralateral tongue, and only the more rostral part devoted to
the ipsilateral tongue. O3 responds to ipsilateral teeth, while O4 responds to ipsilateral
tongue. Face-S1 neurons receive mainly contralateral and some ipsilateral and bilateral
exteroceptive inputs from the peripheral orofacial tissues that are organized in a
somatotopic manner that is essential for precise spatial localization of peripheral
mechanoreceptive inputs (Huang et al., 1989b; Lin et al., 1994a; Toda and Taoka, 2002,
2004, 2006; for review, see Kaas et al., 2006). Face-S1 has equal ratios of both rapid and
slow adapting neurons in response to orofacial mechanical stimuli (Lin et al., 1994), and a
few neurons in the areas 3b, 1, and 2 also respond to deep orofacial stimuli (Huang et al.,
1989b).
Rats lack the 4 distinct cytoarchitectonic areas 1, 2, 3a and 3b that are characteristic
of primate S1. The S1 in rats is divided into primary and secondary regions, based on
differences in thalamocortical and intracortical connectivity, organization, and function
(Burton, 1986; Benison et al., 2007). In rats, the S1 region is characterized by a single
systematic map of the body surface, except for the forelimbs that have multiple
Chapter 1
13
representations. Cytoarchitecturally, the map of the body surface resides in the S1 in a
complex array of “barrels” or “isomorphs”, containing dense aggregates of layer IV
granule cells (granular layer) that are distinctly demarcated by the interspersed septa that
are less granular (dysgranular layer) (Chapin and Lin, 1984; Tutunculer et al., 2006;
Foffani et al., 2008). The overall orientation of the cutaneous map in the rat S1 is similar to
that in the primate area 3b, with the cutaneous submodalities toward the rostral S1, and
“deep” submodalities toward the caudal S1 (i.e., with the head representation lateral; the
tail and rear extremities medial; the trunk caudal; and the distal extremities and perioral
regions rostral). This fact has led to the assumption that area 3b in primates is the exclusive
homologue of S1 in rats, and the dysgranular cortex corresponds to area 3a (for review, see
Kaas, 1983; Kaas et al., 2006).
In rats the arrangement of the large barrels, called the postero-medial barrel sub-
field (PMBSF), mimics the arrangement of mystacial vibrissae on the face (Woolsey and
Van der Loos, 1970; Benison et al., 2007). All of the barrels in the rat are particularly
evident within layer IV of S1. Sinus hairs on the lateral and rostral aspect of the face and
furry buccal pad project to a continuous sheet of barrels in the face area of the contralateral
face-S1 (Welker 1971, 1976; Chapin and Lin, 1984). The pattern of projections in this area
indicates that each barrel receives information from afferents supplying one sinus hair with
afferents supplying the large whisking vibrissae projecting to the largest cell dense barrels
and afferents supplying smaller non-whisking sinus hairs project to smaller barrels that are
relatively cell-sparse. Anatomical experiments in rodents indicate that each large barrel is
related to a specific mystical vibrissa (Van der Loos and Woolsey, 1973; Towal et al.,
2011). The highly specialized and specific representation of the vibrissae within S1
provides the substrate, as shown by anatomical and behavioural studies, for the whisking
vibrissae to play an important role in tactile exploration in the rat (Welker, 1964, Towal et
al., 2011). Barrels are also found beyond the region of the head in regions that receive
projections from the forepaw and hindlimb. There are cell-dense areas of S1 that do not
contain barrels and receive projections from other parts of the body. Barrel subgroups and
other somatotopic areas are separated by narrow barrel-free zones.
Chapter 1
14
A relatively large region of S1 in rats is responsive to light tactile stimulation of oral
structures, such as the incisors, hard palate, and tongue. Nearly one-third of S1 consists of
separate representations of the upper and lower front teeth. The contralateral lower incisors
are represented in an area rostral to the representation of the lower lip in S1. The
contralateral tongue representation is located in an oval area caudolateral to the lower
incisor representation, just rostral to the representation of the furry buccal pad [anterolateral
barrel subfield (ALBSF)] in S1. A narrow region of cortex caudal to the tongue
representation contains neurons responsive to light tactile stimulation of the hard palate and
the upper molars. Neuronal receptive fields (RFs) in this region are contralateral and are
large, encompassing several oral structures in addition to the upper molars and palate.
Neurons for the upper incisors are found in a strip of cortex lateral to ALBSF. This region
is bordered on its rostral, caudal, and medial extent by sites responsive to buccal pad
stimulation; however no upper incisor responsive sites are identified within the ALBSF.
Since the upper incisors contact each other in rats, it is not possible to differentiate between
bilateral, contralateral, and ipsilateral upper incisor responses. Similar to the lower incisors,
RFs for the upper incisors include the entire tooth. Neurons responsive to stimulation of
both the upper and the lower incisors are also present lateral to the upper tooth
representation. This region forms a fractured map of the teeth in which neurons respond to
stimulation of the lower, upper, or both incisors (Catania and Remple, 2002; Remple et al.,
2003; Henry and Catania, 2006; Seelke et al., 2012). Rostral to the barrel subfields for the
lower lip and buccal pad two dense modules termed OM1, with predominant lower incisor
representation, and OM2 with tongue representation are reported. The other three dense
modules, namely OM3 with mixed tooth representations, and the forelimb and the hindlimb
module are considered a part of the S2 (see below) (Hayama et al., 1993; Remple et al,
2003).
Evidence from electrophysiological studies suggests that stimulation of vibrissae in
rats results in the activations of not only neurons in layer IV barrels, but also of neurons in
both the supragranular as well as infragranular layers of the cortex (Welker, 1976; Di et al.,
1990; Brett-Green et al., 2004). Neurons in the vibrissal barrel cortex are functionally
organized into vertical columns of which the barrels in layer IV are morphologically
Chapter 1
15
recognizable correlates. Although the rat vibrissal cortex is topographically arranged in
discrete barrel-related columns, the arrangement of neurons in layers within each column
reflect functional and cytoarchitectural order, with the smallest RFs and response latencies
in the middle granular layer IV and deeper segments of layer III, twice the size of RFs and
longer response latencies in supragranular layers, and even larger RFs and response
latencies in the infragranular layers (Armstrong-James et al., 1992; Petersen and Sakmann,
2001; Schubert et al., 2003). These findings suggest that information may be processed
within each barrel-related column in a spatially and temporally organized sequence along
the laminar axis. Based on unit recordings, sequential information-processing in cortical
layer circuitry has been proposed so that processing within the columns originates in layer
IV and the deep parts of layer III, proceeds to the superficial cortical layers and then to the
infragranular layers (Mitzdorf, 1985; Barth et al., 1989, 1990; Schubert et al., 2003).
Similar layer-dominant processing has been documented in somatosensory cortex of
primates (Mountcastle, 1957; Kulics and Cauller, 1986; Kaas et al., 2006). Furthermore,
sensory signal processing in cortical layer IV for rats utilizes two major morphological
classes of excitatory neurons, spiny stellate and pyramidal cells. Spiny stellate cells receive
monosynaptic excitation and inhibition inputs originating almost exclusively from neurons
located within the same barrel, whereas pyramidal cells display additional excitatory inputs
from nongranular layers and from neighbouring barrels; however, the inhibitory inputs for
pyramidal cells originate mainly from neurons located in the same barrel. This indicates
that spiny stellate cells act chiefly as local signal processors within a single barrel, while
pyramidal cells integrate horizontal and top-down information within a functional column
and between neighbouring barrels (Schubert et al., 2001, 2003; Brecht and Sakmann,
2002).
The secondary somatosensory area of rats consists of two distinct areas, referred to
as S2 and PV (parietoventral). Electrophysiological studies have revealed complete (S2)
and nearly complete (PV) body maps in these areas. The vibrissae and somatic
representation of S2 are upright, rostrally oriented, and immediately lateral to the S1, with a
dominant face area. Area PV is located rostral and lateral to the auditory cortex and has a
rostrally oriented and inverted body representation that is dominated by the distal
Chapter 1
16
extremities, with minimal representation of the face or vibrissae. The evidence of
multisensory interactions within S2 and PV suggests that both secondary areas process
features that are chiefly associated with multisensory integration in parallel with unimodal
processing in the primary areas (Brett-Green et al., 2004; Benison et al., 2007).
In rat S1, multiple intrahemispheric as well as interhemispheric connections have
been reported. The cortical representations of the jaws in S1 of both the hemispheres show
dense callosal connections in the granular zones (Hayama and Ogawa, 1997, 2001),
whereas distal body area representation in S1, e.g. vibrissae, tends to have sparse
connections from thalamic and callosal inputs, with the callosal connections chiefly
restricted to dysgranular zones and the thalamic projections terminating in the granular
areas (Wise and Jones, 1977a, b; Akers and Killackey, 1978; Hayama and Ogawa, 1997,
2001). The lower incisor representation in S1 has reciprocal intrahemispheric connections
to neighbouring S1 orofacial areas (including tongue, buccal pad, and chin), M1, and parts
of lateral parietal cortex related to the S2/PV tooth area. Callosal connections from the S1
lower incisor representation project to contralateral S1 within the rostral portion of lower
incisor module 1, the septal regions related to the tongue and teeth, and a small patch of
cortex rostral to the S1 chin and buccal pad representations (Henry and Catania, 2006).
Direct projections from S1 to M1 have also been demonstrated in rodents with a majority of
the face-M1 projections from the granular zones of face-S1 (Donoghue and Parham, 1983;
Fabri and Burton, 1991a, b; Porter, 1996). In primates, the Brodmann cortical areas 3a, 1,
and 2 project directly to cortical area 4 (M1) (Vogt and Pandya, 1978; Burton and Fabri,
1995; Hoffer et al., 2003). In addition, there are considerable amounts of reciprocal
connections among areas 1, 2, 3a and 3b within each hemisphere and between homotopical
cortical areas. Also, area 2 projects to area 4 and area 3a has reciprocal connections with
area 4 (Iyengar et al., 2007; for review, see Kaas et al., 2006). In rats the thalamic nuclei,
VPM and PO, are reciprocally connected to S1 thereby providing the substrate for the S1 to
modulate the somatosensory inputs to different regions of S1.
The findings of these studies should be assessed with the understanding of the
limitations of the methods utilized in the above mapping studies in general. Firstly, the
Chapter 1
17
basis for defining the organization of the cutaneous RFs has been almost exclusively
limited to low-threshold mechanical stimuli. Secondly, it has to be taken for granted that
whether sensory representations are single or multiple depends on the discreteness and
homogeneity of the RFs recorded within each microelectrode penetration (Chapin and Lin,
1984). Further, it has been suggested that the apparent separation of the S1 into different
body representations may suffer from confounds from the use of general anaesthetics that
markedly reduce the sizes of RFs (McKenna et al., 1981, 1982; Duncan et al., 1982). The
impact of these parameters on the cortical maps is discussed in detail in the section on
ICMS below.
1.2.5. Role of face-S1 in the control of orofacial movements
Sensory inputs from oral structures are essential to the health and survival of
mammals. For example, distinctions can be made between edible and non-edible objects in
the mouth, the amount of masticatory force required to effectively crush the food is thereby
determined, and then the tongue and jaw musculature is engaged in the masticatory process.
This is accomplished based on inputs from receptors in the tongue, PDL of teeth, palate,
masticatory muscles, and TMJ (for review, see Jacobs et al., 1998). The range and
complexity of orofacial movements require sophisticated control and coordination of motor
patterns of the face, which necessitate the presence of complex neural circuits that can
perceive the inputs from the peripheral structures and relay the information to the cortex for
controlling and refining the motor activity of the involved orofacial structures.
Insights into the function of S1 have been derived from studies that use procedures
such as ICMS, recordings of peripherally evoked responses and movement-related activity
patterns of single neurons in the S1 and M1 in primates, cats, and rodents, as well as
stimulation, recording, or imaging studies in humans. Studies have shown that S1 firstly
contributes to the control of limb movements even before the initiation and execution of the
limb movements by M1, secondly also plays an important role in the acquisition of new
motor skills involving the limbs, and thirdly that sensory inputs to S1 from the limbs are
important in the learning, control, and modifications of these movements (for review see
Asanuma, 1989; Sanes and Donoghue, 2000; Nicolelis and Lebedev, 2009). Likewise,
Chapter 1
18
experiments employing ICMS, cortical cold block, or single neuron recordings in face-S1
and cortical masticatory area (CMA)/swallow cortex during elemental, learned tasks, or
semiautomatic (e.g., chewing and swallowing) orofacial movements have documented that
the face-S1 receives extensive orofacial sensory inputs and is involved in the fine control of
these orofacial movements, consistent with the findings of analogous studies of the
subprimates face sensorimotor cortex (Lund et al., 1984; Huang et al., 1989b; Lin and
Sessle, 1994; Hiraba et al., 1997, 2000 ; Martin et al., 1997, 1999; Lin et al., 1998; Sessle et
al., 2007).
Although ICMS studies in primates reported EMG responses of the orofacial areas
only from face-M1 (Huang et al., 1989b; Lin et al., 1998; and Martin et al., 1999), it has
been documented that stimulation within area 3a as well as several sites within area 3b can
elicit orofacial movements of the jaws (Burish et al., 2008). Further, ICMS of face-S1 as
well as the insular cortex can evoke rhythmical jaw movements in primates (Huang et al.,
1989a; Lin et al., 1998). In rats also, ICMS of the granular cortex can also evoke orofacial
movements (Donoghue and Wise, 1982; Neafsey et al., 1986; Avivi-Arber et al., 2010b)
and jaw and tongue EMG responses (Adachi et al., 2007; Lee et al., 2006; Avivi-Arber et
al., 2010a). ICMS can evoke tongue movement also from the insular taste sensory cortex
(Neafsey et al., 1986). Further, jaw movements can be evoked by ICMS of S1 in
anaesthetised rabbits (Lund et al., 1984), cats (Hiraba et al., 1997) and the insular cortex in
rats (Zhang and Sasamoto, 1990).
The above-mentioned findings suggest a role for face-S1 in the control of orofacial
motor activity. This is further supported by anatomical evidence of efferent projections
from S1 to motoneurons in primates and rats, or mostly through premotoneurons via lateral
reticular formation at the levels of the caudal pons and the medulla oblongata and
trigeminal brainstem subnuclei in rats (Rathelot and Strick, 2006; Chang et al., 2009;
Yoshida et al., 2009: Tomita et al., 2012; Haque et al., 2012). Also, face-S1 has extensive
connections with face-M1 through axon collaterals and interneurons (Donoghue and
Parham, 1983; Chakrabarti and Alloway, 2006; Henry and Catania, 2006; Iyengar et al.,
2007). Thus, ICMS-evoked EMG activities from S1 can conceivably be the result of spread
Chapter 1
19
of ICMS currents from face-S1 to face-M1 either directly (Cheney, 2002) or indirectly
through axon collaterals (Henry and Catania, 2006; Chakrabarti and Alloway, 2006;
Iyengar et al., 2007). However, this is unlikely, firstly as the distance between face-M1 and
many of the positive ICMS sites within the face-S1 is much larger than the estimated extent
of ICMS current spread of less than 0.5mm at 60 µA ICMS intensity (Asanuma, 1989;
Schieber, 2001; Cheney, 2002). Secondly, many of the positive ICMS sites within the face-
S1 have short onset latencies (8-12 ms), comparable to those of face-M1, thus suggesting
relatively direct projections to brainstem motoneurons rather than projections via face-M1
(Avivi-Arber et al., 2010a). Thirdly, as mentioned above, efferent projections from face-S1
to motoneurons that provide the anatomical substrate have been documented (Rathelot and
Strick, 2006; Chang et al., 2009; Yoshida et al., 2009; Tomita et al., 2012; Haque et al.,
2012).
1.3. Motor pathways and related mechanisms
Orofacial movements include voluntary movements that are regulated by CNS and
that can be refined through sensory inputs to reflex circuits and to higher brain centres.
These voluntary movements can be short-ranged limited movements involving a select
number of muscles (elemental movements, e.g. jaw opening or tongue protrusion) or
diverse movements, e.g. movements of the tongue to alter its shape and position in the
jaws, or even more complex and sophisticated movements that characterize the function of
speech, which involves an elaborate bilateral integration of sensory and motor pathways
and central programming of muscles of the jaw, face, tongue, palate, pharynx, and larynx
and other parts of the upper alimentary and respiratory tract. The orofacial muscles are also
involved in a vast array of reflexes, some involving relatively “simple” reflex circuits (e.g.,
jaw-opening reflex) while others that are more complicated and involve several brainstem
motor neuron pools bilaterally (e.g., cough, gag), and others are even more complex that
necessitate bilateral activity of numerous muscle groups and highly integrated CNS circuits
(e.g., swallowing). Some of these complex motor events may be cyclic in nature, such as
mastication (semiautomatic) that involves alternating rhythmic jaw opening and closing
that is driven by a CNS program produced by a central pattern generator (CPG). Further,
Chapter 1
20
the activity of the orofacial muscles during chewing is sensitive to afferent inputs acting
through brainstem reflex circuits or CNS centres. Therefore, orofacial functions are not
absolutely “motor” but “sensorimotor” in nature since they depend upon and utilize sensory
feedback to initiate or guide them (for review, see Dubner et al., 1978; Sessle, 2006,
2011b).
M1 plays a critical role in the planning, initiation, and execution of movement of
muscles/body part (for review, see Asanuma, 1989; Sanes and Donoghue, 2000; Barbay et
al., 2005) and can be identified by electrical stimulation techniques and by M1
cytoarchitecture (Donoghue and Wise, 1982). The M1 of the rat is identified as the region
in the CNS from which movements can be evoked by applying electrical stimulation and
the location of this region is correlated with cytoarchitectonic features in the frontal and
parietal cortex.
1.3.1. Cytoarchitecture of M1
In rats, M1 is located medial and rostral to S1, and in primates in Broadman’s area 4
that extends along the precentral gyrus. Cortical efferent neurons with different extrinsic
targets are partially segregated in M1, with those of layers II and III projecting to other
cortical areas, while those of layers V and VI projecting to subcortical structures
(Mountcastle, 1997, 2003; Burish et al., 2008). The large pyramidal cells within layer V are
a characteristic feature of M1 in both rats and primates, and the lowest ICMS threshold to
produce movement corresponds with the deepest part of layer V. The layer IV in M1 lacks
the prominent granular appearance that is noticed in S1, and therefore M1 is termed the
“agranular” cortex (Donoghue and Wise, 1982; Sessle and Wiesendanger, 1982; Burish et
al., 2008). In rats, the agranular cortex has been divided into medial and lateral agranular
areas. The medial agranular cortex, also referred to as the secondary motor cortex (M2), is
differentiated from M1 because microstimulation within this area does not produce
movement in ketamine-anaesthetized rats, and M2 is characterized by a pale-staining layer
III and a relatively compact layer II. The lateral agranular area that is referred to as the M1,
lies between M2 and S1 and is characterized by a more homogenous appearance of
superficial layers and a broader layer V. A part of M1 overlaps with two parts of the
Chapter 1
21
adjacent S1 representation - the region of the S1 hind limb and forelimb representations. At
the border between S1 and M1 there is a transition zone where layer IV gradually thins
(Donoghue and Wise, 1982). Caudally, M1 and M2 are very narrow and at the caudal limit
of the S1 cortex these agranular fields merge with the agranular retrosplenial cortex
(Krettek and Price, 1977).
1.3.2. Somatosensory, intra-cortical and extra-cortical inputs to M1
Somatosensory inputs project through the thalamic motor nuclei (VL) in a
somatotopic manner to layer V of M1. In addition, layer III of M1 receives projections
from a wide area of the thalamus, including ventrobasal complex (for review, see
Donoghue et al., 1979; Cicirata et al., 1986 a, b; Asanuma, 1989; Miyashita et al., 1994;
Rausell and Jones, 1995). In addition to receiving direct somatosensory input through the
thalamus, face-M1 receives indirect somatosensory inputs through face-S1. In rats,
anatomical studies demonstrate that the face-M1 neurons receive direct somatotopically
organized inputs from the vibrissae-S1 (Izraeli and Porter, 1995; Porter, 1996). Although
both proprioceptive and exteroceptive inputs are transmitted to limb-M1 (Donoghue and
Parham, 1983; Chakrabarti and Alloway, 2006), vibrissae-M1 neurons receive primarily
exteroceptive inputs (Welker, 1976; Henry and Catania, 2006). In primates, area 3a neurons
receive direct proprioceptive inputs from muscle spindles and from the orofacial cutaneous
tissue, and indirect exteroceptive inputs via areas 3b and 1 that project largely to area 4
(M1) (Huang et al., 1988; Murray et al., 2001; Hatanaka et al., 2005; Kaas et al., 2006;
Iyengar et al., 2007). Further, there are also other varied somatosensory projections from S1
to M1 through long horizontal interneurons and axon collaterals (Schwark and Jones, 1989;
Keller et al., 1990). The electrophysiological finding that any particular locus within M1
tends to receive somatosensory input from the same body part evoked by ICMS at that
locus (Rosen and Asanuma, 1972; Murphy et al., 1978; Murray and Sessle, 1992a, b, c)
implies that somatosensory inputs to M1 also have a scattered and intermingled distribution
similar to the “complex mosaic” of muscle representations within M1 (see below) (Wong et
al., 1978; Lemon, 1988, 2008). A close spatial match as noted here between the peripheral
location of the ICMS-defined motor output response and the peripheral location of the
somatosensory afferent input has been demonstrated by our laboratory (Huang et al. 1988,
Chapter 1
22
1989b; Murray et al., 2001). Mechanosensory afferent input to neurons at tongue-M1 sites
from superficial receptors in the tongue dorsum and within jaw-M1 and tongue-M1 from
afferents supplying PDL have been documented (Murray and Sessle, 1992a, b). Also, a
ƒMRI study in humans has shown that electrically-induced tooth pain activates a cortical
network that includes the M1 (Jantsch et al., 2005). Both corticocortical and
thalamocortical afferents thus distribute their information widely in the M1. M1 also
receives a generous amount of inputs from other cortical areas such as the cingulate cortex,
insular cortex, orbital cortex, S2 and PV (Jones, 1982; Donoghue and Parham, 1983;
Tokuno et al., 1997; Kaas, 2004; Henry and Catania, 2006; Iyengar et al., 2007). Face-M1
also has reciprocal connections with the cortical masticatory area (CMA), premotor
masticatory area (PMA) and supplementary masticatory area (SMA) (Pandya and Vignolo,
1971; Barbas and Pandya, 1987; Hatanaka et al., 2005). In addition, there are considerable
amounts of inter-hemispheric connections (Donoghue and Parham, 1983; Darian-Smith et
al., 1990; Huntley and Jones, 1991).
1.3.3. Motor outputs from M1
The pyramidal tract, with neurons originating in layer V of the M1, is the prominent
descending system influencing motor activity, including that of the orofacial region. Most
pyramidal tract axons originate in the M1 but many axons also originate from S1 (Wise and
Jones, 1977; Zhang and Sasamoto, 1990). In primates and in rats, neurons within the M1
and S1 consist of pyramidal cells (cortical efferents, Betz cells) and stellate cells
(intracortical interneurons) organized in 6 horizontal layers (layer I- layer VI). Anatomical
studies show that face-M1 efferents and some of face-S1 efferents have their cell bodies
located in layer II-VI but most prominently in layer III and layer V and their axons in the
pyramidal tract (corticobulbar tract) project mainly to contralateral brainstem motor nuclei
to activate cranial nerve motoneurons innervating the orofacial muscles. Almost 40% of
efferent neurons from columns of M1 project to a single motoneuron pool in the brainstem,
the remainder project to the motoneuron pools of muscle groups active in similar
movements (Chase et al., 1973; Jones, 1976; Wise and Jones, 1977; Sirisko and Sessle,
1983; Zhang and Sasamoto, 1990; Grinevich et al., 2005; Hatanaka et al., 2005).
Chapter 1
23
Within the layers of M1, recurrent axon collaterals of pyramidal cells project
vertically into a zone that extends through the cellular layers of M1. This zone in M1 is
responsible for a strong excitatory drive to adjacent neurons with glutamate as the main
neurotransmitter. Also, the axons of double bouquet cells (stellate cells), which are present
predominantly in layer II and layer III, with GABA as the main neurotransmitter, contribute
to the vertical pattern of intrinsic connectivity via a columnar surround inhibition (Keller,
1993; Mountcastle, 1997). Other collaterals of the pyramidal cells of layer III and layer V
project horizontally through the cortex and end in terminal clusters in M1 cortical columns
with efferent linkages similar to those of the layer V pyramids of the column of origin
(Huntley and Jones, 1991; Aroniadou and Keller, 1993). Thus, these intrinsic connections
in M1 form an extensive intracortical network of neurons that can collectively contribute to
the regulation of motor efferents and thereby have an impact on the area of effective motor
outputs (DeFelipe et al., 1986; Kwan et al., 1987; Huntley and Jones, 1991; Aroniadou and
Keller, 1993; Huntley, 1997; for reviews, see Keller, 1993; Mountcastle, 1997; Schieber,
2001).
The brainstem motor nuclei include several motoneuron pools and a motoneuron
pool includes all the motoneurons innervating a specific muscle. Although each pyramidal
tract neuron diverges extensively and a majority of them innervate several motoneurons
pools within a particular brainstem motor nucleus or within different motor nuclei,
neighbouring pyramidal tract neurons converge to innervate most heavily a particular
motoneuron pool that innervates a particular muscle (for review, see Schieber, 2001; Miles,
2004). M1 neurons can project directly to brainstem motor nuclei to activate motoneurons,
however, a majority of projections in primates and rats related to the masticatory
movements are multisynaptic and project to brainstem motoneurons via brainstem
premotoneurons located in the brainstem reticular formation, as well as the V main sensory
nucleus and the subnuclei oralis and interpolaris (Zhang and Sasamoto, 1990; Takada et al.,
1994, Bourque and Kolta, 2001; Luo et al., 2001; Hatanaka et al., 2005; for review, see
Capra, 1995; Sessle, 2000, 2009a). In addition, these M1 projections to premotoneurons
may also project either directly or indirectly through the basal ganglia, red nucleus,
vestibular nuclei, superior colliculus and cerebellum (Takada et al., 1994; Takada et al.,
Chapter 1
24
1999; Hatanaka et al., 2005; Satoh et al., 2006). For voluntary control of jaw muscles, the
premotor neurons are bypassed with neurons projecting directly from the M1 to V motor
nuclei (Vm) (Lund et al., 1998; Miles, 2004). The Vm receive input from CMA and SMA
in primates (Takada et al., 1994; Hatanaka et al., 2005) and the agranular insular cortex in
rats (Zhang and Sasamoto, 1990). Further, the brainstem motor nuclei receive extensive
somatosensory afferents from the skin, teeth, muscles and joints either directly or indirectly
through the premotoneurons (Marfurt and Rajchert, 1991; Tolu et al., 1994; Dessem et al.,
1997; Luo et al., 2001). Finally, there are many brainstem interneurons (for review, see
Dubner and Sessle, 1978; Lund et al., 1999, Sessle, 2009a) and connections between
various brainstem motor nuclei, such as between Vm and hypoglossal motor nuclei (XIIm),
and Vm and facial motor nuclei (VIIm) (Manaker et al., 1992; Zhang et al., 2001; Luo et
al., 2006).
In a motor unit, each muscle fibre is innervated by 1 motoneuron and each
motoneuron innervates many muscle fibres. Muscles involved in fine precise motor
movements, e.g. the tongue, have large number of motor units with a smaller innervation
ratio. The V motoneurons project from the Vm in the brainstem through the motor branch
of the V mandibular nerve to innervate the ipsilateral jaw muscles (anterior digastric,
masseter, temporalis and pterygoids), the XII motoneurons project from XIIm through the
XII cranial nerve to innervate the ipsilateral tongue muscles [extrinsic (genioglossus,
hyoglossus, styloglossus) and intrinsic (superior and inferior longitudinal, verticalis,
transverses)], although the palatoglossus muscle of the tongue is innervated by the X nerve.
The VII motoneurons project from the VIIm through the VII cranial nerve to innervate the
ipsilateral muscles of facial expression, and also the vibrissal muscles in rodents (for
review, see Miles, 2004; Waite, 2004).
1.3.4. Organization of M1
1.3.4.1. Motor map as a motor engram
Since the 1ate 1800s, the M1 has been known to have a very precise somatotopic
organization, with different regions representing the three major body motor regions,
Chapter 1
25
namely, face, forelimbs, and hindlimbs. In the last quarter of the 20th century, however,
experimental evidence especially from monkeys and rats has indicated that somatotopy in
M1 is not spatially discrete or sequentially arranged at the micro-level. Rather, underlying
gradual somatotopic gradients of representations, the representations of different smaller
body parts or muscles are scattered within the face, forelimb, or hindlimb representations,
such that the representations of any two smaller parts overlap extensively. Since cortical
territory for each muscle is extensive enough to preclude spatially separate territories for
each muscle representation in the M1, outputs from large territories of M1 converge on the
motoneuron pool of any particular muscle (Humphrey, 1986; Sato and Tanji, 1989,
Donoghue et al., 1992; for review, see Schieber, 2001). Similar features exist in ICMS
maps of the face representation as well (Huang et al., 1988). Subsequently, several studies
have confirmed that ICMS maps show multiple scattered loci within M1 from which
threshold stimulation evokes movement about a given joint or EMG activity in a given
muscle, and these M1 areas also are threshold loci for not one exclusive but multiple
movements or muscles, thereby forming a “complex mosaic” of multiple muscle
representations scattered in-between (Nudo et al., 1992). In addition, as the stimulus
intensity is increased above threshold, current spreads to adjacent M1 sites so that
movements are produced at additional joints (Sessle and Wiesendanger, 1982), or
contractions are evoked in other muscles (Donoghue et al., 1992), so that the loci for any
given muscle expand and coalesce. This demonstrates the extensive total territory
representing that muscle, which overlaps with the representations of the neighbouring
muscles (Humphrey, 1986; Sato and Tanji, 1989).
M1 is also thought to play a role in the learning of novel motor skills. Motor skill
acquisition occurs through modification and organization of muscle synergies that are
stored in “motor maps” formed in M1. The learning process is reflected
neurophysiologically as a reorganization of movement representations within M1,
suggesting that the motor map is a “motor engram”. The motor map topography reflects the
capacity for skilled movement and forms the basis for the related concept of neuroplasticity
that is discussed in the next section. The specific contribution that motor maps make to
movement is controversial, however, the motor map itself may represent the capacity of the
Chapter 1
26
M1 to both produce and acquire skilled movement through a motor engram (Monfils et al.,
2005). The existence of motor maps in M1 reflects a sufficient extent of maturation within
the corticospinal/corticobulbar system to support the capacity to produce dexterous
movement (Reitz and Muller, 1998; Fietzek et al., 2000; for review, see Monfils et al.,
2005). Thus, disrupting cortical circuitry (e.g. through cold block or ablation) leads to a
loss of motor maps and a degradation of skilled movement (see below). These results
support the concept that motor maps reflect a level of synaptic connectivity within the M1
that is required for the performance of a skilled movement. Further, motor map topography
reflects its predisposition for the activation of particular muscle/group of muscles for
execution of complex movement. For example, primates that display a high degree of digit
dexterity have a proportionately larger area of the motor map dedicated to digit
representation in contrast to rodents with little digit dexterity (Nudo et al., 1992; Kleim et
al., 1998). Conversely, rodents have a higher degree of vibrissal motor control than
primates and therefore have a larger proportion of the motor map dedicated to vibrissal
motor representations (Donoghue and Sanes, 1988; Franchi, 2001; Franchi et al., 2006).
Also, within-subject variations in map topography reflect the motor capacity. For example,
the hand representation of the dominant hand in primates is larger and more complex than
that of the non-dominant hand (Nudo et al., 1992). These findings suggest that M1 is
involved in the acquisition and performance of skilled movements and that the stimulation
derived motor maps represent a motor engram.
1.3.5. Role of face-M1 in the control of elemental and semiautomatic
orofacial motor functions
The orofacial area performs complex functions that involve coordinated bilateral
voluntary movements that include elemental movements, such as jaw opening and closing,
tongue protrusion or retraction, and semiautomatic movements such as mastication and
swallowing as well as vibrissal whisking movements in rodents. It has been well
established for some time that voluntary orofacial motor functions are initiated and
controlled by face-M1. However, it has been only in the last two decades that studies have
suggested the involvement of face-M1 also in the generation and control of the
Chapter 1
27
semiautomatic orofacial movements, e.g. chewing and swallowing, that for many decades
were considered to be under the control of brainstem central pattern generators (CPGs).
Voluntary movements are driven by CNS and can be refined through sensory inputs to
reflex circuits and to the higher CNS centres. In contrast, semiautomatic movements are
orofacial reflex in nature and involve the brainstem motor neuron pools. Other movements
such as chewing involve highly integrated CNS circuits and may involve a feature of
learned motor function (Sessle, 2011b).
Numerous methods have been used to study the role of the M1 in the initiation and
control of these functions (for review, see Toga and Mazziotta, 2002; Nicolelis and
Lebedev, 2009). Electrophysiological methods have employed single neuron recordings
and electrical stimulation of the face-M1. Single neuron recordings used transdural
microelectrodes and recordings of peripherally evoked responses and movement-related
activity patterns of single neurons in the face-M1. Electrophysiological mapping of motor
representations uses electrical stimulation applied to specific sites within face-M1 to
observe the evoked muscle movements and/or record evoked EMG responses. Electrical
stimulation can be applied either by surface cortical stimulation or by subdural
microelectrodes (ICMS) in animals or by transcranial magnetic stimulation (TMS) in
humans. Studies using these techniques, imaging studies in humans, and various central and
peripheral manipulations of the sensorimotor systems have been instrumental in revealing
that the face-M1 plays a crucial role not just in the initiation and control of orofacial motor
functions but also in adaptation and learning processes.
1.3.5.1. The role of face-M1 defined from ICMS studies
1.3.5.1.1. The ICMS technique
The ICMS technique introduced by Hiroshi Asanuma and colleagues in 1967 is a
tool for investigating sensory and motor maps (Asanuma and Sakata, 1967). ICMS consists
of applying currents through a microelectrode to activate clusters of neurons in the vicinity
of the electrode tip. ICMS in the M1 produces activation of centrifugal neurons projecting
to motoneurons and results in movement of the part of body supplied by the activated
motoneurons (Asanuma and Rosen, 1972; Cheney, 2002). ICMS along with other
Chapter 1
28
electrophysiological methods have been used to reveal the properties of individual neurons
or discrete clusters of neurons and their relation to the larger body map. Although ICMS is
considered the technique of choice for many types of mapping experiments (Cheney,
2002), as the procedure is invasive its use is limited to studies in animals. It can only be
used in humans when they are undergoing a neurosurgical procedure that provide access to
the brain, such as is done for patients with Parkinson disease, epilepsy, chronic pain and
brain tumors. The procedure is time consuming since multiple cortical sites of interest must
be determined before a complete map of a body part can be assembled. Further,
subthreshold effects on muscle activity cannot be detected and muscles that are activated
most strongly will dominate the movement that is evoked.
One of the output measures used for M1 mapping is ICMS-evoked EMG activity
related to the muscle/body part under study. Tabulating the topography of evoked
movements from electric stimulation of M1 is the simplest and quickest method for
generating a motor map. ICMS can activate corticospinal and corticobulbar outputs that
stimulate sufficient premotor and motoneurons to produce a movement that can be detected
by recording EMG activity from individual muscles. EMG responses can be averaged over
several stimulation trials to produce a reliable and quantitative end-point measure. Features
of ICMS-evoked EMG response include its threshold (i.e. the lowest ICMS intensity that
evokes an EMG response for more than 50% of the stimulation trials), amplitude, duration,
and onset latency. When constant stimulation parameters (e.g. intensity, frequency and
duration) are used, changes in the features of the ICMS-evoked responses may indicate
changes in the strength of the corticobulbar/corticospinal projections to the motoneurons
(Ranck, 1975; Asanuma, 1989; Greenshaw, 1998). On the other hand, altering the
stimulation parameters (stimulus current, stimulus duration, stimulus polarity, and electrode
dimension) as well as changing other parameters such as the cortical depth at which the
ICMS is applied, state of anaesthesia of the animal, previous stimulation, initial posture of
the body part/muscle, as well as individual variations, can all impact the features of the
ICMS-evoked EMG responses and thereby may influence the overall mosaic of
muscle/movement representation in M1 (Sessle and Wiesendanger, 1982; Asanuma, 1989;
Greenshaw, 1998; Graziano et al., 2002; Tehovnik et al., 2006).
Chapter 1
29
The ICMS-related cortical motor representation for a muscle is defined by the sites
in the cortex where the application of electrical stimulation can trigger a response from that
muscle. Electrical stimulation applied to several cortical areas including the face-M1, face-
S1, SMA, CMA, and the cingulate motor area can evoke orofacial muscle responses.
However face-M1 has the lowest ICMS threshold for evoking a muscle twitch/movement.
(Donoghue and Wise, 1982; Neafsey et al., 1986; Huang et al., 1989b; Lin et al., 1994b;
Martin et al., 1999; Murray et al., 2001; Hatanaka et al., 2005; Avivi-Arbor et al., 2011;
Sessle, 2011b).
The amount of current introduced through a microelectrode that is essential to
directly activate a neuron (i.e., cell body or axon) is proportional to the square of the
distance between the neuron and the electrode tip (Rattay, 1987; Cheney, 2002). The extent
of effective current spread from the stimulating electrode is an important issue for the
interpretation and for defining boundaries within the M1 map. The estimates of current
spread in layer V suggest that a 10-µA stimulus will directly activate 1-12 large pyramidal
tract neurons and 180-2168 small pyramidal tract neurons (Cheney and Fetz, 1985). Most
of the corticospinal neurons that discharge in response to ICMS are excited directly at the
soma or axon hillock. In cat M1, it has been estimated that low amplitude ICMS pulses
directly excite to the order of 28 pyramidal neuron somata within a radius of 88 µm (Stoney
et al. 1968). In baboon, a 0.2 ms 35 µA pulse delivered in layer V has been estimated to
directly excite 90 - 900 small and 1- 5 large pyramidal neurons within a radius of 40 - 125
µm, whereas at 90 µA the effective spread of stimulating current has been estimated to be
only 0.6 mm (Andersen et al., 1975). Thus, direct excitation of neuronal somata or axon
hillocks by ICMS is considered to be reasonably focal (Schieber, 2001).
Apart from the ICMS technique, the introduction of transcranial electrical
stimulation (TES) and magnetic stimulation (TMS) of the cortex has provided new non-
invasive tools to explore cortical function and motor maps in humans (Merton and Morton,
1980). However, the spatial and temporal resolution of these techniques is much less than
that ICMS evokes. Imaging methods such as functional magnetic resonance imaging
(fMRI) and positron emission tomography (PET) can provide indirect measures of brain
Chapter 1
30
activity during a particular behaviour of interest. However, the disadvantages of these
imaging modalities are their relatively low spatial and time resolutions compared to
electrophysiological measures. Time resolution with high channel-density
electroencephalography (EEG) recording and magnetoencephalography (MEG) is excellent
but their relatively low spatial resolution is still a disadvantage. Therefore, combining
electrophysiology and imaging methods constitutes a powerful approach to study the
constitution of motor maps (Cheney, 2002). Since the ICMS technique was used to map the
M1 in the study reported in this thesis, the review will focus on ICMS-related findings for
face-M1.
1.3.5.1.2. ICMS-defined features of face-M1
ICMS studies of face-M1 utilizing low-threshold, short-duration ICMS (e.g. 0.2 ms
pulses, 333 Hz, 35 ms, < 30μA) have revealed extensive topographic organization of the
orofacial muscles within the face-M1. ICMS-defined multiple efferent microzones in the
face-M1 appear to represent the different functional contingencies in which the orofacial
muscles (e.g. tongue and jaw muscles) participate. These microzones are organised in a
mosaic-like region where each region represents a muscle (or movement) or group of
muscles (or movements) with predominantly contralateral representations, although
extensive ipsilateral representations also exist ( Neafsey et al., 1986; Huang et al., 1989b;
Murray et al., 1991; Miyashita et al., 1994; Brecht et al., 2004; Lee et al., 2006; Adachi et
al., 2007; Burish et al., 2008; Tandon et al., 2008; ; Guggenmos et al., 2009; Avivi-Arber et
al., 2010a, b; for review, see Sanes and Donoghue, 2000; Luscher et al., 2000; Sanes and
Schieber, 2001; Schieber, 2001; Avivi-Arber et al., 2011; Sessle, 2011b). Analogous
studies in humans that have used TMS of the cortex and recording of evoked muscle
responses, as well as neuroimaging techniques such as fMRI also reveal extensive
representation of the orofacial region within an area consistent with the presumed location
of face-M1 (Svensson et al., 2003b; Svensson et al., 2006; Boudreau et al., 2007;
Nordstrom, 2007; Sowman et al., 2009; Iida et al., 2010; Arima et al., 2011). Such
extensive bilateral representation of the orofacial region within face-M1 may be suggestive
of its role in the control of bilateral orofacial movements.
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Although some voluntary orofacial movements such as jaw-opening, tongue
protrusion, and vibrissal movements can be evoked by ICMS (Donoghue and Wise, 1982;
Huang et al., 1989b; Murray and Sessle, 1992a; Yao et al., 2002a; Lee et al., 2006; Adachi
et al., 2007; Burish et al., 2008; Avivi-Arber et al., 2010a), other movements such as jaw-
closing are not frequently evoked by similar ICMS parameters (Neafsey et al., 1986; Huang
et al., 1988; Murray et al., 1991; Murray and Sessle, 1992a). Thus, face-M1 may play a
vital role in the generation of many but not all orofacial movements. However, unit
recordings have documented that jaw-M1 neurons discharge more rapidly during a stronger
bite force (Luschei et al., 1971; Hoffman and Luschei, 1980) and also show different
patterns of firing rates during different jaw-closing and biting tasks (Murray and Sessle,
1992b; Murray et al., 2001). This suggests that face-M1 does play a role in the control of
jaw-closing movements. Further, a significant increase in the spontaneous EMG activity of
the masseter muscle subsequent to the cold block of face-M1 has been reported (Yamamura
et al., 2002). Therefore it can be suggested that the masseter representation in face-M1
principally involves inhibitory effects of face-M1 neurons on masseter motoneurons (Chase
et al., 1973). Another viewpoint that is suggested is that antagonistic
corticospinal/corticobulbar neurons are closely grouped or intermingled in M1, and thus
antagonistic motor cortical points are linked by excitatory intracortical connections held in
check by local GABAergic inhibition (Ethier et al., 2007). Therefore the masseter
representation may be masked by an inhibitory effect of intracortical interneurons.
ICMS studies in animals have revealed that the jaw and tongue muscles have a
considerable amount of overlapping motor representations. It has been shown that a
specific site within face-M1 represents both jaw and tongue muscles/movements as
reflected in a similar ICMS threshold for evoking muscle responses (Neafsey et al., 1986;
Huang et al., 1988; Murray and Sessle, 1992a; Murray and Sessle, 1992b; Adachi et al.,
2007; Burish et al., 2008; Avivi-Arber et al., 2010a; for review, see Sanes and Donoghue,
2000; Murray et al., 2001; Sanes and Schieber, 2001; Schieber, 2001; Tehovnik et al.,
2006, Avivi-Arbor et al., 2011; Sessle, 2011b). Long-duration ICMS trains were used in
recent studies in primates in the orofacial representation area to evoke consistent short-
latency (< 33 ms) movements of the mouth, lips and tongue towards a specific orofacial
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posture (Graziano et al., 2002a, b; Graziano and Aflalo, 2007a, b). These series of studies
suggest that long-duration ICMS trains applied to face-M1 can evoke coordinated, complex
movements arranged within the M1 in a map of meaningful body postures in space,
suggesting a combined and selective activation of a group of different efferent areas. These
observations also highlight the role of face-M1 in the control of coordinated orofacial
motor functions. As noted below, long-duration ICMS can also evoke other complex
semiautomatic orofacial movements.
The activities of chewing and swallowing in all mammals as well as vibrissal
whisking in rodents are semiautomatic movements that require coordinated activity of the
bilateral orofacial muscles. These semiautomatic movements are under the control of
subcortical brainstem neuronal networks (CPG) (Gao et al., 2001; Berg and Kleinfeld,
2003; for review, see Dubner et al., 1978; Sawczuk and Mosier, 2001; Lund and Kolta,
2006). However, electrophysiological studies in primates, rats and in ƒMRI studies in
humans collectively suggest that face-M1 also plays a role in the regulation of
semiautomatic orofacial movements (Huang et al., 1989a; Aziz et al., 1996; Yao et al.,
2002a; Martin et al., 2004), also supported by M1 cold block findings (see below) (Murray
et al., 1991; Narita et al., 2002; Yamamura et al., 2002). In primates and rats, long-duration
ICMS trains (e.g. 0.2ms pulses, 50Hz, 3sec, <60μA) can evoke rhythmic masticatory and
swallowing movements from within face-M1 (Huang et al., 1989a; Sasamoto et al., 1990;
Zhang and Sasamoto, 1990; Martin et al., 1999; Yamamura et al., 2002; Yao et al., 2002a;
Satoh et al., 2006). Further, different patterns of ICMS-evoked rhythmic jaw movements
are associated with different patterns of EMG activity (Huang et al., 1989a; Martin et al.,
1997). In addition, ICMS within face-M1 of rats can modulate brainstem and basal ganglia
neuronal activity associated with the rhythmical jaw movements (Zhang and Sasamoto,
1990; Nishimuta et al., 2002; Satoh et al., 2006). Similarly, ICMS of the vibrissal area of
M1 in rats can evoke rhythmic vibrissal movement through its actions on a subcortical CPG
(Berg and Kleinfeld, 2003; Haiss and Schwarz, 2005; Cramer and Keller, 2006).
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1.3.5.1.3. What is exactly being mapped in M1 with ICMS?
ICMS mapping in M1 entails placing the microelectrode tip at a certain intracortical
position in or near layer V, and gradually adjusting stimulus strength until at least 50% of
stimulation trials initiate the discharge of some motor units detected by recording EMG
activity. The recorded EMG activity suggests that the evoked output from M1 to a
particular muscle or group of muscles responsible for the recorded movement activity is
greater than the output to other muscles which was insufficient to cause them to discharge.
The discrete zones of ICMS maps obtained with threshold stimuli thus represent the
quantitatively most effective outputs, not qualitatively exclusive outputs. Since cortical
layer III has mainly horizontal connections and layer V is the main
corticobulbar/corticospinal output layer, ICMS of cortical layer V is associated with the
lowest threshold values and ICMS of layer III evokes responses at higher threshold values
due to indirect activation of or current spread to pyramidal tract neurons (Asanuma et al.,
1976; Sapienza et al., 1981). Further, neural elements perpendicular to the electrode surface
(layer III) are predominantly excited by anodic stimulation while cathodic stimulation
excites those with a direction parallel to its surface (layer V) (Du et al., 2009; Parikh et al.,
2009; Yazdan-Shahmorad et al., 2011a, b).
1.3.5.1.4. Influence of general anaesthesia on ICMS-induced cortical excitability
Acute ICMS experiments often require anaesthetics and the duration of experiments
may be long, often in the range of 12-24 hrs. Many types of general anaesthetics have been
used for the ICMS mapping procedures such as thiopental, propofol, alphaxalone-
alphadolone, tiletamine-zolazepam, and ketamine. Ketamine is often used in human and
veterinary medicine, but there are concerns regarding the use of ketamine in M1 mapping.
Ketamine is a noncompetitive NMDA receptor antagonist (Di Lazzaro, 2003). Since
NMDA receptors have been implicated in synaptic mechanisms of neuroplasticity, the use
of an NMDA receptor antagonist as an anaesthetic during the mapping procedures could be
considered to be problematic. However, ketamine is a relatively weak NMDA receptor
antagonist compared with typical neuroprotective agents and is commonly used in ICMS
mapping studies because it is one of the few general anaesthetics that does not abolish
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ICMS-evoked muscle activity as the NMDA receptor antagonist property of ketamine does
not have an impact on the depolarization of the cortical axon (Nudo et al., 2003).
It has been reported that deeper states of general anaesthesia tend to wax and wane
over the course of several hours during the induction of ketamine and can influence cortical
excitability and in turn can be manifested as changes in the extent of motor representations
within M1 and in S1 (Gioanni and Lamarche, 1985; Nudo et al., 2003; Tandon et al., 2008).
Remarkably, EMG responses in orofacial muscles can be evoked by low ICMS intensities
in anaesthetised as well as in awake animals (Sapienza et al., 1981; Huang et al., 1989b;
Tandon et al., 2008), and almost congruent motor maps have been obtained from different
studies using different anaesthetic agents (Nudo et al., 2003) or different states of
anaesthesia or consciousness (Huang et al., 1989b; Tandon et al., 2008). Further, spike-
triggered averaging has documented similar EMG activity in awake animals and those with
repetitive ICMS in anaesthetised animals (Cheney, 2002).
Therefore, ICMS using ketamine anaesthesia can be an appropriate technique to
reveal the organizational features of face-M1. The results from anaesthetised animals are
generally comparable to those derived from studies with awake animals. Yet it is crucial to
regulate the administration of the anaesthetic and to maintain a stable level of general
anaesthesia that allows for EMG activity evoked by relatively low ICMS intensities (Nudo
et al., 2003).
1.3.5.2. The role of face-M1 defined from movement-related neuronal activity
Single neuron recordings of the ICMS-defined face-M1 areas in primates have
revealed that many face-M1 neurons are active in relation to a tongue or jaw movement
task (Murray and Sessle, 1992b, c; Yao et al., 2002a). This is consistent with fMRI studies
of movement-related activations in face-M1 in humans (Corfield et al., 1999; Hamdy et al.,
1999; Martin et al., 2004). However, this movement-related neuronal activity can also be
related to co-activation of other orofacial muscles associated with the tongue or jaw
movements such as facial muscles (Moustafa et al., 1994). Some of the face-M1 neurons
representing the tongue and the jaw muscles can either increase or decrease their firing rate
even prior to the onset of a tongue or jaw muscle activity; also different trajectories of
Chapter 1
35
tongue movement have different patterns of neuronal firing (Luschei et al., 1971; Murray
and Sessle, 1992b; Murray et al., 2001; Yao et al., 2002a), and some of the M1 neurons
discharge more rapidly during a stronger biting force (Luschei et al., 1971; Hoffman and
Luschei, 1980). Further, many of face-M1 neurons can be activated by mechanical
stimulation of orofacial mechanoreceptive fields (Huang et al., 1989b; Murray et al., 2001;
Murray and Sessle, 1992a; also see below) and since there are extensive intracortical
projections from S1 to M1 as mentioned above, it is possible that some of the movement-
related face-M1 neuronal activity is a reflection of the sensory inputs generated by the
orofacial movement and projected to face-M1, either directly or indirectly, through face-S1
(Yao et al., 2002a). Studies of the vibrissal area of M1 in freely moving rats have shown
task-related neuronal activity whereby the discharge of face-M1 neurons correlates with the
level of a muscle output as measured by vibrissal EMG activity (Carvell et al., 1996).
These various studies suggest that face-M1 does play an important role in the integration of
sensorimotor information and the control of orofacial movements.
Single neuron recordings studies in primates during semiautomatic orofacial
movements have revealed that many face-M1 neurons also have differential activities
dependent on the different phases of the chewing and swallowing cycles (Huang et al.,
1989a; Martin et al., 1997; Yao et al., 2002a; for review, see Murray et al., 2001; Sawczuk
and Mosier, 2001). These movement-related neuronal activities suggest that face-M1 plays
an important role in the generation and control of orofacial semiautomatic movements,
supporting the findings from ICMS studies (see above) and cold block studies noted below.
1.3.5.3. The role of face-M1 defined from cold block and ablation studies
Inactivation of the face-M1 by bilateral lesioning or reversible bilateral cold block
severely impairs the animal’s ability to perform a tongue-protrusion task (Castro, 1972;
Castro, 1975; Murray et al., 1991); however the procedures have a minimal effect on the
animal’s ability to maintain a learned biting task (Luschei and Goodwin, 1975; Murray et
al., 1991). In primates, bilateral but not unilateral ablation of face-M1 alters patterns of
masticatory jaw movement (Larson et al., 1980) and bilateral cold block disrupts but does
not prevent coordinated masticatory movements (Narita et al., 2002; Yamamura et al.,
Chapter 1
36
2002). Similarly in rats, unilateral lesioning of the vibrissal area of M1 disrupts but does
not prevent coordinated rhythmic whisking movements (Gao et al., 2003), and ablation of
M1 can alter rhythmical jaw movements (Sasamoto et al., 1990). These findings suggest
that while face-M1 plays a role in the generation of tongue protrusion and jaw-opening
movements, it also has a limited role in the control of jaw-closing movements, thereby
indicating a possible modulatory role of face-M1 in semiautomatic orofacial movements.
1.3.5.4. The role of face-M1 defined from functional organization of sensory inputs
Primate studies have revealed that face-M1 receives prominent somatosensory
inputs from the orofacial tissues that are characterized by multiple representations of
somatosensory inputs from the same orofacial area to which the outputs of face-M1 are
directed. These inputs can be excitatory or inhibitory and a majority of them are derived
from contralateral orofacial sites, although a considerable amount of M1 neurons receive
ipsilateral or bilateral inputs (Gould et al., 1986; Huang et al., 1989b; Murray and Sessle,
1992a; Yao et al., 2002b). In awake primates, most face-M1 neurons receive primarily
exteroceptive inputs from the orofacial tissues, especially from the upper lip, lower lip, and
tongue and limited proprioceptive inputs (Huang et al., 1989b). However some jaw-M1
neurons receive exteroceptive and proprioceptive inputs from the jaw muscles and PDL
(Luschei et al., 1971; Huang et al., 1989b; Murray and Sessle, 1992a). In anaesthetized
primates, face-M1 neurons receive more proprioceptive inputs (Sirisko and Sessle, 1983;
Huang et al., 1988), whereas face-M1 in awake rats receives somatosensory inputs from the
lips and vibrissae that are primarily exteroceptive (Sapienza et al., 1981; Farkas et al.,
1999). Anatomical studies in anaesthetised rats show that ICMS-defined M1 areas
containing vibrissal and jaw-M1 neurons receive some inputs directly through the thalamic
sensory nucleus (PO) but a majority of the inputs is indirect through the S1 barrel cortex
that are of exteroceptive nature (Izraeli and Porter, 1995; Miyashita et al., 1994; Farkas et
al., 1999; Hoffer et al., 2003, 2005). However, there are no published studies on the
functional organization of somatosensory inputs to face-M1 representing the oral region in
rats.
Chapter 1
37
1.3.5.5. The role of face-M1 defined from functional overlapping of somatosensory
inputs and motor outputs in sensorimotor cortex
Although the M1 and S1 are described in the literature as distinct entities in the
cortex based on the anatomically and physiologically characteristics, it is evident from the
above-mentioned studies that M1 receives sensory inputs and S1 has some motor outputs,
and both have functionally related overlapping representations. M1 neurons project to
motoneurons of a specific muscle to evoke a specific movement and may also receive
somatosensory inputs from peripheral receptors activated by contraction of the muscle to
which those M1 neurons project. Similarly, neurons within S1 receive somatosensory
inputs from a specific peripheral area and may project to a specific muscle to evoke a
specific movement within that same peripheral region from which the somatosensory
afferents to the S1 neurons derive (Cicirata et al., 1986a, 1986b; Huang et al., 1989b;
Murray and Sessle, 1992a; Izraeli and Porter, 1995; Lin et al., 1998). Such spatial
contiguity of sensory inputs and motor outputs underscores the importance of sensory
inputs in modulating M1 functions and further provides the substrate for cortical
neuroplasticity (see below). Furthermore, it has been documented in primates and rats that
although a majority of face-M1 neurons receive exteroceptive inputs from the same
orofacial areas within which movement is evoked by ICMS applied to the same neuronal
recording site receiving the exteroceptive input, a sizable number of face-M1 neurons
receive exteroceptive inputs from distant orofacial areas that have no close spatial relation
with the ICMS-evoked movement area (Sapienza et al., 1981; Huang et al., 1989b; Murray
and Sessle, 1992a; Lin and Sessle, 1994; for review, see Murray et al., 2001). Thus, the
functional organization of somatosensory inputs to face-M1 indicate a role of face-M1 in
sensorimotor integration and highlights the elaborate exteroceptive somatosensory
feedback from a wide peripheral orofacial area that is used for the precise control,
coordination and modulation of the orofacial muscle activities during orofacial movements
(Huang et al., 1989b; for review, see Murray et al., 2001; Avivi-Arbor et al., 2011; Sessle,
2011b).
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38
1.4. Neuroplasticity and its relationship to the role of face-M1
and face-S1 in adaptive and learning processes
Over the last few decades there has been a substantial shift in understanding of the
role of motor cortical areas as areas that are related not only for planning and controlling
movements, but also to learning and cognition (Sanes and Donoghue, 2000). In contrast to
older views it is currently accepted that brain maps are dynamic constructs that are
remodeled throughout life. “Neuroplasticity” refers to changes in neural pathways and
synapses which are due to changes in behaviour, environment and neural processes, as well
as changes resulting from bodily injury (Greenough et al., 1985a, b; Kleim et al., 1996;
Monfils et al., 2004). Neuroplastic alterations may occur at the peripheral, subcortical, or
cortical level and involve molecular, cellular or synaptic transformations. These changes
may be structural or functional in nature, may have a fast (in seconds) or a slow-onset (in
days) and can be either short (for hours) or long-lasting (for months to years).
Clinically, M1 neuroplasticity has been associated most significantly with motor
function recovery following central (e.g. stroke) or peripheral injury. It has also been
associated with the onset of painful conditions leading to chronic conditions, changes in
muscle use or disuse, learning of novel motor skills, and adaptive processes. For example,
neuronal recordings and activation patterns revealed with neuroimaging methods have
shown considerable neuroplasticity of M1 representations and cellular properties following
pathological or traumatic changes and in relation to everyday experience, including motor-
skill learning and cognitive motor actions (Sanes and Donoghue, 2000; Nudo, 2006; for
review, see Nudo et al., 2001; Chen et al., 2002; May, 2008; Kaas et al., 2008; Avivi-Arber
et al., 2011; Kleim, 2011; Nudo, 2011). The dynamic architecture involving convergence,
divergence, horizontal interconnections in the M1, and distributed activation described in
the previous section, provides a substrate conducive to the phenomenon of neuroplastic
reorganization (Schieber, 2001) along with the processes of long-term potentiation (LTP)
and long-term depression (LTD) for changes to occur (Keller, 1993; Hess and Donoghue,
1994; Hess et al., 1996, Hess, 2004). Therefore, M1 is at the centre of a domain in the
cortex where neuroplastic changes can be reflected as a functional reorganization of motor
Chapter 1
39
representations and changes in cortical excitability (for review, see Donoghue, 1995, 1997;
Buonomano and Merzenich, 1998; Sanes and Donoghue, 2000; Sessle, 2006; Sessle et al.,
2007).
The topographic organization of motor representations is highly variable between
individuals and the basis of this individual variability may be due to individual variation in
motor experiences manifested as a use-dependent cortical neuroplasticity (Nudo et al.,
1992, 1996a, b; Nudo, 2003; Monfils and Teskey, 2004; Cramer et al., 2011). This is
further supported by the observations that the representation of the limb in the hemisphere
contralateral to the dominant hand is larger and more complex than the representation of the
non-dominant hand in its hemisphere (Nudo et al., 1992). Further, for an evident chewing-
side preference, the blood-oxygenation-level dependent signal (BOLD) change in a fMRI
study in the S1/M1 is significantly greater on the side contralateral to the preferred chewing
side (Shinagawa et al., 2003; Jiang et al., 2010).
The following review outlines: 1) the manifestations of face-M1 neuroplasticity, 2)
neuroplastic changes in other cortical or subcortical areas, 3) description of some of the
mechanisms thought to underlie the neuroplastic changes, and 4) the possible clinical
applications of the concept of neuroplasticity.
1.4.1. Neuroplasticity of face-M1 associated with altered somatosensory
and motor experience
Numerous studies of humans, monkeys and rodents have revealed that motor
representations in the cortex are altered by changing motor experience such as following
training in a novel limb motor skill (Pascual-Leone et al., 1995; Nudo et al., 1996a, b;
Karni et al., 1998; Kleim et al., 1998; Remple et al., 2001; for review, see Ebner, 2005).
Overall, these studies revealed that there is an increase in the representation of the muscles
involved in the trained movement at the expense of the representations of the less trained
muscles.
In a study in our laboratory, ICMS and neuron recordings were made over a 1 – 2
month period in awake monkeys before and again after a 1 – 2 month period of training on
Chapter 1
40
a novel tongue protrusion task. There was an increase after training in the proportion of
discrete M1 efferent zones for tongue protrusion and an associated decrease in zones for
lateral tongue movement. However, there was a lack of analogous change in
CMA/swallow cortex suggesting that differential expression of task-related neuroplasticity
may occur in these three cortical regions (Sessle et al., 2005, 2007). Analogous findings
have been documented in humans (Svensson et al., 2003; Boudreau et al., 2007). These
findings suggest that face-M1, like limb-M1, may be critical in motor skill learning,
reflecting dynamic and adaptive events modelled in a use-dependent manner by
behaviourally significant experiences.
Neuroplasticity of face-M1 induced by peripheral nerve injury has been
demonstrated in several studies in rats that have documented the effects on face-M1 motor
representations of the vibrissae (for review, see Buonomano and Merzenich, 1998; Sanes
and Donoghue, 2000; Ebner, 2005). These studies have been carried out in the face-M1 by
manipulating the infraorbital nerve and the facial nerve (Semba and Egger, 1986; Sanes et
al., 1990). Transection of branches of the facial nerve that innervate the vibrissae
musculature enlarged the ICMS-defined forelimb and eye/eyelid output areas of the M1
along with a marked decrease in eye/eyelid movement threshold suggestive of an increased
excitability, and these enlargements occupied the former vibrissae area. Reorganization
within the contralateral M1 occurred as early as 1 week following a unilateral facial nerve
transection and was maintained for at least 4 months (Sanes et al., 1990). A similar change
in the face-M1 was noted (Toldi et al., 1996) after unilateral facial nerve transection, with
the primary change being the loss of contralateral vibrissal response, while ipsilateral
vibrissae started to respond to the electrical stimulus within 4 minutes of the transection.
This change was transient and gradually disappeared within hours to days. The secondary
change was that the contralateral movements of forepaw and eye/eyelid muscles could be
evoked from increasing portions of the former vibrissal field and this change was stable for
at least 2 weeks.
In contrast to the above findings, injury to the infraorbital nerve supplying sensory
innervation to the vibrissae has been reported to result in no changes in the vibrissal motor
Chapter 1
41
representations, although facial nerve transection resulted in reorganization of the vibrissal
movement representation in the contralateral face-M1 (Franchi, 2001; Franchi and
Veronesi, 2004, 2006). However, in a study from our laboratory, unilateral transection of
the lingual nerve supplying sensory innervation to the tongue in rats resulted in a time-
dependent change of the genioglossus (GG – tongue-protrusion muscle) representation in
the face-M1 (Adachi et al., 2007). Transmagnetic stimulation (TMS) studies in humans
have reported that lingual nerve anaesthetic block is associated with decreased excitability
of tongue representation in face-M1 (Halkjaer et al., 2006) and local anaesthesia of lower
facial skin is associated with increased excitability of jaw representation in face-M1 (Yildiz
et al., 2004). When trained monkeys that performed the task that required them to protrude
their tongue onto a strain gauge and produce a stable force had the lingual nerve blocked
bilaterally by local anaesthetic to block the orofacial sensory inputs, their ability to protrude
their tongue was markedly impaired suggesting that orofacial sensory inputs were
necessary for successful performance of a tongue-protrusion task, and also suggests that
loss of a major intraoral sensory input may cause the monkeys to use alternative strategies
to adapt to the sensory loss (Lee et al., 2011). In lines with the studies of trimming the
vibrissae (Keller et al. 1996, Huntley, 1997a, b), neuroplastic changes occur in face-M1 as
a result of trimming the incisal edges of the lower incisors in rats to take them out of
occlusion with the upper incisors; this resulted in a decrease in the face-M1 representations
of the GG and anterior digastric (AD - jaw opening muscle) muscles reflected as a
significant decrease in the number of sites from which GG and AD could be activated by
ICMS in face-M1, that were restored to initial baseline levels on eruption of the lower
incisors into occlusion (Sessle et al., 2007). Furthermore, extraction of the right lower
incisor was associated with larger right anterior digastric (RAD) representations and
RAD/GG overlapping representations in face-M1 and a decrease in the onset latency of
ICMS-evoked responses at 1 week after the extraction (Avivi-Arber et al., 2010b). Also,
extraction of maxillary right molars was associated with a larger AD and GG motor
representation in face-M1 1 week after the extraction (Veeraiyan et al., 2011).
Chronic pain conditions, such as backache, phantom limb pain (Dettmers et al.,
2001) or complex regional pain syndrome (Krause et al., 2006) have been associated with
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42
increased limb-M1 excitability and reorganization of the S1 as well as M1, and a regular
use of a myoelectric prosthesis in these patients is associated with cortical reorganization
and a related decrease in phantom limb pain (Lotze et al., 1999; Tsao et al., 2008). Studies
involved with the effects of experimental acute pain on limb-M1 in humans have shown
that noxious stimuli can decrease M1 excitability (Farina et al., 2001; Le Pera et al., 2001;
Svensson et al., 2003a). Peripheral electrical stimulation, known as transcutaneous
electrical nerve stimulation (TENS), is used for pain relief in the management of acute and
chronic pain states such as rheumatoid arthritis, chronic low back pain, post-surgery and
during labour (Brosseau et al., 2003; Nnoaham and Kumbang, 2008; Dowswell et al.,
2009). Although changes occur locally at the level of the muscle and spinal cord, it has also
been demonstrated that TENS induces cortical plasticity (Ridding et al., 2000, 2001;
McKay et al., 2002).
The mechanism of the interaction between acute orofacial pain and face-M1
neuroplasticity is a subject of controversy. Studies applying hypertonic saline or capsaicin
to the masseter muscle, facial skin or tongue in humans have not demonstrated any
association between pain and face-M1 excitability (Romaniello et al., 2000; Halkjaer et al.,
2006). However, experimental pain through topical application of capsaicin to the tongue
does restrict the increased tongue-M1 excitability associated with training in a tongue-
protrusion task and also interferes with successful performance of the task (Boudreau et al.,
2007). Further, topical application of the algesic chemical glutamate to the tongue in rats
reduces tongue-M1 excitability; however injection of hypertonic saline in the tongue does
not affect tongue-M1 excitability (Adachi et al., 2008). This variability in the results of the
different studies may be related to differences in study designs or could be related to the
multidimensional nature of pain and its modulatory effect on face-M1 excitability or to
differential effects of activating different groups of nociceptive afferents.
Thus, the neuroplastic changes in the face-M1 are differential in nature and
dependent on the type of peripheral manipulations applied. Face-M1 also has the potential
to adapt and be modelled in a task-dependent manner.
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1.4.2. Neuroplastic changes in face-S1, other cortical and subcortical
areas
Numerous studies on face-S1 neuroplasticity have focused on the rat barrel cortex
where vibrissae have an extensive face-S1 representation. Changes in somatosensory inputs
to the vibrissal-S1 have been induced by perioral noxious inputs, anaesthetic block,
vibrissal trimming or transection of the infraorbital nerve supplying sensory innervation to
the vibrissae, and are associated with reorganization of vibrissal and neighbouring (e.g.,
limbs) mechanoreceptive fields and changes in the response properties of vibrissal-related
neurones within the vibrissal-S1 (for review, see Feldman and Brecht, 2005; Petersen,
2007; Fox, 2009; Barnes and Finnerty, 2010). Neuronal activities of the vibrissal-S1 were
explored bilaterally in rats with a chronic constriction injury of the infraorbital nerve and
reorganization of the mechanoreceptive fields in the face-S1 have been documented
(Benoist et al., 1999).
A series of studies have focused on the impact of behavioural changes on S1
neuroplasticity induced by a sensory change and have observed a large-scale expansion of a
single whisker’s functional representation following innocuous removal of all neighboring
whiskers. However, a large-scale contraction of the representations is documented if the
animal is removed from its home cage and given a brief opportunity to use its whiskers for
active exploration of a different environment (Polley et. al., 1999, 2004b). Both the
expansion and contraction reverse upon re-growth of the deprived whiskers. This
demonstrates that representation of non-deprived regions can either contract or expand,
depending on the nature of the animal’s interaction with its environment during the period
of sensory deprivation and questions the widely accepted belief that an adjacent intact input
in the cortex will always come to activate areas of the cortex formerly responsive to a
deprived input. Further, studies have shown that when a stimulus is cognitively associated
with reinforcement, its cortical representation is strengthened and enlarged and is not
caused due to the sensory experience alone, but requires learning of the sensory experience,
and is the strongest for reward associated stimuli (Polley et al. 2004a; Blake et. al., 2005,
2006). Studies to understand the impact of environmental modification on the neuroplastic
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changes in M1 subsequent to peripheral alterations need to be considered for their possible
influence on the reorganization of the muscle representations.
In a study in mole-rats, the lower right incisor extraction results in a dramatic
reorganization of the orofacial representation in the former lower tooth zone in face-S1 of
other orofacial tissues including the contralateral upper incisor, ipsilateral lower incisor,
tongue and the buccal pad (Henry et. al., 2005). Recent findings from our laboratory have
documented neuroplastic capabilities of face-S1 in terms of its motor outputs. Extraction of
a mandibular incisor tooth is associated 1 week later with a significant increased AD motor
representation within face-S1 and face M1 in rats (Avivi-Arber et al., 2010a).
Alterations in orofacial somatosensory inputs may induce neuroplastic changes at
cortical levels other than face-S1 and face-M1, and also in the subcortical levels of the
sensorimotor system (e.g. thalamus, brainstem and peripheral nerves) analogous to the
occurrence of neuroplastic changes in limb-S1, limb-M1, as well as in subcortical areas
following limb nerve injury or amputation (for review, see Jones, 2000; Wall et al., 2002;
Kaas et al., 1991, 2008). As mentioned above, face-M1 receives somatosensory inputs
either directly through the thalamus (Rausell and Jones, 1995; Hatanaka et al., 2005;
Simonyan and Jurgens, 2005), or indirectly through face-S1 (Hoffer et al., 2005;
Chakrabarti and Alloway, 2006; Iyengar et al., 2007). Further, ICMS of M1 evokes EMG
responses through activation of brainstem motoneurons that integrate a large number of
sensory and motor inputs (Capra, 1995; Sessle, 2000; Waite, 2004; Miles, 2004).
Reorganization of the face-S1 has been quantified by simultaneously recording from single-
unit neural ensembles in the whisker regions of the VPM nucleus of the thalamus and the
face-S1 cortex in anesthetized rats before, during, and after injecting capsaicin under the
skin of the lip (Katz et al., 1999) and sensory deprivation induced by intraoral local
anaesthesia (Nicolelis et al., 1993). Changes in the basal ganglia and cerebellum subsequent
to facial nerve transection have been reported (Franchi et al., 2006) along with changes
within brainstem in V and VII nuclei (Kis et al., 2004). Tooth pulp deafferentation changes
reflected in V brainstem nuclei have been documented (Hu et al., 1986, 1999; Kwan et al.,
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1993). Thus, any neuroplastic change within face-M1 may conceivably reflect related
changes that occur at other level in the processing hierarchy of the sensorimotor system.
1.4.3. Mechanisms underlying cortical neuroplasticity
Different mechanisms may be involved in different forms of neuroplastic processes
and may operate simultaneously or at different times in the process. The possible
neuroplastic mechanisms that may be involved are: 1) Unmasking of inactive synapses, 2)
Change in synaptic efficiency, 3) Change in gene expression, and 4) Dendritic branching or
axonal sprouting and synaptogenesis. The immediate changes occurring subsequent to
nerve injury in animals and nerve block in humans are based on unmasking of the
previously present, but functionally inactive synaptic connections, and can also be due to
increased excitatory neurotransmitter release, increased density of postsynaptic receptors,
changes in membrane conductance thereby enhancing the postsynaptic effects of weak
inputs, or withdrawal of inhibitory influences that control excitatory inputs. The removal of
inhibition of excitatory synapses by reducing GABAergic inhibition has been suggested
strongly as the predominant mechanism for short-term plastic changes (Jacobs and
Donoghue, 1991; Farkas et al., 2000; Chen et al., 2002). The basis for this claim is that
GABAergic neurons constitute approximately 25 to 30% of the neuronal population in the
M1 and their horizontal connections can extend up to 6 mm or more (Jones, 1993). Further,
it has been shown that administration of GABA receptor antagonists enhance evoked
responses to stimulation and expansion of the receptive fields of S1 neurons (Chowdhury
and Rasmusson, 2002). Also, topical application of the GABA antagonist bicuculline to the
forelimb-M1 in rodents allows for a rapid ICMS activation of forelimb muscles from the
vibrissae area in the M1 (Jacobs and Donoghue, 1991). In humans, systemic administration
of GABA agonist drug decreases motor excitability as tested by TMS-evoked motor
activity (Ziemann et al., 1996, 2001). The decreased distribution of GABA in the cortex is
evident for several months after nerve injury (Garraghty et al., 1991).
Another mechanism that is important in short-term reorganization is a change in
synaptic efficacy, both at the central axons and axons of the neuromuscular junction.
Potentiation of synapses through high-frequency stimulation resulted in an increase in the
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amplitude of the EPSPs in the target neurons for minutes to hours or even up to a year
following the cessation of the stimulation (Abraham, 2003). This increase in synaptic
efficacy is termed as LTP, and requires high-frequency stimulation of excitatory afferents
(Bliss and Lomo, 1973; Lau and Zukin, 2007, Bliss and Cooke, 2011). The potentiation of
the synapses could be due a combination of the following suggested changes: 1)
presynaptic modifications which result in an increase in the amount of glutamate released
per impulse, 2) postsynaptic modifications, such as an increase in the number of receptors
or a change in their functional characteristics, 3) an extrasynaptic change, such as reduction
in uptake of glutamate by glial cells that leads to increased neurotransmitter availabilty at
the receptors, or 4) morphological modifications (Bliss and Collingridge, 1993; for review,
see Bliss et al., 2003). In contrast, a decrease in synaptic efficacy can also be induced with
lower frequencies of stimulation, and is referred to as LTD (Dudek and Bear, 1992).
Activity-dependent potentiation of synapses can broadly be divided into potentiation that is
either N-methyl-D-aspartate (NMDA) receptor-dependent or NMDA receptor-independent.
There is evidence that another class of glutamate receptor - the kainite receptor - plays a
pivotal part in the NMDA receptor-independent potentiation (Bliss and Collingridge, 1993;
Bliss et al. 2003). The NMDA receptor is a ligand-gated (glutamate) ion channel that is
normally blocked by Mg²⁺ in a voltage-dependent manner, and for induction of LTP a
threshold level of stimulation of the neuron is to be attained to unblock NMDA channels by
expelling the Mg²⁺, which is a complex function of the intensity and pattern of the high
frequency stimulation.
Neuroplastic changes of a longer onset likely involve more stable functional or
structural mechanisms, such as enhanced gene expression (Kleim et al., 1996) and
increased neuronal excitability during early stages of learning (Aou et al., 1992); dendritic
branching (Greenough et al., 1985; Jones et al., 1996; Monfils et al., 2004) or formation of
new connections through axonal sprouting and synaptogenesis (Kaas, 1991; Kleim et al.,
1996; 2002a; 2004) during later phases of learning; LTP and LTD may play a role in early
as well as late phases of the learning process (Hess and Donoghue, 1994; Rioult-Pedotti et
al., 1998; Rioult-Pedotti and Donoghue, 2003; Monfils and Teskey, 2004a; Teskey et al.,
2007; for review, see Bi and Poo, 2001; Boroojerdi et al., 2001; Chen et al., 2002; Bliss et
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al., 2003; Navarro et al., 2007). Deafferentation or nerve lesions may remove the associated
normally dominant excitatory and inhibitory inputs and weaker and previously latent
existing synapses can become strengthened over time, thereby stabilizing and reinforcing
their post-synaptic effects on the basis of the new activation patterns. It has been reported
that reorganization following deafferentation or nerve lesions may occur in two phases.
During the initial phase, some new responses may become evident immediately after the
nerve cut; changes in synaptic efficacy, e.g. “unmasking”, may be responsible for this
phase. This immediate reorganization is followed by a more protracted second phase,
lasting several weeks, during which the neurons throughout virtually all of the remaining
deprived cortex become responsive to neighbouring peripheral fields with intact
innervations (Merzenich et al. 1983; Myers et al., 2000); further changes in synaptic
efficacy and/or to the sprouting of new connections are implicated for this phase.
In vitro studies have documented that when the activity of neuronal cultures is
inhibited, the neurons become hyperexcitable, whereas when an increase in neuronal
activity is induced for long durations, the firing rates of the neurons in the culture dropped
(Turrigiano et al., 1998). It has been suggested that in order to control neuroplasticity
changes from becoming rampant on a long term basis, a stabilizing mechanism exists that is
termed “homeostatic plasticity” (Turrigiano and Nelson, 2004). LTP induces a higher firing
rate in post-synaptic neurons and in absence of a homeostatic mechanism, this would result
in a downstream saturation of the neuron efficacy, whereas depression of neuronal activity
associated with LTD would result in quiescence (Turrigiano and Nelson, 2004). Ideally, the
intrinsic properties of a neuron should be arranged to make the most of the dynamic range
of a neuron's firing rates to encode information, thus acting as a homeostatic mechanism
(Stemmler and Koch, 1999). One of the mechanisms suggested to preserve the dynamic
range of a neuron is through a process termed synaptic scaling. The homeostatic form of
plasticity restores neuronal activity to its normal 'baseline' levels by changing the post-
synaptic response of all the synapses of a neuron as a function of activity, i.e. the same
level of scaling is done to each synapse, to either strengthen or weaken all of a neuron’s
connections (Turrigiano et al., 1998; Turrigiano and Nelson, 2000; Perez-Otano and Ehlers,
2005; Turrigiano, 2008).
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Five basic components of experience and training-dependent neuroplasticity have
been identified to be applicable in S1 and may be applicable to M1. The first component is
a rapid depression of responses to deprived inputs; the second is a slower response
potentiation to the spared inputs. These components of neuroplasticity are hypothesized to
utilize classical Hebbian weakening and strengthening of deprived and spared pathways,
and are modulated by competition between active and inactive inputs. The third component
is potentiation of responses to active inputs during normal sensory use and also to temporal
correlation between inputs. The fourth component is potentiation of responses paired with
reinforcement in adults. The third and fourth components are both based on Hebbian
strengthening of active inputs but vary on the inclusion of factors such as attention or a
reward. The fifth component is homeostatic regulation of cortical activity in response to a
marked increase or decrease in sensory inputs (for review, see Feldman, 2009).
Recently, sensitive physiological and anatomical techniques have revealed many
novel sites and mechanisms for neuroplasticity, and at the same time many similarities and
differences in these mechanisms across cortical areas have also been revealed. These
discoveries have lead to the view that cortical neuroplasticity involves multiple
molecular/cellular mechanisms, each working at distinct neuronal sites, time scales, and
developmental stages (Bi and Poo, 2001; Boroojerdi et al., 2001; Chen et al., 2002; Wall et
al., 2002; Bliss et al., 2003; Turrigiano, 2008; Feldman, 2009).
1.4.4. Potential applications of neuroplastic mechanisms in a clinical
setting
Injury or disease causes extensive biochemical, anatomical and physiological
changes that modify the brain to what might be considered a very different brain. This
adapted brain is forced to reacquire behaviours lost as a result of the injury or disease and
relies on neuroplasticity within the residual neural circuits. The same fundamental neural
and behavioural signals driving neuroplasticity during learning in the intact brain are relied
upon during relearning in the damaged/diseased brain. The flexibility through
neuroplasticity in the use of brain circuits enables compensation to various extents for brain
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injuries, and thereby maintains capabilities as brain impairments progress or recover
capabilities lost after injuries or disease of peripheral or CNS tissues (Kaas, 2007).
Although major strides have been made in the understanding of neuroplasticity this
knowledge has not yet been translated into many established clinical interventions. In many
cases, the clinical interventions represent application of neuroscience knowledge to
rehabilitation techniques. Neuroplasticity can be adaptive when associated with a gain in
function (Cohen et al., 1997) or maladaptive when associated with negative consequences,
such as function loss or injury potentiation (Nudo, 2006). Further, adaptive neuroplasticity
is different from compensatory behaviours, whereby behavioural patterns alter prior to
disease onset (Levin et al., 2009).
1.4.4.1. Stroke, trauma and spinal cord injury
An area in which the concept and mechanisms of neuroplasticity have been
extensively studied is motor recovery subsequent to stroke. Motor deficits are predominant
in a majority of patients with stroke (Rathore et al., 2002; Nudo, 2011). Injury to a region
of the motor network can result in spontaneous intra-hemispheric changes, such as in the
size and extent of representational maps, e.g. the hand area can shift dorsally to invade the
shoulder region (Nudo et al., 1996; Muellbacher et al., 2002) or face region (Weiller et al.,
1993; Cramer and Crafton, 2006). Inter-hemispheric balance can shift at the same time such
that the uninjured hemisphere performs hyperactively in relation to movement (Chollet et
al., 1991; Murase et al., 2004). Motor recovery after stroke may present many forms of
neuroplasticity that can be ongoing in parallel.
Similar forms of adaptive neuroplasticity have been reported following different
forms of acute CNS injury such as traumatic brain injury (Munoz-Cespedes et al., 2005;
Belanger et al., 2007) and spinal cord injury (Topka et al., 1991; Cramer et al., 2005;
Rosenzweig et al., 2010). The similarity of neuroplastic mechanisms at work across
divergent forms of CNS injury suggests that neuroplasticity uses a limited repertoire of
events across many contexts (Cramer et al., 2011).
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Unfortunately, all neuroplastic changes do not have a positive impact on clinical
status, e.g. cerebral trauma can lead to a delayed onset of new onset epilepsy. Progressive
brain changes, such as axonal sprouting and the generation of new connections, produce
changes in neuronal signalling and disinhibition that may initiate the induction of seizures
(Prince et al., 2009). Other examples reported in the literature suggestive of maladaptive
plasticity include phantom limb pain following injury to a limb (such as amputation),
dystonia subsequent to CNS injuries and autonomic dysreflexia after spinal cord injury
(Karl et al., 2001). Therefore recovery from trauma may result in both adaptive and
maladaptive neuroplasticity that can occur simultaneously.
The concept of neuroplasticity has also found applications in the fields of paediatric
and developmental disorders, and neurodegeneration, aging and neuropsychiatric disorders
(for review, see Cramer et al., 2011).
1.4.4.2. Neuroplasticity-based interventions
1.4.4.2.1. Non-invasive brain and peripheral stimulation
Several forms of non-invasive brain stimulation have been suggested as a means to
alter brain function to induce neuroplasticity (Webster et al., 2006; Plow et al., 2009), and
TMS is chief amongst them. Although TMS activates both inhibitory and excitatory
cortical interneurons, in general, continuous trains of repetitive low frequency (51 Hz)
TMS lead to suppression of cortical excitability in healthy subjects, while intermittent,
bursting high frequency (41 Hz) trains lead to facilitation (Wagner et al., 2007). Along
with the aid of neuroimaging and physiological measures TMS can be applied specifically
and selectively to defined cortical regions (Neggers et al., 2004; Kleim et al., 2007). TES
induces low-amplitude direct currents that are strong enough to penetrate the layers of the
brain and modify membrane potentials and influence neuronal excitability, but without
triggering the depolarization of neurons (Wagner et al., 2007). Combination therapies using
concomitant pharmacological and peripheral nerve stimulation have the potential to drive
Hebbian neuroplasticity (Conforto et al., 2010).
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Repetitive TMS to the prefrontal region for several weeks can have significant
antidepressant effects (O’Reardon et al., 2007; Padberg and George, 2009) and has also
been used to treat schizophrenia. Low-frequency stimulation of temporoparietal cortex has
been suggested to treat treatment-resistant auditory hallucinations (Hoffman et al., 2007;
Stanford et al., 2008; Bagati et al., 2009; Vercammen et al., 2009).
1.4.4.2.2. Brain-machine interface (BMI)
Advances have suggested that, with prolonged usage, an artificial actuator can be
effectively incorporated in the internal representation of the subject’s body (Head and
Holmes, 1911; Gurfinkel et al., 1991; Maravita et al., 2003; Nicolelis, 2003). Such
neuroplastic adaptations then function to optimize neuronal representation of new
behavioural goals (Todorov and Jordan, 2002). With the use of neuronal ensemble
recordings, predictions can be made for motor parameters from neuronal activity and used
to control a brain–machine interface (BMI) (Chapin et al., 1999; Wessberg et al., 2000;
Nicolelis, 2001; Serruya et al., 2002; Taylor et al., 2002; Carmena et al., 2003; Patil et al.,
2004; Wessberg and Nicolelis, 2004). Further, BMIs can be driven by the planning and
motivation signals that are selected from cortical activity (Musallam et al., 2004). Studies
on able-bodied monkeys have used a neural interface system to control a robotic arm
(Lebedev et al., 2005; Velliste et al., 2008) and recent studies in human subjects with
profound upper extremity paralysis or limb loss that have used cortical neuronal ensemble
signals to direct neutrally controlled robotic arm to perform useful arm actions suggest
exciting clinical applications in the field of BMIs (Hochberg et al., 2012).
1.5. Orthodontic tooth movement (OTM) –- its neural regulation
Orthodontic force applied to a tooth immediately displaces it in the direction of the
forces to traverse the width of the PDL thereby creating a zone of compression and a zone
of tension in the PDL. This initial physical effect of force is followed closely by a
biological response in extracellular matrix and cells of the alveolar bone, PDL, gingiva, and
associated blood vessels and neural elements, (Kerrigan et al., 2000; Krishnan and
Davidovitch, 2006; Masella and Meister, 2006; Meikle, 2006; Henneman et al., 2008; Wise
and King, 2008). OTM comprises of an initial phase in which the tooth displaces almost
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instantaneously through the width of the PDL, followed by a lag phase in which the
necrotic tissue, which is produced due to the compression of the vessels of the PDL and the
alveolar bone, is removed and almost none or minimal tooth movement results, and a final
linear phase of OTM characterized by bone formation on the tension side and bone
resorption on the pressure side of the loaded tooth (King et al., 1991; Shirazi et al., 2002;
Ren et al., 2004; von Bohl et al., 2004; Krishnan and Davidovitch, 2006).
The traditional orthodontic view regarding normal growth and development of the
orofacial region is that the sensory and motor functions of the orofacial region are
genetically controlled to serve as a template for the development of normal orofacial
behaviour (Moyers, 1988; Linder-Aronson and Woodside, 2000). Contrary to this
traditional thinking are current concepts of neuroplasticity in which sensory experiences
modify the structure and function of the brain and thereby influence the sensorimotor
function of the orofacial region. Therefore, it is important to consider OTM in terms of
neural changes, especially the role of PDL receptors since the PDL is the structure that is
intimately involved and altered by the process of OTM.
1.5.1. Periodontal mechanoreceptors (PMRs) and their role in orofacial
motor control
The PDL provides support to the teeth and contains low-threshold
mechanoreceptors that provide for sensory perception. These PMRs are concentrated
between the fulcrum and the apex of the tooth, respond to stretch and provide tactile
information that is critical for regulating motor control of the mandible during function
(Linden, 1990). The PMRs with cell bodies in the V ganglion are distributed with the
highest concentration around the middle of the root (Byers and Dong, 1989). Through the
inputs of PMRs to the main sensory nucleus and spinal nucleus of the VBSNC they help in
the perception of touch and pressure from the teeth (Sessle, 2000; Miles, 2004; Trulsson
and Essick, 2004). PMRs with cell bodies in the mesencephalic nucleus of the V nerve are
in smaller numbers and have a distribution localized to the apex of the root (Byers, 1985,
Byers et al., 1986; Millar et al., 1989; Linden et al., 1994) and have prominent projections
to the Vm. These projections relay information from the PDL to the jaw muscles to
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facilitate brainstem reflexes during mastication. PDL fibres, whose cell bodies are located
in the V ganglion and project to the VBSNC and then to the neurons of the Vm, also
influence the activity of the jaw muscles through reflexes operational at the segmental level
(Sessle, 2000; Trulsson and Essick, 2004).
The number of PMRs decreases from the anterior teeth to the molar in humans
(Trulsson, 1993; Johnsen and Trulsson, 2003). This finding is supported by several
histological animal studies (Passatore et al., 1983; Byers and Dong, 1989; Hassanali, 1997).
This fact, when taken together with the increased number of mechanoreceptors in the tip of
the tongue (Trulsson and Essick, 1997), emphasizes the importance of highly developed
mechanoreceptive mechanisms in the highly innervated anterior part of the mouth to
effectively manipulate the food in the initial stages of chewing before being moved to the
molars. In the horizontal plane, the optimal tuning of the receptors supplying the anterior
teeth and the premolars is quite evenly distributed around the circumference of the tooth.
However, in the molar area the receptors are optimally tuned for the distal-lingual direction.
In the vertical plane, there is optimal tuning for downward-directed forces in the anterior
teeth, with few receptors responding optimally in this direction in the posterior teeth. The
preference shift from a high sensitivity to most directions at the anterior teeth to the distal-
lingual direction at the molars caters to the functional demands of mastication (Trulsson,
1992; Trulsson and Gunne, 1998; Johnsen and Trulsson, 2003; Svensson and Trulsson,
2009; for review, see Trulsson, 2006, 2007). In rats, the predominant PMRs of the incisors
are the unencapsulated Ruffini-like ending (Sato et al., 1988; Byers and Dong, 1989;
Takahashi-Iwanaga et al., 1997) that are directionally sensitive (Tabata et al., 2002).
The primary motor activity for mastication is initiated in the brainstem. However,
the PMRs encode information about the temporal, spatial and intensive aspects of tooth
loads that then regulates the muscle activity that generates masticatory forces and jaw
movements (Lund, 1991; Trulsson and Johansson, 1996; Turker et al, 2007; Svensson and
Trulsson, 2009). Due to their high sensitivity at low forces, most PMRs, along with the
information concerning the movement of the jaw, encode detailed temporal changes during
the early contact phase of each chewing cycle which then aids the selection of the most
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appropriate motor signals to effectively handle the mechanical food properties. This cycle
occurs in a predictive feed-forward manner that is guided by learned relationships between
patterns of afferent signals and appropriate efferent signals, similar to that documented in
studies of precise hand movements (Johansson and Westling, 1987). A disruption of this
coordination in chewing is observed following denervation of intraoral receptors, including
PMRs (Lavigne et al., 1987; Inoue et al., 1989). ‘Additional muscle activity’ is required
from the closing muscles of the mandible during the power phase of mastication to
overcome the resistance of the food and is largely parameterised in advance on the basis of
sensory experiences during the preceding chewing cycle (Ottenhoff et al., 1992 a, b), the
timing of which may be controlled mainly by information from muscle spindles, while the
magnitude may be under the control of both muscle spindles and PDL receptors (Komuro
et al., 2001a, b; for review see, van der Glas et al., 2007).
The regulatory function of the mechanoreceptors of the PDL, bony socket, and
other orofacial tissues in masticatory muscle contraction has vital orthodontic implications
since the activation of these receptors, through excitatory and inhibitory reflex effects and
more complex integrative mechanisms involving CNS centres, may normalize and/or
strengthen the forces of masticatory muscles (Trulsson and Essix, 2004; Miles, 2004; for
review, see, Turker, 2002; Turker et al, 2007). Further, neurophysiological studies of dental
malocclusion suggest that the abnormal occlusal contacts of teeth may result in lack of
appropriate sensory inputs to the cerebral cortex, that result in sensorimotor integration
dysfunction and leads to compromised oral perception and modified motor behaviour
(Ahlgren, 1966, 1967; Subtelny, 1970; Bakke et al., 1992; Yashiro and Takada, 1999;
Trovato et al., 2009). Further, the concept that PMRs modulate jaw movement to occlude in
maximum intercuspal position supports the Cybernetic Model proposed by Petrovic and
Stutzmann (1977) describing the influence of physiological occlusal relationship of the
teeth on the growth of the maxilla and the mandible (Section 5.4.1.2).
1.5.2. Receptor function during and after OTM
OTM affects the number, functional properties and distribution of the PMRs and
PDL nociceptors. The responses of the PDL receptors have been reported to become
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progressively impaired during the course of OTM. PMRs derived chiefly from myelinated
fibres, demonstrate higher thresholds, reduced conduction velocities, and reduced numbers,
even after the removal of the orthodontic appliance (Loescher et al., 1993; Okazaki, 1994;
Long et al., 1996; Ogawa et al., 2002). However, once the teeth are in their desired position
and the tissues have healed, the response of the PMRs improves and returns to normal
levels (Nakanishi et al., 2004). The cause of the low response levels during treatment has
been linked to disorganization of collagen fibers and direct injury to the receptors and their
axons, and is also supported by histological studies (Long et al., 1996; Nakanishi et al.,
2004). The clinical significance of altered PMR responses during OTM is unknown.
However, there may be an adaptive modification of jaw function and oral motor behaviour
to accommodate the altered pattern of chewing during the active stage of OTM. Thus, the
brain may learn to interpret the altered response from the PDL. The functional impacts of
altered response properties of the PMRs during and after OTM have not been studied.
Apart from the influence of OTM on PMRs, receptors in the skin of the face, oral
mucosa, gingival tissues, alveolar bone, dental pulp, tongue and TMJ (see above) may be
affected by either the physical presence of the orthodontic appliance or by the forces
exerted by the orthodontic appliances to induce orthopedic or orthodontic therapy.
However, the effects of OTM on receptors in orofacial tissues other than the PDL are not
known. OTM is considered to involve a neurogenic inflammatory process. Mild
inflammatory changes due to the release of inflammatory products have been reported on
the receptors in the alveolar bone, dental pulp, and gingival tissues (Kvinnsland et al.,
1989; Goto et al., 2001; Yamaguchi and Kasai, 2005, 2007; Yamaguchi et al., 2006;
Vandevska-Radunovic et al., 1997; for review see Vandevska-Radunovic, 1999; Wise and
King, 2008; Krishnan and Davidovitch, 2009). A decrease in the masseter activity during
OTM has been reported (see below), and alteration in the response properties of TMJ
mechanoreceptors due to reduced masseter activity during the growth period have been
reported (Ishida et al., 2009). Also, OTM results in altered chewing patterns of the
mandible (Ahlgren et al., 1967; Sohn et al., 1997), and an alteration of the response
properties of TMJ mechanoreceptors subsequent to a functional lateral shift of the mandible
has been suggested (Kokai et al., 2007). Functional appliances that are used in orthodontics
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may induce a more robust influence on the receptors in the orofacial tissues, and especially
TMJ due to the influence of jaw repositioning from functional appliances. However, since
this project studies changes in the face-M1 and face-S1 due to OTM, the changes inflicted
by orthodontic functional appliances on receptors of the orofacial region and TMJ are
beyond the scope of this review.
1.5.3. OTM-induced pain and motor functions
Pain during OTM is a complex experience and is amongst the most reported
negative experiences of orthodontic treatment (Kluemper et al., 2002; Asham, 2004; Keim,
2004). Pain is a subjective response with large individual variations and has a non-linear
relationship with factors such as age, gender, individual pain threshold, present emotional
state and stress, cultural differences, and past pain experiences (Ngan et al., 1989; Brown
and Moerenhout, 1991; Scheurer et al., 1996; Firestone et al., 1999; Bergius et al., 2000;
Krishnan 2007; Polat, 2007). The pain experienced by patients may not be directly related
to the magnitude of force exerted by the appliance but is strongly influenced by the state of
the individual (Brown and Moerenhout, 1991; Sergl et al., 1998; Bergius et al., 2000;
Otasevic et al., 2006; Krishnan 2007).
Almost all orthodontic procedures, such as separator and band placement,
application of orthopaedic forces, archwire placement and activations, and debonding
produce pain. Pain assessment in patients has been evaluated by using verbal and numerical
rating scales, and visual analogue scales (for review, see Melzack and Katz, 2006). Pain
was experienced by the majority of patients 4 hours after orthodontic appliance placement,
which reached peak levels at 24 hours (Jones, 1984; Ngan et al., 1989, 1994; Scheurer et
al., 1996; Firestone et al., 1999; Erdinc and Dincer, 2004; Polat et al., 2005; Polat, 2007),
and usually lasted for 2-3 days and then gradually decreased in its intensity by 5-6 days
(Jones and Chan, 1992a, b). There is no co-relation between tooth positions, applied force
levels, and experienced pain (Jones and Richmond, 1985; Otasevic et al., 2006).
OTM creates tension and compression zones in the PDL space and reduces the
proprioceptive and discriminative abilities of the patients for up to 4-5 days, which result in
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lowering of the pain threshold and disruption of normal mechanisms associated with altered
proprioceptive input from PMRs (Soltis et al., 1971; Loescher et al., 1993), along with the
initiation of pressure, ischemia, inflammation, and oedema in the PDL space (Furstman and
Bernick, 1972; Wise and King, 2008). The mechanisms of pain resulting during OTM are
not fully understood. The pain perceived has been related to the levels of prostaglandins
(PGEs) and substance P in the PDL, and is associated with an inflammatory response
(Kamogashira et al., 1988; Marklund et al., 1994). An immediate and slightly delayed
painful response after orthodontic force application has been reported (Burstone, 1962;
Krishnan, 2007). The initial response has been attributed to compression of the PDL and
the resulting input from activated nociceptors, and the delayed response to a hyperalgesic
state. Hyperalgesia has been related to PGEs, which make the PDL sensitive to released
algogens such as histamine, bradykinin, PGEs, serotonin, and substance P (Ferreira et al.,
1978; Sessle, 2000; Polat et al., 2005). The orthodontic forces may also injure the PDL
nerve endings, thereby also affecting the force thresholds and discharge frequencies of the
PMRs (Loescher et al., 1993; Okazaki, 1994); the distribution of nerve growth factor
receptors is also altered during OTM (Saito et al., 1993; for review, see Vandevska-
Radunovic, 1999; Krishnan, 2007; Wise and King, 2008).
Mechanical allodynia, reflected for example as a decrease in pressure pain threshold
(PPT) for masseter and anterior temporalis muscles along with a decrease in the frequency
of tooth contacts have been reported during the early stages of OTM (Michelotti et al.,
1999). Algometric measurements reflect the combined effects of peripheral nociception and
central pain processing. Therefore, a lowering of pain thresholds may also be related to
central mechanisms, in addition to the peripheral neural changes described above. For the
orofacial region, nociceptive neurons in the trigeminal nucleus caudalis receive extensive
convergent inputs from both superficial and deep tissues, and can be modulated by nerve
injury and by the inflammatory conditions. There is evidence that neuroplastic changes and
neuro-anatomical convergence of the trigeminal nociceptive inputs may account for the
local tenderness, spread, and referral of pain (Reid et al., 1994; Lund et al., 2009; Sessle et
al., 2011a, b). Another explanation suggested for the reduction in the EMG activity of the
masseter and the temporalis muscles on initiation of OTM is that the nociceptive input
Chapter 1
58
arising from the teeth, PDL, and perioral structures provide a protective reflex with
inhibition of the jaw-closing muscles (Sohn et al., 2000; Mason et al., 2004). Yet another
reason suggested is that PMR inputs to the brain stem are capable of inducing both
facilitatory and inhibitory effects on jaw-closing motoneuron activity (Lund and Lamarre,
1973; Lund and Sessle, 1974, Miles, 2004). Further, a net increase in the number of
chewing strokes before swallowing was reported during OTM and was related to subjects
having to make more strokes to prepare the food in the presence of pain to attain a particle
size that is suitable to swallow (Goldreich, 1994). Thus, the short-term occurrence of
orthodontic pain is associated with motor and sensory changes of the masticatory muscles,
represented by a decrease of the motor output and of the PPT of the jaw-closing muscles.
These changes are probably mediated by the CNS and may reflect a protective mechanism
against further damage to an injured part of the masticatory system.
In order to evaluate the degree of discomfort during OTM and its impact on muscle
activity, EMG studies have also been carried out on orthodontic patient groups (Goldreich
et al., 1994; Miyamoto et al., 1996; Miyawaki and Takada, 1997; Ferrario et al., 1999a,
1999b, 2002; Michelotti et al., 1999). These studies have shown that masseter muscle
activity decreased substantially in the first 24 hours following appliance placement, and for
11 hours after archwire changes during follow-up appointments. Normal EMG levels of
activity were attained from 6 months into treatment to the completion of the treatment,
suggestive of an adaptation to the treatment. This was followed with a decreased EMG
activity again when the appliance was removed, with a return to normal levels 6 months
after the end of the treatment (Goldreich et al., 1994; Miyamoto et al., 1996). Other EMG
studies support a significant decrease of temporal and masseteric muscle activity during
maximal clenching and chewing during OTM (Goldreich et al., 1994; Michelotti et al.,
1999; Tanaka et al., 2003).
Patients undergoing orthodontic treatment report an impairment of chewing and
biting ability (Miyamoto et al., 1996). Thus, it has been suggested that jaw-closing EMG
activity may decrease during OTM because discomfort or pain causes changes in the
frequency and duration of occlusal contacts and/or due to alterations in the occlusal
Chapter 1
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relationship produced by the OTM (Goldreich et al., 1994; Michelotti et al., 1999). As
suggested by the ‘Pain Adaptation Model’ (Lund et al., 1991), the nociceptive afferents
from the peripheral tissues can affect the motor control of the jaw muscles through an
interaction between the peripheral nociceptive inputs with motor neurons along reciprocal
pathways, resulting in a decrease in the motoneuron output when the muscle is acting as an
agonist, and an increase in the output when the muscle is acting as an antagonist. Thus, the
reduction of the masseteric and temporalis motor output has been explained by the
interaction between PDL nociceptive afferents and inhibitory inter-neurons supplying the
jaw-closing motor neurons (Michelotti et al., 1999). However, a decrease in the activity in
both agonist and antagonist muscles after placement of orthodontic functional appliances
has been reported in EMG animal studies (Sessle et al., 1990; Voudouris et al., 2003a, b).
The simultaneous decrease in the muscular activity of both the agonist and antagonist
muscles may be a process to reduce pain and protect the painful tissues, and may be
explained on the basis of recent theories on motor adaptation to pain (Murray and Peck,
2007; Turker, 2010; Hodges and Tucker, 2011).
It has been reported that a small occlusal interference in the intercuspal position can
change the coordination of orofacial muscular activity (Riise and Seikholeslam, 1984,
Tanaka et al., 2003). For example, in healthy subjects a 250-μm-thick experimental
occlusal interference provoked an altered pattern of contraction of masseter and temporal
muscles, and also, altered occlusal relationships such as a dental crossbite, modify jaw
muscle activity (Ferrario et al., 1999a, 1999b, 2002) and reduce muscular efficiency, which
returns to the norm after orthodontic correction (Goldreich et al., 1994; Alarcon et al.,
2000). Masseteric EMG responses to experimental occlusal interference results in reduced
recorded EMG clenching activity on the side opposite to the interference (Christensen and
Rassouli, 1995). Further, several studies have reported altered jaw and lip muscle activity
depending on the consistency of the food, and also that by improving occlusal contacts,
OTM promotes a change in the masticatory muscle EMG patterns to the normal levels
during swallowing and chewing (Takada et al., 1994; Miyawaki and Takada, 1997; Yashiro
and Takada, 1999).
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1.5.4. Animal models used to study OTM
According to a systematic review of animal studies used to study OTM in
orthodontic literature until 2003, 175 studies on tooth movement were performed in rats,
which comprised 55% of the 320 animal investigations. Rats and dogs together comprised
71% of the studies; primates were used in 10%, cats in 7%, mice in 5%, and rabbits in 4%.
Other animals were used less often (Ren et al., 2004). Thus, the rat seems to be the animal
of choice for studying OTM.
There are many advantages in using the rat model to study OTM (Ren et al., 2004).
Firstly, they are relatively inexpensive, which facilitates the use of large samples. Secondly,
the histological preparation of rat tissue is easier than other animals. Thirdly, most
antibodies required for cellular and molecular biological techniques are readily available
currently for mice and rats. Finally, transgenic strains are almost exclusively developed
currently in small rodents. Further, since mice are small in size and pose a technical
challenge to place an effective orthodontic appliance, rats have been the first choice as
experimental animals to be used to study the various facets of OTM.
1.6. Models used to study orofacial nociceptive behaviour
In contrast to the characteristics of pain that can be described and graded by
humans, pain in awake animals can be estimated only by observing their reactions or
behaviours. Studies in awake animals most commonly involve monitoring the response
threshold to a stimulus that would be a nociceptive stimulus if applied to humans. Such
responses typically involve flexion reflexes and/or vocalization. In such experimental
designs, as soon as a response is elicited the stimulus is stopped. A number of animal
models of chronic pain that mimic conditions in humans have been developed, and these
have facilitated the study of the mechanisms of chronic pain (for review, see Ren and
Dubner, 1999). In these animal models, pain condition develops following nerve or tissue
injury. These include behavioural hyperalgesia, which is interpreted as such by an
exaggerated nociceptive response to a noxious stimulus, and allodynia presumed from
nociceptive responses induced by a normally nonnoxious stimulus. The various tests used
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to evaluate nociceptive behaviour are based on the nature of the stimulus (electrical,
thermal, mechanical, or chemical) and then for the behavioural parameters that are
measured (Le Bars et al., 2001). An elaborate behavioural model for facial pain assessment
in rats has been reported (Vos et al., 1994, 1998), another rat model to assess TMJ pain has
also been developed (Roveroni et al., 2001), and a rat model for persistent orofacial muscle
pain and hyperalgesia has been documented (Ro, 2005). An animal model that can allow
direct correlation between change of pain characteristics during OTM and a quantifiable
behaviour change will aid in elucidating the mechanisms of OTM-related pain.
Quantification of behavioural responses reflecting cutaneous hyperalgesia in
animals has relied upon mechanical testing that was introduced in 1957 (Randall and
Selitto, 1957). This method in its modified form was then popularly used for testing
behavioural responses to orofacial nociception (Rosenfield et al., 1978; Morris et al., 1982;
Clavelou et al., 1989; Vos et al., 1994, 1998). However, this method suffers from
limitations, such as, mechanical stimuli activate both low and high threshold
mechanoreceptors in cutaneous and non-cutaneous tissues. Hence the relative contribution
of each receptor to the elicited behaviour response by this test is unknown. Further, the
mechanical method measures only the nociceptive threshold and uses a non-automated
detection of the behavioural end-point which requires intense experimentor interaction
(Dubner et al., 1974; Ferreira et al., 1978; Meyer et al., 1985; Willis, 1985; Hargreaves et
al., 1988). To overcome these limations of the mechanical testing procedures, thermal
testing procedures for quantifying thermal nociception in animal models of hyperalgesia
have been used (Mor and Carmon, 1975; Carmon and Frostig, 1981; Hargreaves et al.,
1988). Comparison of thermal to the mechanical method indicates that both methods
provide a quantitative measurement of the behavioural correlates of hyperalgesia and
allodynia. However, amongst both the methods, thermal methods have been reported to
have a higher sensitivity to detect hyperalgesia and allodynia as the test is done on an
unrestrained rat that is exposed to minimal environment cues which, unlike mechanical
testing, do not signal the commencement of a testing session thereby facilitating a learning
effect (Vinegar et al., 1978; Hargreaves et al., 1988). Further, the factor that contributes to
different sensitivity for both the methods could be the development of hyperalgesia which
Chapter 1
62
differs in responsiveness to thermal and mechanical stimuli (Benoist et al., 1985;
Handwerker et al., 1987).
1.7. Statement of the problem, hypothesis, and objectives
1.7.1. Statement of the problem
The ICMS technique has been extensively used to reveal the organizational features
of face-M1 (Huang et al., 1988; Adachi et al., 2007; for review, see Avivi-Arber et al.,
2011; Sessle, 2011b). Further, jaw and tongue movements can be evoked by ICMS of face-
S1, disruption of the functioning of face-S1 can severely restrict orofacial behaviours
(Neafsey et al., 1986; Murray et al., 2001; Hiraba et al., 2007; Burish et al., 2008), and
manipulations of orofacial somatosensory inputs can result in neuroplastic changes in the
face-S1 representations (Henry et al., 2005). Neuroplasticity of the limb-M1 (for review,
see Adkins et al., 2006; Nudo 2006; Graziano and Aflalo, 2007; Navarro et al., 2007) and
face-M1 (for review, see Sessle, 2007; Avivi-Arber et al., 2011; Sessle, 2011b) has been
demonstrated in several studies subsequent to peripheral manipulations. Recent studies in
our laboratory have revealed that alterations in orofacial sensory inputs into the CNS may
be associated with neuroplastic changes in motor representations in both face-S1 and face-
M1 (Adachi et al., 2007; Avivi-Arber et al., 2010a).
OTM is a clinical therapeutic procedure that alters intraoral tissues and related
sensory inputs into the CNS. OTM affects the number, functional properties and
distribution of the PMRs and nociceptive periodontal nerve fibres, and the responses of the
PDL receptors become progressively impaired during OTM (Loescher et al., 1993; Long et
al., 1996; Ogawa et al., 2002). The regulatory function of the mechanoreceptors of the
PDL, bony socket, and other orofacial tissues in masticatory muscle contraction has
important orthodontic implications since the activation of these receptors, through
excitatory and inhibitory reflex effects and more complex integrative mechanisms
involving CNS centres, may normalize and/or strengthen the forces of masticatory muscles
(Trulsson and Essix, 2004; Miles, 2004; for review, see, Turker, 2002; Turker et al, 2007).
Also, studies of dental malocclusion suggest that the abnormal occlusal contacts of teeth
Chapter 1
63
may result in lack of appropriate sensory inputs to the cerebral cortex, that result in
sensorimotor integration dysfunction and leads to compromised oral perception and
modified motor behaviour (Ahlgren, 1966, 1967; Subtelny, 1970; Bakke et al., 1992;
Yashiro and Takada, 1999; Sonnesen and Bakke, 2005; Trovato et al., 2009; Sonnesen and
Svensson, 2011). Also, PMRs of the teeth are involved in the guidance of the jaw
movement to occlude in maximum intercuspal position and have a physiological influence
on the occlusal relationship of the teeth and the growth of the maxilla and the mandible. It
has been suggested by a classical theory on growth and development [Cybernetic Model
proposed by Petrovic and Stutzmann (1977)] that the growth of the mandible may be
regulated by the CNS. However, it is not known if OTM produces neuroplastic changes in
the face-M1 and face-S1. Such information will help in understanding the adaptive
sensorimotor cortical mechanisms that regulate muscle activity and jaw function during and
after orthodontic interventions, and thereby address the contribution of the face-M1 and
face-S1 in regulating orofacial muscle activity that may influence growth of the jaws. Such
insights also may help in the design of new or improved orthodontic biomechanical
treatment approaches that could have a positive impact on the oral neuromuscular
behaviour and significantly enhance achievement of orthodontic treatment goals.
This thesis project developed a new OTM rat model to study the impact of OTM on
face-M1 and face-S1 that would simulate orthodontic forces applied at clinical levels in
humans and with orthodontic forces that were calibrated in proportion to the smaller teeth
of rats. In addition, the orthodontic spring was attached in a manner that minimizes any
additional nociceptive inputs that previous experimental animals were subjected to when
the orthodontic spring was secured to the tissues. Additional measures were taken for its
long-term retention and to prevent its dislodgment due to the continuous eruption of the rat
incisors. Consideration was also given to OTM-induced nociceptive inputs since pain
occurs during OTM in humans (Jones, 1984; Ngan et al., 1989, 1994; Jones and Chan,
1992a, b; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and Dincer, 2004; Polat et al.,
2005). However, there has not been any animal OTM model that has evaluated nociceptive
behavioural changes induced during OTM. Thus, the OTM-induced behavioural change
was tested in the rat model by applying orofacial thermal and mechanical stimuli to assess
Chapter 1
64
facial hypersensitivity. Therefore, this new model could provide insights into whether any
face-M1 and face-S1 neuroplastic changes are associated with nociceptive inputs induced
by OTM.
1.7.2. General hypothesis
OTM is associated with neuroplasticity of the rat’s face-M1 and face-S1 motor
representations and with mechanical and thermal hypersensitivities in the orofacial region.
1.7.3. Objectives
1. a) To develop an OTM rat model.
b) To design and manufacture a cephalostat for standardized radiographic measurement
of the amount and the rate of OTM.
2. To use ICMS and EMG recordings to test if neuroplastic changes occur in the ICMS-
defined motor representations of anterior digastric (LAD, RAD), masseter (LMa, RMa),
buccinator (LBu, RBu), and genioglossus (GG) muscles within the rat’s face-M1 and
face-S1 during OTM; the analyses to include any alterations in the number of ICMS
sites representing these muscles and in the onset latencies of ICMS-evoked responses in
the muscles.
3. To test if orofacial mechanical and thermal hypersensitivities occur in rats during OTM.
CHAPTER 2
DEVELOPMENT OF A RAT MODEL FOR STUDYING
NEUROPHYSIOLOGICAL CHANGES DUE TO
ORTHODONTIC TOOTH MOVEMENT
Chapter 2
66
2.1. Abstract
Objectives: To develop an orthodontic tooth movement (OTM) rat model, and design and
manufacture a cephalostat for standardized radiographic measurement of the amount and
the rate of OTM.
Material and Methods: Sprague-Dawley rats (140-160 g), 6 weeks old, were divided into
an experimental (E) group (n=8) and a sham (S) group (n=8). The E group had an activated
NiTi closed-coil spring (10 cN) stretched from a stainless-steel ligature wire tied around the
right maxillary molars to a stainless-steel ligature wire around the maxillary incisors, while
the S group had a similarly placed inactivated orthodontic spring. A cephalostat was
designed and radiographs were taken in both lateral and transverse views immediately
before placing the orthodontic spring and subsequently at 1 day, and 7, 14, 21, and 28 days
to measure OTM and the distal drift (DD) inherent in rats in the left (control) side due to
growth and development of the jaws. Statistical analyses involved mixed model repeated
measures ANOVA (MMRM ANOVA).
Results: A constant force of 10 ± 4 cN applied for 28 days produced OTM of the right
three maxillary molars and maxillary incisors of 1.75 ± 0.23 mm. The rate of OTM was
uniform for the 28 days of the experiment. The amount of DD of the maxillary molars for
the control side of the E group was 0.18 ± 0.03 mm after 28 days. The experimental
animals demonstrated a significant transient weight loss on day 1 after orthodontic spring
placement, but they then rapidly gained normal weight.
Conclusions: This new OTM rat model reflects the advantages of using orthodontic force
parameters that are well defined and within the physiological limits when applied to the
small teeth of rats and that correlates well with the orthodontic forces applied clinically in
humans. Trauma inflicted to the tissues to aid in the anchorage of the orthodontic spring
could alter conditions at the molecular level in the intraoral tissues and can alter
interpretation of the data. Hence orthodontic spring attachment and maintenance for the
length of the experiment was designed to produce minimal nociceptive input other than that
generated by the force of the orthodontic spring on the teeth. The standardization and the
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67
convenience of taking both the lateral and transverse radiographic views of the head of the
rat by using the cephalostat is helpful in identifying landmarks that can be customized to
the requirements of the study.
2.2. Introduction
The concept that OTM involves resorption and deposition of bone of the tooth
socket has been suggested as early as 1839 (Harris, 1839). Since then there have been
numerous studies using different animal models to improve our understanding of the
biological responses to OTM and to elucidate fundamental clinical parameters of an
optimal orthodontic force. According to a systematic review of animal studies documented
in the orthodontic literature (Ren et al., 2004), 175 studies on OTM had been performed in
rats and comprised 55 per cent of the 320 animal investigations published until 2002.
Although there are many advantages to studying OTM in a rat model (see chapter 1), the
previous rat studies have several shortcomings related to the physiological aspects of OTM
and to the design of the orthodontic appliances used (Ren et al., 2004). Growth and
development with superimposed functional loads may cause posterior lengthening of the rat
jaw (Kraw and Enlow, 1967; Vignery and Baron, 1980; Herber et al., 2012) and can
contribute to PDL turnover and bone remodeling during the lifespan of a rat. These growth
and adaptation features result in an inherent DD of the rat molars that have not been
adjusted for in most of the studies in calculating the amount of OTM (Kraw and Enlow,
1967; Vignery and Baron, 1980; King et al., 1991; Tsuchiya et al., 2013). Also, the rat
incisor teeth erupt continuously at the rate of 1-2 mm daily to compensate for their gnawing
behaviour (Sessle, 1966; Burn-Murdoch, 1995; Risnes et al., 1995), and this may result in
changes in force direction. These factors confound the interpretation of data acquired from
the previous OTM studies in rats.
Interrupted force application and changing force magnitude may compromise the
interpretation of the relationship between force and tooth displacement. Thus, constant and
continuous forces are recommended for experimental research (van Leeuwen et al., 1999).
However, the small size of the rat teeth make it difficult to design and fabricate an efficient
orthodontic appliance that is suitable for producing a constant and continuous force with an
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68
acceptable force range. The Waldo technique (Waldo and Rothblatt, 1954) has been the
most common method for inducing OTM in rats, and consists of placing an intermaxillary
elastic between the rat molars. Other studies have used custom-designed and non-
standardized springs as force delivery systems, but these have resulted in uncontrolled and
unknown force magnitudes and force decay rates. Since the molar tooth of rats is up to 50
times smaller than that of a human tooth (Ren et al., 2004), the force applied to the molar
teeth in rat OTM models should be adjusted in proportion to mimic a clinical OTM
situation. It is also noteworthy that as soon as orthodontic force is applied to a tooth, the
tooth immediately displaces in the direction of the force to traverse the width of the
periodontal ligament (PDL) thereby creating a zone of compression and a zone of tension
in the PDL. Thus, since it may take from a few days to a few weeks to reach the linear
phase of OTM (see chapter 1), studies aimed at studying the characteristics of orthodontic
force and biological responses in the linear phase of OTM should have an experimental
period of at least 14 days (Ren et al., 2004).
Other rat OTM models have been introduced (King et al., 1991; Ren et al., 2004) to
overcome most of the shortcomings and considerations. However, some of the features to
secure the orthodontic spring to the teeth warranted either extracting opposing molars
(King et al., 1991), or anchoring the anterior end of the orthodontic spring by drilling a
transverse hole through the maxillary alveolar bone and both incisors at the mid-root level
with a dental bur (Ren et al., 2004). Both these procedures can lead to trauma of the PDL
and alveolar bone, and in one rat model (Ren et al., 2004) irreversible pulpitis of the
maxillary incisors, thereby likely activating pulpal nociceptive inputs into the central
nervous system (CNS) independent of the OTM process. Hence there is a merit in
developing a new rat model of OTM that would reflect changes introduced by OTM
exclusively, without any cofounding effects resulting either from extracting opposing
molars or from nociceptive pulpal inputs that are induced by securing the anterior end of
the orthodontic spring by passing the ligature wire through the maxillary incisor pulp and
bone. Furthermore, precise quantification of OTM in the rat model is essential as it can
influence the interpretation of the data. The cephalometric technique to measure OTM has
been recommended as it is reliable, reproducible, and sensitive enough to register small
Chapter 2
69
increments in OTM, and allows taking multiple radiographs during different time frames in
a longitudinal study (King et al., 1991; Bresin and Kiliaridis, 2002; Maki et al., 2002;
Bryndahl et al., 2004; VandeBerg et al., 2004; de Carlos et al, 2006; Singleton et al., 2006;
Gonzales et al., 2008). Therefore, the objectives of this study were: 1) to develop an OTM
rat model, and 2) to design and manufacture a cephalostat for standardized radiographic
measurement of the amount and the rate of OTM.
2.3. Material and Methods
2.3.1. Animal preparation
Experiments were performed on 16 6-week-old adult male Sprague-Dawley rats
(140-160 g) that were housed in cages (27cm X 45 cm X 20 cm) in a temperature (21 ±
1°C) - and humidity (50 ± 5%) - controlled environment under a 12 h light/dark cycle
(lights on at 07:00 a.m.) and that received water and mashed diet (Rodent diet #2018M,
Harlan Teklad) ad libitum. The rats were acclimatized to the environment for 1 week before
the initiation of the study. The animals were monitored on a daily basis to assess body
weight and food consumption, abnormal behaviour (e.g., standing in a corner of the cage
for prolonged periods, strange eating or chewing behaviour, swaying their heads back and
forth, vibrating their tails, or vocalizing), and any post-operative complications (e.g.,
infection and ulceration) that could ensue from tissue injury. All experimental procedures
were approved by the University of Toronto Animal Care Committee, in accordance with
the Canadian Council on Animal Care Guidelines and the regulations of the Ontario
Animals for Research Act (R.S.O. 1990). All experimental procedures were carried out by
one investigator to ensure consistency in the experimental procedures while to minimize
the possibility of experimental bias, the data analyses were performed by another
investigator who was blinded to the animal groups.
2.3.2. Study groups and dental procedures
The rats were separated into 2 groups, an experimental (E) (n = 8) group that
received a NiTi closed-coil orthodontic spring (coil diameter 1.15 mm, eyelet diameter 2.45
mm, force on activation 10 cN, GAC, NY, U.S.A.) that was activated to induce OTM, and
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70
a sham (S) group (n = 8) that received the orthodontic spring but in an inactive state. Under
general anaesthesia (inhalation isoflurane 5% induction, 2~2.5% maintenance), a stainless-
steel ligature wire (0.009 inch, American Orthodontics, WI, U.S.A.) was tied around the
right three maxillary molars of the rat to attach one eyelet of the orthodontic spring (Fig. 2-
1). The ligature wire was then covered with flowable light-cured composite resin
(Transbond Supreme LV, 3M-Unitek, CA, U.S.A.) on the lingual and buccal crown
surfaces of the right maxillary molars. For the E group, the orthodontic spring was then
stretched so that the other end of the orthodontic spring reached the maxillary incisors; the
eyelet of this side was attached to a stainless-steel ligature wire (0.009 inch, American
Orthodontics, WI, U.S.A.). The anterior ligature wire was tied around the circumference of
the right and left maxillary incisors at the gingival end and flowable light-cured composite
resin was then placed on the ligature wire all around the incisor teeth except for the incisal
edges, to secure the ligature wire to the incisors. For the S group, the orthodontic spring
was similarly placed on the right maxillary molars, except the orthodontic spring remained
passive with no stretch of its coil. The anterior ligature wire was then twisted on itself to
reach the maxillary incisors and anchored to the incisors, as in the E group. Before
anchoring the anterior ligature to the maxillary incisors in the E and S groups, the force
magnitude was measured by using a Correx tension gauge (range 3-30 g, Accuracy ± 0.01x,
Haag-Streit AG, Switzerland) 3 times and the average of the 3 readings was recorded as the
force magnitude of the orthodontic spring for that day.
Since the rat incisors normally erupt continuously at a rate of approximately 1-2
mm per day (Sessle, 1966; Burnmurdoch, 1995; Risnes et al., 1995) as a compensatory
mechanism to tooth wear resulting from their gnawing behaviour, to prevent the
dislodgement of the anterior ligature wire from the impact of the animal’s biting forces and
to maintain the direction of the orthodontic force parallel to the occlusal plane, the ligature
wire was reattached gingivally under brief general anaesthesia (inhalation isoflurane 5%
induction, 2~2.5% maintenance) to the maxillary incisors every third day and covered with
flowable composite resin. Before repositioning the orthodontic spring gingivally every third
day the force magnitude was measured 3 times and the average of the 3 readings was
recorded as the force magnitude of the orthodontic spring for that day.
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71
2.3.3. Cephalostat and radiographic measurement of OTM
A cephalostat was designed and manufactured in collaboration with the
Bioengineering department of the Hospital for Sick Kids, Toronto, to permit positioning the
head of the rat in two different planes, the lateral and the transverse planes so that
standardized radiographs could be taken for cephalometric evaluation. The cephalostat
assembly consisted of a base platform with built-in positioning guides that held the
radiographic film (Kodak dental size 4), and an independent rat-holding unit that had
movable ear rods and a maxillary incisor bar to stabilize the head. This rat-holding unit was
positioned on the base platform with the positioning guides built into the base platform to
allow for a standardized alignment and distance between the x-ray source, head of the rat,
and the radiographic film for the x-ray exposures (Fig. 2-2). The distance between the x-ray
source and the midsagittal plane of the rats head was 5 feet. After the radiograph in the
lateral view was taken, the rat-holding unit was rotated counter-clockwise by 90° and
repositioned on the base with the help of the built-in guides on the base platform to take the
transverse view (kV 80, mA 15, exposure time 1.5 sec.). During the radiographic
procedure, the rat was under brief general anaesthesia (inhalation isoflurane 5% induction,
2~2.5% maintenance). The radiographs were taken immediately before the placement of
the orthodontic spring, and subsequently at 1 day, and 7, 14, 21, and 28 days after
orthodontic spring placement. The radiographs were digitized using a film scanner (600
dpi, Epson Expression 1680, U.S.A). The digital images of the radiographs were evaluated
and used to measure the Incisor-molar (I-M) distance with the software Viewbox 3 (dHAL
software, Greece). The I-M distance on the transverse radiograph was measured from the
most mesial point on the mesial surface of the maxillary first molar to the most lingual
point on the distal surface of the ipsilateral maxillary incisor at the orthodontic spring and
the control sides for both the E and the S groups; 3 measurements were made and the
average of the 3 readings was recorded as the I-M distance for both the sides. To calculate
the error of the measurement from the radiographic method, 20 randomly selected occlusal
radiographs were used to measure the I-M distance twice in a random order in a blind test
by a single investigator. Error was calculated by using Dahlberg’s equation: Error =
√∑d²/2n (where, d is the mean difference between two measurements made for the 20
Chapter 2
72
occlusal radiographs, and n is the total number of paired measurements). The error of the
measurement of the I-M distance from the radiographs was found to be 0.006 mm and was
thus considered to be of no significance.
The measured OTM (M-OTM) was defined as the difference of the I-M distance
measured before placing the orthodontic spring and the I-M distance measured during
various post-operative time periods, namely 1 day, and 7, 14, 21, and 28 days. The adjusted
OTM (A-OTM) in the E group on the orthodontic spring side included an adjustment for
the inherent DD of the maxillary molars. The DD was calculated for the E group based on
the difference between the measured I-M distance in the control side before and after
placing the orthodontic spring at the different time periods stated above. The DD value
was added to M-OTM in the orthodontic spring side of the E group to attain A-OTM {M-
OTM (orthodontic spring side) + DD (control side) = A-OTM}. The rationale to include
this adjustment was that the OTM induced by the orthodontic spring in the E group also
included a component of distal drift of the maxillary molars that the orthodontic spring
overcame inherently but the value of which could not be measured directly on that side and
was obtained from the control side for the E group.
2.3.4. Statistical Analyses
Mixed model repeated measures ANOVA (MMRM ANOVA) followed by post-hoc
Sidak-adjusted pairwise comparisons as appropriate were used to assess within and across
groups differences in mean daily gain of body weight and the difference in amount and rate
of OTM. In all analyses a probability level of P < 0.05 was considered statistically
significant. All data is reported as mean ± SEM. Data was analyzed by a statistician using
the SAS statistical software program (version 9.3).
2.4. Results
2.4.1. Weight gain during the course of the experiment
Before placing the orthodontic spring, both E and S groups had a daily gain in body
weight of 6.5 ± 0.73 g and 7.12 ± 0.66 g respectively, however after the placement of the
Chapter 2
73
orthodontic spring, the E group had a small but significant loss of weight for day 1 as
compared to the S group, but thereafter gained weight again at a similar rate to that of the S
group (P < 0.001) (Fig. 2-3).
2.4.2. Force characteristics
The force levels of the orthodontic springs were maintained at a consistent level of
10 ± 4 cN during the course of treatment for the E group. This level was consistent with a
force of 10 cN for an activation range of 1 - 15 mm for this NiTi closed-coil spring as
suggested by the manufacturer in the load-deflection curve of the orthodontic spring (Fig.
2-4). In the Fig. 2-4, the graph shows the initial loading phase (upper curve) when the
spring is stretched to activate it, and the discharge phase (lower curve) that indicates the
dissipation of the force of the orthodontic spring as its activation length decreases (Miura et
al., 1986; Lombardo et al., 2012).
2.4.3. OTM
2.4.3.1. Amount and rate of OTM
The change in I-M distance (A-OTM) on the orthodontic spring side for the E group
at day 28 was 1.75 ± 0.23 mm. The A-OTM on the orthodontic spring side for the E group
was significantly larger than that on the control side for 7 (P = 0.037), 14, 21, and 28 days
(P < 0.001), and compared to the orthodontic spring side in the S group for 7 (P = 0.047),
14, 21, and 28 days (P < 0.001) (Fig. 2.5). The change in A-OTM for the E group on the
orthodontic spring side was significantly larger for day 28 compared to day 1, and days 7
and 14 (P < 0.001), but not for day 21. Likewise, the change in A-OTM for the E group was
significantly larger for day 21 compared to day 1, and days 7 and 14 (P < 0.001), for day 14
compared to day 1 (P < 0.001), and day7 (P = 0.002), and for day 7 compared to day 1 (P <
0.001).
OTM on the orthodontic spring side in the E group progressed at a similar rate over
the course of 28 days of the experiment length. A trend of a slow OTM rate in the first 7
days (0.37 ± 0.03 mm), a higher rate from day 7 to day 21 (0.52 ± 0.07 mm from day 7 to
Chapter 2
74
day 14, and 0.56 ± 0.07 mm from day 14 to day 21), and slower rate after day 21 (0.29 ±
0.03 mm) was apparent, but was not statistically significant (Fig. 2-6). A slight palatal
tipping of the incisors was inevitable due to the reciprocal pull of the orthodontic spring,
however without any noticeable rotation of these teeth. The lateral radiographs
demonstrated that the line of force application by the orthodontic spring was maintained
parallel to the occlusal plane (Fig. 2-2).
2.4.3.2. Amount of DD of the maxillary molars
The amount of DD of the maxillary molars for the control side of the E group was
0.18 ± 0.03 mm after day 28, and along with the orthodontic spring side and the control
side of the S group, stayed relatively constant from day 1 until day 28 (Fig. 2-5). There was
no significant difference in DD for the control side of the E group compared to the
orthodontic spring and control sides of the S group at any of the corresponding time points.
However, the DD was significantly different between the day 1 and day 28 time points for
the control side of the E group (P < 0.001), and at day 1 and day 28 in the S group for the
orthodontic spring side, indicating that the inactivated orthodontic spring did not restrict the
inherent DD of the right maxillary molars in the S group (P < 0.001).
2.5. Discussion
This study established an OTM rat model for studying tooth movement and
associated neurophysiological changes induced by OTM. Radiographic analyses revealed
that the A-OTM of the three maxillary molars and maxillary incisors produced in a span of
28 days was 1.75 ± 0.23 mm. The rate of OTM was uniform throughout the 28 days of the
experiment. The experimental animals demonstrated a significant transient weight loss on
day 1 after orthodontic spring placement, but they then rapidly assumed a normal weight
pattern thereafter for the rest of the experiment.
Chapter 2
75
2.5.1. Force system
2.5.1.1. Force delivery method
Due to the need for using standard parameters for the rat OTM model, the force
delivery method of attaching an orthodontic spring on the rat’s teeth had defined and
measurable force parameters with force levels that were of constant magnitude and in a
direction that was maintained parallel to the occlusal plane for the duration of the
experiment. Also, the quantitative measurement of OTM used standard radiographic
techniques, and the intraoral manipulation for placement and the maintenance of the
orthodontic spring was given a high priority in the experimental design to simulate a
clinical scenario in humans.
The “Waldo technique” of promoting OTM has been used in over 25% of the
publications on animal OTM studies that were conducted before 2002 (Ren et al., 2004). In
most of the other animal OTM studies, non-standardized force parameters or orthodontic
springs with varying designs were chosen as the force delivery method. The use of elastics
or orthodontic springs suffered from the drawback that the force delivery parameters were
not defined in the studies. Some studies reported only the force applied initially and did not
report the force decay rate along the course of the study. A major experiment design flaw
with the “Waldo technique” is the application of inconsistent force that has a rapid force
decay rate. The initial force of 45 N sharply decreases to 15 N, 5 N, and then to almost
negligible levels in a short duration (Ren et al., 2004). A majority of the studies that used
orthodontic coil springs have documented the force magnitude measured only at the time of
placement of the orthodontic spring, or the value specified by the manufacturer of the
spring, rather than testing the force levels in their laboratory setting. This uncontrolled
experimental situation may produce unreliable data on the relationship between force and
tooth movement. The use of NiTi closed-coil orthodontic spring in this study with force
levels that were recorded during the initial stage of force application and then followed up
by checking the force magnitude every 3 days assured that force levels were monitored
during the entire course of the experiment and produced a constant level of 10 ± 4 cN
throughout the experimental period.
Chapter 2
76
2.5.1.2. Force parameters
The appliance design used in the present study anchored the orthodontic spring with
a stainless-steel ligature wire around the maxillary incisors and was distinct from other
studies that have also used closed-coil orthodontic springs to study OTM (Kawasaki et al.,
2000; Ren et al., 2004; de Carlos et al., 2006; de Carlos et al., 2007; Sprogar et al., 2007,
2008; Yamaguchi et al., 2007; Fujita et al., 2008; Drevensek et al., 2009; Yoshida et al.,
2009). Some of these studies had tied the ligature wire around all three maxillary molars to
make them a single unit (Ren et al., 2004), while others tied the ligature wire around only
the first maxillary molar (Kawasaki et al., 2000; de Carlos et al., 2006; 2007; Sprogar et al.,
2007, 2008; Fujita et al., 2008; Kriznar et al., 2008; Drevensek et al., 2009; Yoshida et al.,
2009). The magnitude of the forces used for OTM also varied extensively for the studies, as
did the methods used for measuring OTM. Because of these differences, direct comparison
between OTM amounts and rates across studies are not feasible.
Since the molar teeth of rats are approximately 50 times smaller than that of human
molars, the force magnitude applied to the rat teeth should be adjusted to this difference in
tooth size to maintain the force applied to the rat molars proportional to orthodontic forces
applied to human teeth (Ren et al. 2004; Tan et al., 2009). It is estimated that an application
of 20 cN force on a rat molar is comparable to a force of 1000 cN (1 kg) on a human molar.
In the literature review on animal studies of OTM, only approximately 20 per cent of the
studies reported use of forces of 20 cN or less (Ren et al., 2004). In the present study, a
constant and continuous force of approximately 10 ± 4cN over a range of 1 - 15 mm
activation was distributed over all three maxillary right molars. If calculated for human
teeth, the effect could be estimated to be the same as a force of 170 cN (10/3 cN × 50),
which is within the force range recommended for the retraction of maxillary canine teeth
(Storey and Smith, 1952; Owman-Moll et al., 1996a, b). The application of the orthodontic
spring force on only the first right upper molar would be comparable to a force of 500 cN
(10cN × 50), which would be excessive compared to the recommended clinical levels (Ren
et al., 2004). Further, the regular reattachment of the anterior ligature wire on the incisors
gingivally every 3 days compensated for the continuous eruption of the incisors and
maintained the direction of the orthodontic spring force parallel to the occlusal plane.
Chapter 2
77
2.5.2. Amount and rate of OTM
The amount and rate of OTM compared well with values reported by other studies
that have used a similar NiTi closed-coil orthodontic spring on the maxillary teeth in rats
(Ren et al., 2003; Ren et al., 2004; Rozman et al., 2010). The amount and the rate of OTM
in the present study was equivalent to the distal movement of 1.22 to 1.91 mm reported for
the maxillary canine in human studies for 28 days (Daskalogiannakis and McLachlan,
1996; Iwasaki et al., 2000, 2009; Hayashi et al., 2004). Further, although the OTM
documented in the present study at 7 days for the E group compared well with the OTM
reported for 40 and 60 cN forces in the study reported by King et al. (1991), the present
study found a higher rate of tooth movement compared to that reported by King et al.
(1991) by day 14. The 20 cN force applied in their study resulted in no significant mesial
movement of the maxillary molar teeth by day 7; on the contrary, they reported a distal
movement of the molar teeth from day 3 until day 7 of orthodontic spring attachment. This
was attributed to deactivation of the orthodontic spring immediately after the initial
activation and also on the physiological DD of the maxillary molars. The difference in
OTM that was achieved in the present study firstly may be attributed to the use of younger
rats; higher levels of OTM have been reported in the younger age rats when compared to
older rats (Bridges et al., 1988; Takano-Yamamoto et al., 1992; Kyomen et al., 1997; Ren
et al., 2003; Misawa-Kageyama et al., 2007). Secondly, the difference could be related to
the type of orthodontic spring used and the load deflection characteristics of the orthodontic
spring used in the two studies. The optimal level of force required to induce maximum rate
of OTM in the shortest duration has eluded the orthodontic profession. Studies in rats
indicate that forces of less than 10 cN may be the ideal force magnitude in terms of
inducing an optimal rate of OTM (Kohno et al., 2002; Ren et al., 2004; Gonzales et al.,
2008). This level of force correlates well with studies on the activity in the
microvasculature of PDL during OTM (Oppenheim, 1944; Kondo, 1969, Vandevska-
Radunovic et al., 1994; Noda et al. , 2009) that suggest that a light force maintains the
patency of the blood vessels of the PDL and thereby promotes biological processes of
OTM, while a heavier force causes partial or total occlusion of the blood vessels resulting
in degeneration or necrosis of the PDL (Schwarz, 1932; Storey, 1952, 1973; Reitan, 1957,
Chapter 2
78
1964, 1967; Gianelly, 1969 ; Gaengler and Merte, 1983; for review, see Meikle, 2006;
Wise and King, 2008; Krishnan and Davidovitch, 2009).
A non-linear rate of OTM may be expected due to the different phases of OTM
suggested in the literature (Reitan, 1967; Storey, 1973; King et al., 1991; Pilon et al., 1996;
van Leeuwen et al., 1999). The suggested normal width of the PDL of a rat molar is
approximately 0.12 - 0.15 mm (Lasfargues and Saffar, 1993; Tengku et al., 2000), and
rapid initial movement on force application has been suggested as the tooth traverses the
width of the PDL. A uniform rate of OTM was however documented in the present study
until the end of day 28. A similar trend of a constant rate of OTM without a lag phase has
been reported in other studies on rats (Kohno et al., 2002; Ren et al., 2004; Gonzales et al.,
2008; Rozman et al., 2010) and humans (Iwasaki et al., 2000, 2009). This may be attributed
to a lower magnitude of forces applied in the present study that promoted physiological
OTM without undermining resorption as a distinct separate phase and thereby
circumventing the lag phase of tooth movement described in the literature (Reitan, 1967;
Storey, 1973; Burstone, 1984; King et al., 1991; Pilon et al., 1996; van Leeuwen et al.,
1999). Thus, OTM progressed from the initial stage when the PDL was compressed and the
root was adjacent to the alveolar bone and direct bone resorption lead to the recovery of the
width of the PDL (Kohno et al., 2002). The lag phase of OTM has been explained on the
basis of the time taken to recruit cells to eliminate the hyalinized tissue in the PDL
(Burstone, 1984; King et al., 1991). However, recent studies that have used low levels of
orthodontic force have reported traces of hyalinized tissue not only in the initial but also in
the linear phase of experimental OTM (Kohno et al., 2002; von Bohl et al., 2004a, b; Iino et
al., 2007), suggesting that the development and removal of hyalinized tissue is a continuous
process instead of a exclusive single phase event during OTM (von Bohl and Jagtman,
2009). Hence, the OTM process induced by low level of forces may escape a distinct lag
phase during which the hylanized tissue is removed before the initiation of the linear phase
of tooth movement. The regional morphological differences in the alveolar bone as the first
molar moves mesially into the lateral diastema of the rat has been suggested as a reason for
the slower rate of OTM after day 21 (Ren et al., 2004). The amount and rate of OTM
documented in the present study was slightly lower at the end of 28 days than that
Chapter 2
79
measured in the study by Ren et al. (2004), and probably if the OTM was continued beyond
day 28 in the present study the rate of tooth movement would have slowed down as the first
molar may have moved into the lateral diastema region of the alveolar bone. A trend
towards a slower rate of OTM from day 21 to day 28 was documented in the present study,
although the change was not statistically significant.
Inherent DD of the molar teeth in rats has been documented in relation to the
posterior lengthening of the jaw (Kraw and Enlow, 1967; Vignery and Baron, 1980; King
et al., 1991; Tsuchiya et al., 2013). The amount of DD of the maxillary molars in the
present study progressed at a constant rate and correlated with the levels described in the
studies reported by King et al. (1991) and Ren et al. (2004). Although the change was not
statistically significant between the different animal groups during the corresponding time
points in the present study, it was statistically significant between the day 1 and day 28 time
points in the control side of the E group. Lack of inclusion of the DD of the maxillary
molars in other studies tends to underestimate the total amount of maxillary molar
movement induced by the orthodontic spring in rats. Hence, it seems reasonable to adjust
the decrease of the I-M distance by factoring-in the inherent DD of the maxillary molars
that the orthodontic spring has to overcome on the orthodontic spring side in the E group.
The amount and rate of OTM in the present study were similar to those reported in
humans (Daskalogiannakis and McLachlan, 1996; Iwasaki et al., 2000, 2009; Hayashi et
al., 2004). However, the teeth were moved into a state of malocclusion from normal
occlusion, whereas in clinical practice in humans the goal of the orthodontic treatment is to
resolve an existing dental malocclusion. Thus approaches such as gene manipulation or jaw
growth modification could be used to first create a state of malocclusion in the rat and then
an active orthodontic spring could be placed to treat that malocclusion to simulate the
clinical situation in humans.
A laboratory animal’s relative weight loss during an experimental period may be
related to either subjecting the animal to pain, distress and discomfort through a stressful
environment or a physical limitation that restricts ingestion of food by the animal, whereas
a significant weight loss is an important sign of deterioration of the animal’s condition
Chapter 2
80
(Olfert et al., 1998; Balcombe et al., 2004). The animals with the orthodontic springs in the
study reported by King et al. (1991) had a tendency to lose weight during the entire length
of the experiment. In order to eliminate extraneous forces from occlusion and tissue
impingement from the appliance, the mandibular first and second molars were extracted in
their study. Further, barbed broaches were placed in the tissue of the palate to serve as
implants as reference markers for radiographic measurements of OTM. In the study by Ren
et al. (2004), to prevent the eruption of the maxillary incisors, a transverse hole was drilled
through the alveolar bone and both maxillary incisors at the mid-root level with a drilling
bur and a stainless-steel ligature wire was inserted in the hole and tied through and through.
This accomplished their goal of preventing the continuous eruption of the maxillary
incisors, but it very possibly resulted in trauma to the dentin and irreversible pulpitis, and
probably eventually a necrotic pulp of the maxillary incisors. Further, it has been reported
that neuroplastic changes in the face-M1 and face-S1 or trigeminal brainstem sensory
nuclear complex are induced by peripheral intraoral manipulations, such as tooth extraction
(Henry et al., 2005; Avivi-Arber et al. 2010a), lingual nerve injury (Adachi et al., 2007), or
pulp extirpation (Hu et al., 1986, 1999; Linden and Scott, 1989). The above-mentioned
procedures may have introduced nociceptive inputs to the CNS and inflammatory changes
that are detrimental for the long-term welfare of the animal and may have induced changes
in the nervous system other than those initiated by the OTM. On the contrary, the approach
in the present study to tie the anterior ligature wire gently on the maxillary incisors and
cover it with flowable composite resin, and then reattach the ligature wire gingivally on a
regular basis, was associated with a weight loss only at day 1 after orthodontic spring
placement. It served the purpose of eliminating any additional nociceptive inputs beyond
those that are generated by OTM, while still maintaining the desired direction of the
orthodontic spring parallel to the occlusal plane. Further, animal welfare for long-term
studies is enhanced by preventing any damage to the orofacial tissues. While the findings in
animal experiments should be extrapolated with caution from species to species, OTM
kinetics and biological responses reported for rats seem to be similar to those reported with
other OTM models, including humans (Storey, 1973; Davidovitch et al., 1980; Yamasaki et
al., 1982; King et al., 1991; Keeling et al., 1993; Daskalogiannakis and McLachlan, 1996;
Chapter 2
81
Pilon et al., 1996; Iwasaki et al., 2000, 2009; Hayashi et al., 2004; von Bohl et al., 2004a, b;
von Bohl and Jagtman, 2009).
2.6. Conclusions
The present study developed a new “OTM rat model” that produced 1.75 ± 0.23 mm
of A-OTM of the three maxillary molars and maxillary incisors in a span of 28 days. The
experimental animals demonstrated only a transient weight loss after orthodontic spring
placement, but thereafter gained a normal weight pattern for the duration of the experiment.
Modifications were made in the present study to OTM rat models used in previous studies
and reflect the advantages of using orthodontic force parameters that are well defined and
within the physiological limits when applied to teeth of rats and that reflect well the
orthodontic forces applied clinically in humans. Trauma inflicted to the tissues to aid in the
anchorage of the orthodontic spring, as suggested in the other rat models of OTM, could
alter conditions at the molecular level in the intraoral tissues and can alter interpretation of
the data. The standardization and the convenience of taking both the lateral and transverse
radiographic views of the head of the rat by using the cephalostat is helpful in identifying
landmarks that can be customized to the requirements of the study. The use of a standard
rat model of OTM can benefit cross-study comparisons to make evidence-based
conclusions as the trend for animal studies increases to use rat as the animal of choice.
Chapter 2
82
2.7. Figures and Graphs
Fig. 2-1. NiTi closed-coil orthodontic spring activated and stretched between the stainless-steel ligature wire
around the right three maxillary molars and the around the maxillary incisors with the aim of moving the right
maxillary molars forward. The DD of the maxillary molars on the control side is shown.
DD
Molars
Incisor
sss
Molars
Incisor
ss
Chapter 2
83
A.
Fig. 2.2. For legend, see below
Rat-holding unit
Radiographic
film
Tube for the
anaesthetic
gas Incisal pin
Ear rods
Positioning guide
Base
platform
Chapter 2
84
B. a. Lateral view
b. Transverse view
Fig. 2-2. A. The cephalostat assembly to position the head of the rat for taking the radiographs. It consists of a
base unit with built-in positioning guides for the rat-holding unit. The cephalostat assembly allows for taking
radiographs first in the lateral view (B. a.), and then by rotating the rat-holding unit counter-clockwise by 90°,
in the transverse view (B. b.). The lateral view was used to confirm the line of force application by the
orthodontic spring by maintaining it parallel to the occlusal plane, while the transverse view was used to
measure OTM.
Incisors Molars
Molars Incisors
Chapter 2
85
Time after spring placed (days)
-1 0 1 2 3 4 5 6 7 14 21 28
Chan
ge
in w
eight
(g)
-50
0
50
100
150
200
250
E group
S group
*
Fig. 2-3. The mean ± SEM change in weight gain for the duration of the experiment in the E and the S groups.
The gain in weight in the E group was significantly less than the S group only on day 1 (*MMRM ANOVA, P
< 0.001).
Chapter 2
86
Fig. 2-4. Force deflection curve of the NiTi closed-coil orthodontic spring (Courtesy GAC Inc., NY, U.S.A.).
The graph shows the initial loading phase (upper curve) when the spring is stretched to activate it, and the
discharge phase (lower curve) indicates the dissipation of the force of the orthodontic spring as its activation
length decreases. The curve depicts that the force application of 10cN (gf on the y-axis) is constant for a 1 -
15 mm range of activation of the NiTi closed-coil orthodontic spring used in this study.
Chapter 2
87
Fig. 2-5. The mean ± SEM change in the I-M distance during the course of 28 days is shown in the
orthodontic spring side and the control side in the E and the S groups. The A-OTM on the orthodontic spring
side for the E group was significantly larger than on the respective control side for 7 (*MMRM ANOVA, P =
0.037), 14, 21, and 28 days (*MMRM ANOVA, P < 0.001), and compared to the orthodontic spring side in
the S group for 7 (†MMRM ANOVA, P < 0.05), 14, 21, and 28 days (†MMRM ANOVA, P < 0.001). The
change in A-OTM for the E group on the orthodontic spring side was significantly larger at day 28 compared
to day 1, and days 7 and 14 (#ANOVA, P < 0.001), but not for day 21.
Time after spring placement
0 day 1 day 7 days 14 days 21 days 28 days
Chan
ge
in I
-M d
ista
nce
(m
m)
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
E group (n=8) - orthodontic spring side; M-OTM
E group (n=8) - total OTM; A-OTM = (M-OTM + DD)
E group (n=8) - control side; DD
S group (n=8) - orthodontic spring side
S group (n=8) - control side
*†#
*†#
*†#
#
*†
Chapter 2
88
Duration of spring placement (days)
0 7 14 21 28
A-O
TM
per
wee
k (
mm
)
0.0
0.2
0.4
0.6
0.8
1.0
Fig. 2-6. The mean ± SEM rate of OTM per week on the orthodontic spring side in the E group for the 28
days duration of the experiment. OTM progressed at a similar rate over the course of the 28 days.
CHAPTER 3
NEUROPLASTIC CHANGES IN THE FACE-M1 AND FACE-
S1 ASSOCIATED WITH ORTHODONTIC TOOTH
MOVEMENT
Chapter 3
90
3.1. Abstract
Objectives: Orthodontic tooth movement (OTM) is associated with transient pain and
change in dental occlusion, and this may lead to altered somatosensory inputs and patterns
of mastication. The objective of this study was to use intracortical microstimulation
(ICMS) and electromyographic (EMG) recordings to test if neuroplastic changes occur in
the ICMS-defined motor representations of anterior digastric (LAD, RAD), masseter (LMa,
RMa), buccinator (LBu, RBu), and genioglossus (GG) muscles within the rat’s face-M1
and face-S1 during OTM; the analyses to include any alterations in the number of ICMS
sites representing these muscles and in the onset latencies of ICMS-evoked responses in the
muscles.
Materials and Methods: A total of 42 male Sprague-Dawley rats were divided into
experimental (E), Sham (S) and Naive (N) groups. In order to move the right maxillary
molars forward the E group had a closed-coil (Ni-Ti) orthodontic spring (10 cN) stretched
from the right maxillary molars to the maxillary incisors. The S group had a similarly
placed inactivated orthodontic spring, and the N group (n = 6) did not have any orthodontic
spring. The E and the S groups were further subdivided into three subgroups based on the
duration that the orthodontic springs were maintained in the oral cavity before cortical
mapping was performed: 1-day (E1, n = 6; S1, n = 6), 7-day (E7, n = 6; S7, n = 6), and the
28-day (E28, n = 6; S28, n = 6). EMG wires were implanted in the LAD, RAD, LMa, RMa,
LBu, RBu, and GG muscles. ICMS (60 and 20 µA) was applied to define the motor
representations of these muscles in the right and left face-M1 and face-S1. The ICMS-
evoked EMG activities of the muscles were analyzed by a customized software program
developed in LabVIEW. The data was analyzed using multivariate [mixed model repeated-
measures (MMRM) ANOVA] analyses, followed by post-hoc Sidak pairwise comparisons.
Results: OTM resulted in significant changes in the number of positive sites in the E
subgroups for motor representations of the LAD, RAD, and GG muscles in the ICMS-
defined face-M1 and face-S1 at time points of days 1, 7, and 28 of continuous orthodontic
force application and in the number of sites in face-M1 from which ICMS could
simultaneously induce EMG activity in different combinations of LAD, RAD, and GG.
Chapter 3
91
There was no significant difference in the shortest onset latency for any of the muscles
across the groups, and between face-M1 and face-S1 within each group. Analyses of the
extent of the face-M1 and face-S1 territory for LAD, RAD, and GG representations for
each time point during OTM revealed significant changes in the number of positive
penetrations in the anteroposterior (AP) and the mediolateral (ML) planes and suggested a
trend in the shift of the centre of gravity (CoG).
Conclusions: This is the first study to document neuroplastic changes induced in the face-
M1 and face-S1 by OTM. These changes may reflect adaptive sensorimotor changes in
response to the altered environment in the oral cavity induced by OTM.
3.2. Introduction
The primary motor cortex (M1) is a part of the sensorimotor cortex and is involved
in the acquisition and performance of sensorimotor behaviours (for review, see Nelson,
1996; Sanes and Donoghue 2000; Schieber, 2001; Graziano et al. 2002; Monfils et al. 2005;
Nudo and McNeal, 2013) through modification and organization of muscle synergies that
are stored in “motor maps” formed in M1. These cortical maps are dynamic constructs that
are remodeled throughout life through “neuroplasticity” (for review, see Kaas, 1991; Sanes
and Donoghue, 2000; Monfils et al., 2005; Nudo, 2006; Sessle, 2011b). M1 is
topographically organized and its most lateral part is the face-M1 which controls the
muscles of the face, mouth and jaws. The ICMS technique has been extensively used to
map the motor representation of the orofacial region in face-M1 in several species and has
revealed the organizational features of face-M1 and its capacity for neuroplasticity (for
review, see Avivi-Arber et al., 2011; Sessle, 2011a). Like the limb-M1 (for review, see
Adkins et al., 2006; Nudo, 2006; Navarro et al., 2007; Graziano and Aflalo, 2007), face-M1
can undergo neuroplasticity in association with motor skill acquisition and also following
peripheral manipulations (for review, see Sessle et al., 2007; Avivi-Arber et al., 2011;
Sessle, 2011a). For example, trigeminal nerve injury can induce neuroplastic changes in the
face-M1 motor representations of the vibrissae (for review, see Buonomano and Merzenich,
1998, Sanes and Donoghue, 2000) or the tongue and jaw muscles in rats (Adachi et al.,
2007), and face-M1 neuroplastic changes have also recently been documented following
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changes to the dental occlusion or loss of teeth in rats (Sessle et al., 2007; Avivi-Arber et
al., 2010a). These findings are consistent with studies in humans showing face-M1
neuroplasticity in association with motor skill acquisition, chewing and swallowing
patterns, and peripheral manipulations including dental implants and occlusal splints
(Tamura et al., 2002, 2003; Shinagawa et al., 2003; Svensson et al., 2003b, 2006; Boudreau
et al., 2007; Kordass et al., 2007; Yan et al., 2008; Baad-Hansen et al., 2009; Byrd et al.,
2009; Martin, 2009; Shibusawa et al., 2009, 2010; Arima et al., 2011, Trulsson et al.,
2012). M1 neuroplasticity has clinical significance since it has been suggested that these
interventions can lead to either adaptive M1 neuroplasticity associated with a gain in
function, e.g. motor recovery after stroke, or maladaptive M1 neuroplasticity associated
with either functional loss or injury potentiation, e.g. chronic pain following peripheral
tissue injury (Cohen et al., 1997; Nudo, 2006).
The primary somatosensory cortex (S1) also contributes to motor control and can
undergo neuroplastic changes in association with motor skill acquisition and also following
peripheral manipulations. For example, limb-S1 plays an important role in the acquisition
of new motor skills involving the limbs, and alterations to sensory inputs to S1 from the
limbs produce neuroplastic changes in limb-S1 (for review, see Asanuma, 1989; Sanes and
Donoghue, 2000; Nicolelis and Lebedev, 2009). Likewise, experiments employing ICMS,
cortical cold block, or single neuron recordings in face-S1 during elemental, learned tasks,
or semiautomatic orofacial movements have documented that the face-S1 receives
extensive orofacial sensory inputs and is involved in the fine control of these orofacial
movements (Neafsey et al., 1986; Huang et al., 1989b; Lin and Sessle, 1994; Martin et al.,
1997, 1999; Lin et al., 1998; Murray et al., 2001; Hiraba et al., 2007; Sessle et al., 2007;
Burish et al., 2008). ICMS of the face-S1 can also evoke orofacial movements (Lund et al.,
1984; Neafsey et al., 1986; Hiraba et al., 1997) and jaw and tongue EMG responses (Lee et
al., 2006; Adachi et al., 2007; Avivi-Arber et al., 2010a). Manipulations of orofacial
somatosensory inputs can result in neuroplastic changes in the face-S1 representations in
rodents and humans (Polley et al. 2004a, b; Blake et. al., 2005, 2006; Feldman and Brecht,
2005; Henry et al., 2005; Petersen, 2007; Teismann et al., 2007; Yan et al., 2008; Fox,
2009; Avivi-Arber et al., 2010a; Barnes and Finnerty, 2010).
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Orthodontic tooth movement (OTM) is used clinically to correct dental
malocclusion and is a response of the tooth to applied mechanical force that is propagated
by remodelling of the alveolar bone supporting the tooth subjected to the orthodontic force.
OTM occurs as a consequence of a series of biological responses and various theories of
the mechanisms underlying OTM have been suggested (Vandevska-Radunovic, 1999;
Krishnan and Davidovitch, 2006; Masella and Meister, 2006; Meikle, 2006). One of the
theories suggests that OTM results from an inflammatory process in the periodontal
ligament (PDL) and is associated with transient pain (Vandevska-Radunovic, 1999; Wise
and King, 2008; Krishnan and Davidovitch, 2009). Also, during OTM the occlusion is
being modified on a continual basis. The PDL and surrounding oral tissues have specialized
mechanoreceptors and their afferents project to the central nervous system (CNS) and
transmit sensory information essential for perceptual as well as reflex and other
sensorimotor responses to intraoral stimuli during orofacial sensorimotor function (Linden,
1990; Trulsson and Essick, 2004). For example, the information encoded by PDL
mechanoreceptors (PMRs) about the temporal, spatial and intensive aspects of tooth loads
contributes to the regulation of the muscle activity generating masticatory forces and jaw
movements (Lund, 1991, 2011; Trulsson and Johansson, 1996; for review, see Turker,
2002; Turker et al., 2007; Trulsson et al., 2012). A lack of coordination in chewing has
been reported following denervation of PMRs (Lavigne et al., 1987; Inoue et al., 1989).
Also, the properties of the PMRs, such as their mechanical thresholds and conduction
velocities, have been reported to become progressively impaired during the course of OTM
(Loescher et al., 1993; Okazaki, 1994; Long et al., 1996), although once the teeth are in
their desired position and the tissues have healed, the response properties of the PMRs
improve and return to normal levels (Nakanishi et al., 2004). Thus, it is possible that the
changes induced by OTM in the properties of the PMRs and in the dental occlusion that,
along with OTM-induced activation of intraoral nociceptors, may result in sensory inputs to
sensorimotor cortex that produce neuroplastic changes in the face-M1 and face-S1.
However, the influence of OTM on the face-M1 and face-S1 has not been
previously determined, yet its study is needed to clarify the adaptive mechanisms that
regulate muscle activity and jaw function during and after orthodontic interventions.
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Therefore, the objective of this study was to use ICMS and EMG recordings to test if
neuroplastic changes occur in the ICMS-defined motor representations of anterior digastric
(LAD, RAD), masseter (LMa, RMa), buccinator (LBu, RBu), and genioglossus (GG)
muscles within the rat’s face-M1 and face-S1 during OTM; the analyses to include any
alterations in the number of ICMS sites representing these muscles and in the onset
latencies of ICMS-evoked responses in the muscles. Some of the findings have been
published briefly in abstract form (Sood et al., 2009, 2010, 2012).
3.3. Materials and Methods
3.3.1. Animal preparation
Experiments were performed on 42 young adult male Sprague-Dawley rats (150-
250 g on arrival at the animal facility, 300-400 g on the day of cortical mapping) that were
housed in cages (27cm X 45 cm X 20 cm) in a temperature (21 ± 1°C) and humidity (50 ±
5%) controlled environment under a 12 h light/dark cycle (lights on at 07:00 a.m.) and
received water and mashed diet (Rodent diet #2018M, Harlan Teklad) ad libitum. It has
been documented that feeding rats a mashed diet in contrast to hard chow does not induce
any neuroplastic changes in the rat’s sensorimotor cortex (Avivi-Arber et al., 2010b). The
animals were monitored on a daily basis to assess body weight and food consumption,
abnormal behavior (e.g., standing in a corner of the cage for prolonged periods, strange
eating or chewing behaviour, swaying their heads back and forth, vibrating their tails, or
vocalizing), and any post-operative complications (e.g., infection and ulceration) that could
ensue from tissue injury. All experimental procedures were approved by the University of
Toronto Animal Care Committee in accordance with the Canadian Council on Animal Care
Guidelines and the regulations of the Ontario Animals for Research Act (R.S.O. 1990). All
experimental procedures were carried out by one investigator to ensure consistency in the
experimental procedures. Also, to minimize the possibility of experimental bias, the data
analyses were performed online by another investigator who was blinded to the animal
groups.
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3.3.2. Study groups and dental procedures
OTM has been reported to comprise three phases: an initial phase, which lasts
approximately 24 hours, in which the tooth is displaced almost instantaneously through the
width of the PDL. This phase is followed by the lag phase in which the necrotic tissue,
which is formed due to the compression of the vessels of the PDL and the alveolar bone, is
removed and almost no tooth movement results in this lag phase that lasts approximately
about 5 to 7 days. The final phase is the linear phase of tooth movement and is
characterized by bone formation on the tension side and bone resorption on the pressure
side of the loaded tooth (Shirazi et al., 2002; von Bohl et al., 2004a, b; Krishnan and
Davidovitch, 2006; Yoshimatsu et al., 2006). To include all the phases of tooth movement
in the study design, rats were divided into seven groups – three experimental (E, n = 18),
three sham (S, n = 18), and one naive (N, n = 6). The E and S groups received a closed-coil
(Ni-Ti) orthodontic spring (GAC, N.Y., U.S.A.) (Fig. 3-1). Under general anaesthesia
(inhalation isoflurane; 5% induction, 2~2.5% maintenance), a stainless-steel ligature wire
(0.009 inch, American Orthodontics, WI, U.S.A.) was tied around the right three maxillary
molars of the rat to attach one eyelet of the orthodontic spring. The stainless-steel ligature
wire was then covered with flowable light-cured composite resin (Transbond Supreme LV,
3M-Unitek, CA, U.S.A.) by taking the precaution of limiting the flow to the lingual and
buccal crown surfaces of the right maxillary molars. In the E groups, the orthodontic spring
was then stretched so that the eyelet at the other end of the orthodontic spring reached the
maxillary incisors and was tied to the incisors by passing the stainless-steel ligature wire
interdentally in a figure of eight shape between the incisors. Flowable light-cured
composite resin was then placed on the ligature wire all around the incisor teeth except the
incisal edges, to secure the ligature wire to the incisors (see chapter 2). A mild constant
force of 10 cN was applied by the orthodontic spring to the maxillary right molars and
maxillary incisors, and the tooth movement was confirmed by taking radiographs in the
lateral and occlusal views in a standardized cephalostat (see chapter 2). In the S groups, the
orthodontic spring was placed on the right maxillary molars as in the E groups, but the
orthodontic spring was attached passively to the maxillary incisors with no stretch of its
coil. The stainless-steel ligature wire was twisted on itself to reach the maxillary incisors
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and anchored to the incisors in a figure of eight configuration passing interdentally between
the maxillary incisors, and then covered by flowable composite resin similar to the
experimental groups. For the N group no orthodontic spring was placed. The E and the S
groups were further subdivided into three subgroups based on the duration the orthodontic
springs were maintained in the oral cavity before cortical mapping was performed: 1-day
(E1, n = 6; S1, n = 6), 7-day (E7, n = 6; S7, n = 6), and the 28-day (E28, n = 6; S28, n = 6)
(Fig. 3-1).
Since the rat incisors normally erupt continuously at a rate of approximately 1-2
mm per day (Sessle, 1966; Burn-Murdoch, 1999; Risnes et al., 1995) as a compensatory
mechanism to tooth wear resulting from their gnawing behaviour, while the animal was
under brief general anaesthesia the stainless-steel ligature wire that was attached to the
maxillary incisors in the E and the S groups was removed and reattached gingivally to the
maxillary incisors every third day and secured with flowable composite resin to prevent its
dislodgement from the impact of the animal’s biting forces.
3.3.3. ICMS and EMG recordings
ICMS mapping was carried out as previously described (Avivi-Arber et al., 2010a,
b) within the face-M1 and face-S1 of rats to define the motor representations of the
orofacial muscles. Mapping was performed on day 1 (E1 and S1), day 7 (E7 and S7), and
day 28 (E28 and S28) after the placement of activated and non-active orthodontic springs in
the E and S groups of rats and also on day 0 in the N group of rats (Fig. 3-1).
Rats were maintained throughout the ICMS experiments under a stable level of
general anaesthesia that has previously been shown to be achieved with ketamine (Gu and
Fortier, 1996; Kita and Kita, 2011; Ordek et al., 2013). A cannula was inserted into the
femoral vein under general anesthetic ketamine (175 mg/kg, i.m., Ketaset®, Ayerst
Veterinary Laboratories, Ontario, Canada). Ketamine anaesthetic solution was then injected
through the femoral vein at a regulated rate with a pump (model 11 Plus, Harvard
Apparatus, Inc., Holliston, MA, USA) to maintain the depth of anaesthesia. For the
implantation of EMG electrodes and for the craniotomy to expose the cortex, ketamine (25
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mg/ml) was infused at a rate of 75 mg/kg/h; subsequently, the infusion rate was reduced to
25-50 mg/kg/h for the ICMS mapping. Also, local anesthetic lidocaine HCl (2%) was
injected in the subcutaneous space below the planned incision line prior to making the
cutaneous incisions needed to place the femoral vein cannula, EMG electrodes and carry
out the craniotomy. Body temperature was maintained at physiological levels of 37-38°C
by a feedback control blanket (Model 73A, YSI, Ohio, USA) and the heart rate was
maintained at 300-350 beats/min. Bipolar EMG electrodes (40-gauge, single-stranded,
Teflon-insulated stainless-steel wires) were used to record EMG activity from LAD, RAD,
LMa, RMa, LBu, RBu, as well as GG muscles. Their location was confirmed by electrical
stimulation (12 × 0.2 ms pulses, 333 Hz, 200-400 µA)-induced observable movements (i.e.,
jaw closing for the Ma, jaw opening for the AD, cheek pad movement for the Bu and
tongue protrusion for the GG muscle) immediately after EMG electrode implantation as
well as at the end of the experiment. The rat was then placed in a stereotaxic apparatus
(model 1340, David Kopf, Tujunga, CA, USA) and a bilateral craniotomy was performed
to expose the M1 and S1 and neighbouring areas. The duramater was kept intact and was
covered with warm mineral oil (37°C). Glass-insulated tungsten microelectrodes (0.5-5.0
MΩ impedance at 1 kHz, 10 µA manufactured by Alpha-Omega Engineering, Nazareth,
Israel) were used for ICMS with a computer-triggered stimulus isolator (model A365,
World Precision Instruments, Stevenage, UK) which generated monophasic, cathodal, five
constant-current ICMS trains (at 333 Hz, 33.2 ms, 12 pulses of 0.2 ms, 2.8 ms inter-pulses
intervals) that were delivered at 1 Hz at an ICMS intensity of 60 μA, as previously
described (Neafsey et al. 1986; Sanes and Donoghue 2000; Adachi et al., 2007; Avivi-
Arber et al., 2010a, b). If ICMS could effectively evoke EMG activities, then a second
series of ICMS trains was applied at an ICMS intensity of 20 µA to analyze any change
induced by OTM in the range of subthreshold intensity (20 µA), and finally a second series
of five ICMS trains at 60 µA was delivered to confirm the presence of the response. Based
on an estimated extent of ICMS current spread of less than 0.5 mm at 60 µA ICMS
intensity (Neafsey et al., 1986; Asanuma, 1989; Schieber, 2001; Cheney, 2002),
microelectrode penetrations had a horizontal spatial resolution of 0.5 mm. The
microelectrode was lowered vertically by a micropositioner (model 660, David Kopf, CA,
USA) until it reached the first positive response site, i.e., where EMG responses could be
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evoked. At each anteroposterior (AP) plane in the left and the right cortex, a series of
microelectrode penetrations were made in the mediolateral (ML) plane until no more
ICMS-evoked EMG activities could be evoked. ICMS mapping started at AP3.0 (3.0 mm
anterior to bregma) and ML3.0 (3.0 mm lateral to midsagittal suture) in the left cortex and
after completion of all ML planes in AP3.0 was continued to the right cortex, followed by
mapping of all the series of sites in the ML plane at AP3.5 and AP4.0 for the left and the
right cortex. If ICMS at AP4.0 evoked EMG activities in jaw or tongue muscles, then
AP4.5 was mapped as well, followed by mapping of AP2.5. If ICMS at AP4.0 did not
evoke EMG activities, then mapping included plane AP2.5 and AP2.0. This sequence
allowed for mapping the anterior, posterior, medial and lateral borders of the ICMS-defined
face-M1 and face-S1.
Many studies mapping the limbs-M1 and vibrissal-M1 have assessed the size of the
cortical region devoted for each type of ICMS-evoked movement by using microelectrode
penetrations perpendicular to the cortical surface and parallel to the assumed cortical motor
columns, and have limited ICMS to only one depth within layer V (Gioanni and Lamarche,
1985; Neafsey et al., 1986; Nudo et al., 1990, 1992; Huntley, 1997; Franchi, 2001; Kleim et
al., 2002). However, it has been documented that ICMS can evoke muscle activities from
the entire depth of layers V and VI (Sapienza et al., 1981; Neafsey et al., 1986; Aldes,
1988; Asanuma, 1989; Brecht et al., 2004, Avivi-Arber et al., 2010a, b). Further,
perpendicular penetrations for mapping the laterally positioned face-M1 and face-S1 in the
present study would have required damaging the orbital and temporal bones and their
associated soft tissues and muscles that can lead to profuse bleeding due to the presence of
a rich plexus of veins located in the vicinity of these structures and could compromise the
activity and ICMS study of some of the muscles. Therefore, to reveal the three-dimensional
extent of face-M1 and face-S1 motor representations, vertical microelectrode penetrations
were used, and within each penetration ICMS was applied in 0.2 mm incremental steps of
penetration depth until no ICMS-evoked EMG activity could be observed (Figs. 3-3, 3-4).
This methodology revealed both the number of “positive ICMS penetrations” that reflected
the approximate size of cortical territory for a motor representation and the number of
“positive ICMS sites” within each positive penetration (see below).
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Electrolytic lesions (Fig. 3-3) were placed within every positive ICMS penetration
for subsequent histological verification of positive ICMS sites (see below) within the gray
matter of the cytoarchitectonically defined granular (sensory) or agranular (motor) cortex.
The rats were sacrificed and fixed by transcardial perfusion of 10% buffered formalin
(Fisher Scientific, Fairlawn, NJ, USA). The cross-sections of the brain (50 µm thickness;
Adachi et al., 2007; Avivi-Arber et al., 2010a, b) were processed with Nissl or Hematoxylin
and Eosin stain. The boundaries between the frontal agranular and the immediately adjacent
granular cortex were delineated in coronal sections by using previously described
cytoarchitectonic criteria for the rat frontal cortex in which the granular cortex is
characterized by a prominent granular layer IV of densely packed cells, and the agranular
cortex is characterized by the absence of a granular layer (Donoghue and Wise, 1982;
Neafsey et al., 1986; Paxinos and Watson, 1998; Brecht et al., 2004; Swanson, 2004;
Avivi-Arber et al., 2010a, b; for review, see Palomero-Gallagher and Zilles, 2004). Based
on these descriptions, the frontal agranular cortex representing the M1 can extend rostral up
to ~ 4.2 mm anterior from bregma. Therefore in the present study, at AP 4.0-4.5, the ICMS-
mapped agranular cortex coincided with the face-M1, whereas at AP 2.5-3.5, the ICMS-
mapped agranular cortex (face-M1) was medial to the immediately adjacent lateral granular
cortex that coincided with the face-S1; the cortex immediately ventrolateral to S1 coincided
with the insular gustatory cortex (Fig. 3-3). Digital images of histological sections were
evaluated and corrected for 3D coordinates of the electrode penetration tracks with ImageJ
(National Institute of Mental Health, Bethesda, USA).
3.3.4. Data acquisition and analysis
EMG activity was amplified [gain 1000×, filtered (bandpass 300-5 kHz); model
1700, A-M system, Washington, USA] and digitized (5 kHz; CED 1401 plus, Cambridge
Electronic Design, Cambridge, UK). Data were analyzed offline by another investigator
with the use of customized software written in LabVIEW (National Instrument, Austin, TX,
USA). For any ICMS site, the seven ICMS-evoked EMG waveforms were rectified and
then averaged, and the onset latency of the ICMS-evoked response in the muscle at 60 µA
was also noted. An ICMS site was defined and counted as a “positive ICMS site” if at least
three of the five ICMS (60 or 20 µA intensities) trains evoked an EMG response in a
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muscle with an onset latency ≤ 40 ms and a peak activity exceeding the mean value of the
initial 10 ms of the EMG response plus 2 SDs (Fig. 3-2D). For each muscle, an ICMS (60
µA) penetration was defined and counted as a positive ICMS penetration if it had at least
one positive ICMS site. Very few positive sites for Ma and Bu muscles were found (e.g.,
Table 3-2), so the analyses focussed on the AD and GG data. The positive ICMS sites were
labeled with a color code to reflect the muscles activated at each site and transferred on a
drawing of the reconstructed histological section to illustrate the representation areas of
LAD, RAD, and GG muscles within the sensorimotor cortex of rats from each of the E and
S groups (e.g., Fig. 3-4A, B, C). Within each penetration, the AP, ML, and depth (De)
positions of the positive ICMS (60 µA) sites with the shortest onset latency of evoked
EMG activities in LAD, RAD, or GG were noted. The shortest onset latency, for all the
positive penetrations for the cortical side, was then calculated for LAD, RAD, and GG for
each animal group. To illustrate the AP and ML frequency distribution of the positive
ICMS penetrations in the face-M1 and face-S1, the mean numbers of positive ICMS (60
μA) penetrations within each AP coordinate were grouped together irrespective of the ML
coordinates, and within each ML coordinate were grouped together irrespective of the AP
coordinates, and plotted as a function of the AP position from the landmark bregma and
ML position from the midsagittal suture (Fig. 3-9, 3-10). In order to identify the shifts in
muscle representations, the centre of gravity (CoG), which defines the mean three-
dimensional centre position of the motor representations, was calculated for each of the
LAD, RAD, and GG muscles by taking into account the mean number of positive ICMS
(60 µA) sites obtained at each ML, AP, and De coordinates in the face-M1 and face-S1,
thereby providing the position of the motor maps weighted relative to the extent of the
motor representation (adapted from Ridding et al., 2000; Svensson et al., 2006). The
following equation was used: XCoG = ∑aiXi / ∑ai, where ai is the number of positive ICMS
sites at a cortical AP coordinate, Xi. In a similar manner, the ML coordinates Yi and the De
coordinate Zi were determined (Fig. 3-11).
3.3.5. Statistical Analyses
Statistical differences between groups and the effects of the independent variables
(study group, cortical side, muscle) on the dependent variables (number of positive ICMS
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sites, onset latency, the AP and ML position of the positive ICMS penetrations, and the
CoG for motor representation of LAD, RAD, and GG) were determined using multivariate
[mixed model repeated-measures (MMRM) ANOVA] analysis, followed by post-hoc
Sidak-adjusted pairwise comparisons as appropriate. A probability level of P < 0.05 was
considered statistically significant. All values were expressed as mean ± SEM. Data was
analyzed by a statistician using the SAS statistical software program (version 9.3).
3.4. Results
Rats were monitored on a daily basis and demonstrated no abnormal behavior and
orthodontic spring related complications, e.g., standing in a corner of the cage for
prolonged periods, strange eating or chewing behaviour, swaying their heads back and
forth, vibrating their tails, vocalizing, tissue lacerations or infection. As noted in chapter 2,
before placing the orthodontic spring, E and S group animals had a similar daily gain in
body weight, however after the placement of the orthodontic spring, the E group had a
small but significant loss of weight for day 1 as compared to the S group, but thereafter
gained weight again at a similar rate to that of the S group (P < 0.001) (see chapter 2).
3.4.1. General features of face-M1 and face-S1 motor representations
The LAD, RAD and GG muscles had extensive motor representations within the
left and right face-M1 and face-S1 mapped areas at intracortical depths ranging from
approximately 1.8 mm to 4.8 mm but there were a very few positive ICMS sites for the
LMa, RMa, LBu, and RBu (Table 3-2) at an ICMS intensity of 60 µA. At AP4.0, the
positive ICMS sites were located within the agranular cortex coinciding with face-M1
while those at AP3.5, AP3.0, and AP2.5 were found both within the agranular cortex (face-
M1) and the adjacent granular cortex (face-S1) (Fig. 3-3, 3-4). Further, consistent with
Avivi-Arber et al. (2010a, b), ICMS at an intensity of 20 µA could evoke LAD, RAD, and
GG muscle activities from only a very small number of sites (Table 3-2). Therefore, the
ICMS data for the Ma, Bu, as well as the data obtained for 20 µA were not included in the
extensive data analyses.
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For all the study groups, either individual muscles or combinations of muscles could
be activated from the same ICMS site (Figs. 3-4, 3-5, 3-6, 3-7, 3-8). The mean number of
positive ICMS penetrations in the AP and the ML planes from which ICMS evoked EMG
activities in LAD, RAD and/or GG reflects two dimensionally the approximate cortical
territory representing each muscle within the mapped area (Fig. 3-9, 3-10). The CoG
reflects the mean three-dimensional centre positions of the motor representations for LAD,
RAD, and GG (Fig. 3-11). Note that ICMS in the left and right face-M1 and face-S1
evoked EMG activities in ipsilateral and contralateral LAD and RAD, with a contralateral
predominance (Fig. 3-5, 3-6, 3-7, 3-8).
3.4.2. Effects of OTM
There were significant differences between E and S and N groups for the numbers
of positive ICMS sites in face-M1 and face-S1 for LAD, RAD, GG, and overlapping
representations of these muscles (Figs. 3-4, 3-5, 3-6, 3-7, 3-8). Further, there were
significant differences in the number of positive ICMS penetrations in the AP and ML
planes for LAD, RAD and GG for various groups suggesting a shift in the territory of
motor representations in the face sensorimotor cortex (Fig. 3-9, 3-10). However, no
significant differences across groups were documented for the mean shortest onset latencies
of evoked EMG activities for LAD, RAD or GG in either the left or right face-M1 and face-
S1, and between left and right face-M1 and face-S1 (Table 3-1).
3.4.2.1. LAD, RAD and GG motor representations in face-M1
3.4.2.1.1. Number of positive ICMS sites
MMRM ANOVA revealed significant differences in treatment (P = 0.008), time (P
< 0.0001), muscle (P < 0.001), and also in treatment * time (P < 0.001), time * cortical side
(P = 0.034), time * muscle (P = 0.029), and cortical side * muscle interaction effects (P <
0.0001). Also, significant differences were revealed in treatment * time * muscle (P =
0.009) and time * cortical side * muscle interaction effects (P = 0.036). Post-hoc analysis of
the total number of positive ICMS sites for LAD, RAD, GG and the sites with overlapping
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representations for the different combinations of these muscles showed no significant
differences across the various S groups and the N group.
Multivariate analysis for each time point revealed that in the left and right face-M1
of the E1 group, there was a decrease in the number of positive ICMS LAD sites compared
to the S1 group (P = 0.034, < 0.001, respectively), whereas there was a decrease in the
number of positive ICMS RAD sites only in the left face-M1 (P = 0.007). However in the
left and right face-M1 of the E7 group, there was a significant increase in the number of
positive ICMS RAD sites compared to the S7 group (P < 0.001, <0.001, respectively),
whereas there was a significant increase in the number of positive ICMS GG sites only in
the left face-M1 (P = 0.026). In the left and right face-M1 of the E28 group there was a
significant decrease in the number of positive ICMS RAD sites compared to the S28 group
(P < 0.001, = 0.045, respectively) (Figs. 3-5A, B, C). For details of significant differences
across various groups (in comparison to the S and N groups and within the E groups) in the
number of positive ICMS sites for LAD, RAD and GG, see Fig. 3-5.
Further, since LAD and RAD had a contralateral predominance in this cortical
representation, the significant changes in the number of positive ICMS sites for LAD and
RAD were more profoundly reflected in the contralateral cortical side (P < 0.0001) (Fig. 3-
5A, B). However, for GG in the E7 group there was a significant increase in the number of
positive ICMS sites only in the left face-M1 compared to the right face-M1 (P < 0.001)
(Figs. 3-5C). Also, cortical side * muscle interaction for the E groups revealed that in the
E7 group, in the left face-M1 there was a significant increase for RAD compared to LAD
and GG (P < 0.001, < 0.001, respectively), and in the right face-M1 for RAD compared to
GG only (P < 0.001) (Fig. 3-5D).
In addition to the above significant differences for individual muscles (i.e., LAD,
RAD, and GG), significant differences in the number of positive ICMS sites in the left-face
M1 and the right-face M1 were found for combinations of muscles (RAD + LAD, LAD +
GG, RAD + GG, and RAD + LAD + GG) (P < 0.001) (Fig. 3-6). In the left and the right
face-M1 of the E7 group, there was a significant increase in the number of positive ICMS
RAD + LAD sites compared to the S7 group (P = 0.004, = 0.013, respectively). In the left
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and the right face-M1 of the E28 group, there was a significant decrease in the number of
positive ICMS RAD + GG sites compared to the S28 group (P < 0.001, = 0.005), whereas a
decrease only in the right face-M1 for RAD + LAD (P = 0.013). In addition, there was a
significant increase in the number of positive ICMS RAD + GG sites in the left face-M1 of
the E7 group compared to the right face-M1 (P = 0.004) (Fig. 3-6B). Also, time * cortical
side * muscle interaction for the E groups revealed that in the left face-M1 for the E7
group, there was a significant increase in the number of positive ICMS RAD + GG sites
compared to LAD + GG and RAD + LAD + GG (P = 0.04, 0.006, respectively), for RAD +
LAD compared to LAD + GG and RAD + LAD + GG (P = 0.007, < 0.001, respectively),
whereas in the right face-M1 there was a significant increase in the number of positive
ICMS RAD + LAD sites compared to LAD + GG , RAD + GG and RAD + LAD + GG (P
< 0.001, < 0.001, < 0.001, respectively). Also, in the right face-M1 of the E28 group, there
was a significant decrease in the number of positive ICMS RAD + GG sites compared to
LAD + GG (P = 0.018), and RAD + LAD + GG compared to LAD + GG (P = 0.003).
Further, visual observations of ICMS-evoked muscular activities of forelimb, neck,
and vibrissae were documented in the left and right AP2.5 coordinate. These topographical
features were observed in all the groups, and were considered to be beyond the territory of
the face-M1 and face-S1 in M1 as their representations were not enclosed within and did
not overlap with those of LAD, RAD, or GG.
3.4.2.1.2. Onset latencies of ICMS-evoked EMG activities in face-M1
In all groups, ICMS within face-M1 evoked EMG activities in LAD, RAD, and GG
with a wide range of onset latencies (Table 3-1). There were no significant differences
across the groups in the mean shortest onset latencies of evoked LAD, RAD, or GG
activities evoked from either left or right face-M1, although the mean shortest onset
latencies for LAD or RAD activities were significantly shorter for responses evoked from
the contralateral face-M1 compared to those evoked from the ipsilateral face-M1 (P <
0.001).
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3.4.2.2. LAD, RAD and GG motor representations in face-S1
3.4.2.2.1. Number of positive ICMS sites
MMRM ANOVA revealed significant differences in treatment (P < 0.001), time (P
< 0.0001), muscle (P < 0.001), and also in treatment * time (P < 0.001), time * cortical side
(P = 0.007), treatment * muscle (P = 0.042), time * muscle (P = 0.019), and cortical side *
muscle interaction effects (P < 0.0001). Finally, significant differences were revealed in
treatment * time * muscle (P = 0.035) and treatment * time * cortical side * muscle
interaction effects (P = 0.014). Post-hoc analysis of the total number of positive ICMS sites
for LAD, RAD, GG and the sites with overlapping representations for the different
combinations of these muscles showed no significant differences across the various S
groups and the N group.
Multivariate analysis for each time point revealed that in the left face-S1 of the E1
group, there was a decrease in the number of positive ICMS RAD and GG sites compared
to the S1 group (P < 0.001, < 0.001, respectively), whereas in the right face-S1 there was a
decrease in the number of positive ICMS LAD and GG sites (P < 0.001, < 0.001,
respectively) (Fig. 3-7). In left and right face-S1 of the E7 group, there was a significant
increase in the number of positive ICMS RAD sites compared to the S7 group (P < 0.001, =
0.006, respectively), whereas there was a significant increase in the number of positive
ICMS GG sites only in left face-S1 (P = 0.006). Also, in the left face-S1 of the E7 group, a
significant increase in the number of positive ICMS GG sites was revealed compared to
right face-S1 (P < 0.001) (Fig. 3-7C). In addition, in the left face-S1 for the E28 group,
there was a significant reduction in the number of positive ICMS RAD and GG sites
compared to the S28 group (P = 0.048, < 0.001, respectively). For details of significant
differences across various groups (in comparison to the S and N groups and within the E
groups) in the number of positive ICMS sites for LAD, RAD and GG, see Fig. 3-7.
Further, cortical side * muscle interaction for the E groups revealed that in the left
face-S1 of the E1 group, there was a decrease in the number of positive ICMS LAD sites
compared to GG (P = 0.003), whereas in right face-S1, RAD was significantly decreased
compared to GG (P = 0.038) (Fig. 3-8A). In the left face-S1 of the E7 group, there was an
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increase in the number of positive ICMS RAD sites compared to LAD (P < 0.001), and GG
was significantly increased compared to LAD and RAD (P < 0.001, <0.001, respectively),
whereas in the right face-S1, GG was significantly increased compared to LAD only (P =
0.006) (Fig. 3-8B). In the left and right face-S1 of the E28 group, there was a significant
decrease in the number of positive ICMS LAD and RAD sites compared to GG (left - P <
0.001, = 0.021; right - = 0.021, < 0.001, respectively) (Fig. 3-8C).
The number of positive sites in left and right face-S1 for combinations of muscles
(RAD + LAD, LAD + GG, RAD + GG, and RAD + LAD + GG) was significantly lower
than those in the face-M1, and the difference in number of positive ICMS sites across all
the groups for each combination of muscles was not significant.
3.4.2.2.2. Onset latencies of ICMS-evoked EMG activities in face-S1
There were no significant differences across the groups in the mean shortest onset
latencies of evoked LAD, RAD, or GG activities evoked from either left or right face-S1,
although the mean onset latencies for LAD or RAD activities were significantly shorter for
responses evoked from their contralateral face-S1 compared to those evoked from the
ipsilateral face-S1 (P < 0.001) (Table. 3-1). Also, there were no significant differences in
the mean shortest onset latencies of evoked LAD, RAD, or GG activities evoked from the
face-S1 when compared to face-M1.
3.4.2.3. Distribution of positive ICMS penetrations in face-M1 and face-S1
The number of positive penetrations in the AP coordinates, irrespective of the
muscle and the ML position, gives the AP frequency distribution of the positive ICMS
penetrations within the left and right face-M1 and face-S1, and was the highest in the
AP2.5 - AP3 mm coordinates from the landmark bregma in all the groups (Fig. 3-8).
Statistical analysis revealed no significant differences between the S groups and the N
group in the number of positive ICMS penetrations in each AP and ML coordinate. In the
left face-M1 and face-S1, multivariate analysis indicated that in the coordinate AP2.5 (most
posterior coordinate), the number of positive penetrations significantly decreased in the E1
group compared to the S1 group (P < 0.001), and decreased in the E28 group compared to
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the S28 group (P = 0.001) suggesting a decrease in the posterior extent of the number of
positive ICMS penetrations in the E1 and E28 groups; the difference was not significant
between the E7 and S7 groups. In the coordinate AP4 (most anterior coordinate) there was
no significant difference between the E groups compared to their analogous S groups and
within the E groups. In the right face-M1 and face-S1, in the coordinate AP2.5, the number
of positive penetrations was significantly decreased in the E28 group compared to the S28
group (P = 0.017), suggesting a decrease in the posterior extent of the number of positive
ICMS penetrations in the E28 group. In the coordinate AP4, decreased in the E28 group
compared to the S28 group (P < 0.001), suggesting a decrease in the anterior extent of the
number of positive ICMS penetrations in the E28 group (for details of significant
differences across groups in each coordinate in the AP plane see Fig. 3-9).
The number of positive penetrations in the ML coordinates, irrespective of the
muscle and the AP position, gives the ML frequency distribution of the positive ICMS
penetrations within the left and right face-M1 and face-S1, and was the highest in the ML3
- ML3.5 mm coordinates from the midsagittal suture in all the groups (Fig. 3-10). In the left
face-M1 and face-S1, multivariate analysis indicated that in the coordinate ML2 (most
medial coordinate) there was no significant difference in the number of positive
penetrations in any of the groups. In the coordinate ML5 (most lateral coordinate) and in
the coordinate ML2.5, there was no significant difference between the E groups compared
to their analogous S groups and within the E groups. In the right face-M1 and face-S1, in
the coordinate ML2.5, there was no significant difference between the E groups compared
to their analogous S groups and within the E groups. In the coordinate ML5, decreased in
the E28 group compared to the S28 group (P < 0.001), suggesting a decrease in the lateral
extent of the number of positive ICMS penetrations in the E28 group compared to the S28
group (for details of significant differences across groups for each coordinate in the ML
plane see Fig. 3-10).
3.4.2.4. CoG of positive ICMS sites in face-M1 and face-S1
In the AP plane, in the left and right face-M1 and face-S1, the CoGs for LAD,
RAD, and GG were located between the coordinates AP3 and AP3.5, with no significant
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differences across the groups or between the left and right face-M1 and face-S1. In the ML
plane, in the left and right face-M1 and face-S1, the CoGs for LAD, RAD, and GG were
located between the coordinates ML2 and ML3.5. However, for LAD, within the right face-
M1 and face-S1, there was a significant medial shift in the CoG in the E1 group compared
to the S1 group (P = 0.022) (Fig. 3-11A). There were no statistically significant differences
in the ML positions in the CoG for LAD in the other groups and in all the groups for RAD
and GG. In the De plane, in the left and right face-M1 and face-S1, the CoGs for LAD,
RAD, and GG were located between the coordinates De2 and De3.2. There were no
statistically significant differences in the De positions in the CoG in all the groups for
LAD, RAD and GG (Fig. 3-11).
3.5. Discussion
This is the first study to document neuroplastic changes induced in the sensorimotor
cortex by OTM. OTM resulted in significant changes in the motor representations of the
jaw-opening (LAD, RAD) and tongue protrusive (GG) muscles in the ICMS-defined face-
M1 at days 1, 7, and 28 of continuous orthodontic force application in OTM-treated groups
compared to the sham-treated and naive groups. Analogous neuroplastic changes were also
revealed in the ICMS-defined motor representations within face-S1. These neuroplastic
changes in the face-M1 and face-S1 may reflect adaptive sensorimotor changes in response
to the altered environment in the oral cavity induced by OTM.
3.5.1. Features of jaw and tongue motor representations in the face-M1
M1 plays a critical role in planning, initiation, and execution of movement of
muscles/body parts (for review, see Asanuma, 1989; Sanes and Donoghue, 2000) and can
be identified by its cytoarchitecture and by techniques including ICMS (Asanuma and
Rosen, 1972; Donoghue and Wise, 1982; Murray et al., 2001; Schieber, 2001; Cheney,
2002; Hatanaka et al., 2005; Avivi-Arbor et al., 2011; Sessle, 2011a). The area of face-M1
mapped in this study consisted of the lateral agranular area and a part of face-M1 that
overlaps with the face-S1. The location of the positive ICMS sites was confirmed
histologically to be in the face-M1 based on cytoarchitecture features of the agranular
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cortex (Donoghue and Wise, 1982; Paxinos and Watson, 1998). This is the same area that
was mapped in other studies (Donoghue and Wise, 1982; Neafsey et al., 1986; Satoh et al.,
2006, Adachi et al., 2007, Avivi-Arber et al., 2010a, b). Some of the characteristic features
of face-M1 found in other studies, such as sites from which LAD, RAD and GG could be
activated individually with significant contralateral face-M1 predominance for RAD and
LAD, and overlapping muscle representations in face-M1 of LAD, RAD, and GG were also
documented in this study. It has been proposed that such overlapping of motor
representations, along with the extensive bilateral representations of LAD, RAD and GG
muscles, may form an essential substrate for the dynamic coordination of bilateral orofacial
movements involving the complex synergistic activities of these muscles, and for face-M1
neuroplastic mechanisms manifested as reorganization of motor representations within
face-M1 (for review, see Sanes and Donoghue, 2000; Sanes and Schieber, 2001; Avivi-
Arber et al., 2011).
3.5.2. Features of jaw and tongue motor representations in the face-S1
The study revealed that ICMS applied to the face-S1 evoked LAD, RAD and GG
EMG activity. This is consistent with previous studies in which short-train ICMS within the
face-S1 evokes orofacial movements and EMG activity in jaw and tongue muscles in
rodents and monkeys (Sapienza et al., 1981; Donoghue and Wise, 1982; Neafsey et al.,
1986; Huang et al., 1989a; Lin et al., 1994; Burish et al., 2008, Avivi-Arber et al., 2010a,
b); long-train ICMS evokes rhythmic jaw or tongue movements and EMG activity in rats,
cats, rabbits, and monkeys (Lund et al., 1984; Zhang and Sasamoto, 1990; Huang et al.,
1989a; Murray et al., 2001; Satoh et al., 2006; Hiraba et al., 2007). These findings support
the view that face-S1 as well as face-M1 may play a role in the generation and control of
orofacial movements (Murray et al., 2001; Yao et al., 2002). However, the observed ICMS-
evoked EMG activities could conceivably have been the result of spread of ICMS currents
from face-S1 to face-M1 either directly (Cheney, 2002) or indirectly through axon
collaterals (Henry and Catania, 2006; Chakrabarti and Alloway, 2006; Iyengar et al., 2007).
However, this is unlikely, firstly as the distance between face-M1 and many of the positive
ICMS sites within the face-S1 was much larger than the estimated extent of ICMS current
spread of less than 0.5mm at 60 µA ICMS intensity (Asanuma, 1989; Cheney, 2002;
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Schieber, 2001). Secondly, many of the positive ICMS sites within the face-S1 had short
onset latencies (8-12 ms), comparable to those of face-M1, thus suggesting relatively direct
projections to brainstem motoneurons rather than projections via face-M1. Thirdly, efferent
projections from rat face-S1 to motoneurons have been documented (Rathelot and Strick,
2006; Chang et al., 2009; Yoshida et al., 2009; Tomita et al., 2012; Haque et al., 2012).
3.5.3. Neuroplasticity associated with altered jaw and tongue motor
representations induced by OTM
M1 neuroplasticity has been associated with motor function recovery following
central or peripheral injury, changes in muscle use or disuse, learning of novel motor skills,
and adaptive processes (Kaas, 2007; Martin, 2009; Avivi-Arber et al., 2011, Kleim, 2011;
Nudo, 2011). Earlier studies have shown that alterations in somatosensory inputs induced
by deafferentation (Franchi, 2001; Yildiz et al., 2004; Halkjaer et al., 2006; Adachi et al.,
2007) or sustained somatosensory stimulation (Hamdy et al., 1998; Adachi et al., 2008)
may result in neuroplastic changes within face-M1 (Toldi et al., 1996; Farkas et al., 2000;
Sanes and Donoghue, 2000; Adachi et al., 2008, Avivi-Arber et al., 2010a) and face-S1 (for
review, see Feldman and Brecht, 2005; Petersen, 2007; Fox, 2009; Barnes and Finnerty,
2010), as reflected in an altered cortical excitability or motor representations. In the present
study OTM for 1, 7 and 28 days resulted in significant neuroplastic changes in the LAD,
RAD, and GG motor representations in the face-M1 and face-S1. Analogous long-term
changes have been previously documented in face-M1 in rats as a result of trimming the
vibrissae (Keller et al., 1996), or lower incisors to take them out of occlusion with the upper
incisors (Lee et al., 2006; Sessle et al., 2007), lingual nerve transection (Adachi et al.,
2007), in face-M1 and face-S1 subsequent to incisor or molar teeth extraction (Avivi-Arber
et al., 2010a, Veeraiyan et al, 2011), and in the face-S1 after extracting the incisor (Henry
et al., 2005). Changes in the M1 of humans have been documented subsequent to
deafferentation of the lower face region (Yildiz et al., 2004) and tongue (Halkjaer et al.,
2006), although local anesthesia and nociceptive stimulation of the PDL were insufficient
to cause face-M1 changes measured at 5, 30, and 60 min post application by TMS in
humans (Zhang et al., 2010).
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The oral tissues, including the PDL, tooth pulp, mucosa, muscle, gingival tissue, the
alveolar bone and TMJ are characterized by rich neural innervations (for reviews, see Miles
et al., 2004; Waite, 2004). The PDL mechanoreceptors (PMRs), for example, encode
information about the temporal, spatial and intensive aspects of tooth loads that contributes
to the regulation of the muscle activity that generates masticatory forces and jaw
movements (Lund, 1991; Trulsson and Johanson, 1996; Trulsson and Essix, 2004, Trulsson
et al., 2012). There are prominent orofacial mechanoreceptive representations within face-
S1 (Catania and Remple, 2002; Catania and Henry, 2006; Miyamoto et al., 2006; Iyengar et
al., 2007) and also motor outputs from face-S1 to specific motoneurons that activate
muscles that produce specific movements (Murray and Sessle, 1992a; Izraeli and Porter,
1995; Lin et al., 1998; Yoshida et al., 2009; Tomita et al., 2012; Haque et al., 2012).
Further, face-M1 also receives somatosensory inputs from orofacial tissues, including the
teeth (Huang et al., 1989b; Murray and Sessle, 1992a; Miyashita et al., 1994; Farkas et al.,
1999; Murray et al., 2001; Avivi-Arber et al., 2011). The functional organization of the
somatosensory inputs to face-M1 and face-S1 indicates a role for face-M1 and face-S1 in
sensorimotor integration and highlights the elaborate exteroceptive somatosensory
feedback from a wide peripheral orofacial area that is used for precise control, co-
ordination and modulation of the orofacial muscle activities during orofacial functions
(Murray et al., 2001; Johansson et al., 2006; Sessle, 2011b).
There is abundant evidence suggesting that neurovascular mechanisms play
important roles in OTM through the development of an inflammatory reaction (Saito et al.,
1991; Brain, 1997; Vandevska-Radunovic et al., 1997; Vandevska-Radunovic, 1999; Wise
and King, 2008). Because pain is one of the cardinal signs of inflammation and has been
reported during OTM (Asham and Southard, 2004; Keim, 2004; Krishnan, 2007), biting
and chewing are reported to be sources of discomfort and can lead to impaired jaw
function. Thus, during OTM the possible influence of postoperative pain in the E groups
with activated orthodontic springs could be a factor in the neuroplastic changes observed in
the present study. However, OTM-induced pain in humans has its peak intensity after day 1
and thereafter there is a gradual reduction in the pain intensity by day 7 (Ngan et al., 1989,
1994; Jones and Chan, 1992a, b; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and
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Dincer, 2004; Polat et al., 2005). We have also documented that OTM is associated with
only short-lasting orofacial mechanical and thermal hypersensitivities that are considered to
reflect nociceptive behaviour (see chapter 4). In the present study, a significant decrease in
the number of positive ICMS sites for LAD, RAD, and GG was noted in the E1 group,
consistent with earlier findings that acute intraoral nociceptive inputs to the brain evoked
by injection of the algesic chemical glutamate into the tongue in rats (Adachi et al., 2008)
and application of capsaicin to the tongue in healthy humans (Boudreau et al., 2007)
resulted in decreased face-M1 excitability suggestive of decreased motor representations
(Ridding and Rothwell, 1997; Monfils et al., 2005). These findings raise the possibility that
the neuroplastic changes observed in the initial phase of OTM (E1 group) may have been
related to acute postoperative pain during OTM. However, the short-time course of the
OTM-induced nociceptive behaviour suggests that the long-term (E7, E28 groups)
neuroplastic changes induced by OTM in the present study are unlikely a reflection of the
presence of maintained pain, although it cannot be ruled out that these neuroplastic changes
were not dependent on nociceptive inputs for their initiation.
The significant OTM-induced changes noted in the E7 and E28 groups compared to
N and their analogous S groups may be related to the initiation and the maintenance of the
linear phase of OTM. The radiographic analysis revealed that the amount of tooth
movement measured for the E group was 0.34 ± 0.07 mm at day 7, and 1.75 ± 0.23 mm at
day 28 (see chapter 2). While there was no significant change in the rate of tooth movement
induced by OTM, the difference in the amount of tooth movement between the two time
points was significant and it leads us to speculate that factors involving differences in the
occlusal contacts of the tooth inclines with the teeth in their opposing arch could be related
to the differences for the two time points during OTM. The radiographic assessment
indicated that in the E7 group the molar relation between the right maxillary to right
mandibular molars on the day of the ICMS procedure was a “cusp to fossa” relation,
whereas for the E28 group, since the maxillary right molars had shifted forward in relation
to the mandibular right molars, an “end to end” cusp relation was noticed on the day of the
ICMS procedure. Therefore, the alterations in the contacts of the cuspal and incisal inclines
of the teeth in the two different types of occlusions in the E7 and E28 groups may have
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provided very different inputs to the cortical areas and thereby could have promoted the
respective changes documented in the face-M1 and face-S1 in these two time points during
OTM. In the E7 group, the altered PMR inputs and/or inputs from other orofacial receptors
may have induced changes in the face-S1 and face-M1, as the change in occlusion could
initiate the process of adaptation to an altered pattern of jaw and tongue function that may
require repetition of the novel motor movements which is analogous to learning novel
motor skills (Lee et al., 2013). This adaptation is consistent with the use-dependent
neuroplasticity and could therefore result in an increase in RAD and GG motor
representations from the E1 to E7 time points. Also, the responses of the PMRs have been
reported to become progressively impaired during the course of orthodontic treatment
(Loescher et al., 1993; Okazaki, 1994, Long et al., 1996; Ogawa et al., 2002). Hence, such
a change in the properties of the PMRs subjected to OTM may be hypothesized to be a
major factor contributing to the decrease in the jaw opening and tongue representations
documented in the E28 group.
The neuroplastic changes observed within face-M1 and face-S1 also could have
been influenced by the changes in the other cortical or subcortical areas (for review, see
Wall et al., 2002; Kaas et al., 2008). Face-M1 receives somatosensory inputs from orofacial
tissues either directly through the thalamus or indirectly through face-S1 (Hatanaka et al.,
2005; Simonyan and Jurgens, 2005; Hoffer et al., 2005; Chakrabarti and Alloway, 2006;
Iyengar et al., 2007). Peripherally induced neuroplastic changes within the vibrissal-M1
may involve mechanisms such as disinhibition and unmasking of latent inputs from S1 to
M1 (Farkas et al., 1999; Toldi et al., 1999), and dental manipulations have been associated
with reorganization of mechanoreceptive fields within the trigeminal mesencephalic
nucleus (Linden and Scott, 1989), trigeminal brainstem sensory nuclei (Hu et al., 1986,
1999), and face-S1 (Henry et al., 2005). Thus, it is possible that changes within face-S1 or
other cortical or subcortical areas may have influenced the face-M1 neuroplastic changes
observed in the present study.
Several studies have confirmed that ICMS maps show multiple scattered loci within
M1 from which ICMS evokes movement about a joint/joints or EMG activity in
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muscle/groups of muscles, thereby forming a “complex mosaic” of multiple muscle
representations scattered in-between M1 (Nudo et al., 1992, 1996, Nudo and McNeal,
2013). Neuroplastic changes in these sites with overlapping muscle representations have
been considered to form the substrate for learning novel skills (for review, see Sanes and
Donoghue, 2000; Sanes and Schieber, 2001). In the present study, OTM also resulted in
changes in the number of positive ICMS sites in the face-M1 from which ICMS could
activate various combinations of LAD, RAD, and GG from the same site, suggesting that
unilateral OTM (of the maxillary right teeth) might have induced changes in oral motor
behaviours as the rat adapted to the altered oral state and perhaps adopted novel oral motor
behaviours to execute oral motor functions such as chewing, swallowing and gnawing.
However, there is no indication in the current study that OTM changes in the face-M1
induced any encroachment into the face-M1 region of LAD, RAD, and GG representations
of adjacent motor representations (e.g. vibrissae or forelimb) as reported with acute facial
nerve transection (Sanes et al., 1990; Toldi et al., 1996).
It is also noteworthy that the impact of OTM for all the time points studied (day 1,
day 7 and day 28) was the most robust for RAD in the left face-M1 (Fig. 3-3B), and for GG
in the left face-S1 (Fig. 3-5C). It can be speculated that this effect on RAD and GG motor
representations in the left sensorimotor cortex may be reflective of the adaptation process to
the OTM in the right side of the oral cavity. Further, it is possible that OTM induced the
initial changes in orofacial motor behaviour as an adaptive mechanism to the altered
somatosensory inputs which then contributed to the face-M1 and face-S1 neuroplasticity.
Alternatively, it is possible that the initial neuroplastic changes in the face-M1 and face-S1
to the altered somatosensory inputs allowed for the motor behavioural changes to occur
later. A limitation that the present study shares with many analogous studies in which
peripheral manipulations are carried out (Toldi et al., 1996; Farkas et al., 2000; Sanes and
Donoghue, 2000; Adachi et al., 2008, Avivi-Arber et al., 2010a) was that the sensorimotor
behaviour before and during OTM was not monitored. Thus, the cause-effect relations
between OTM-induced altered somatosensory inputs, modified behaviour and face-MI and
face-S1 neuroplasticity are unclear and require further investigation.
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3.6. Conclusions and clinical implications
This is the first study to document OTM-induced neuroplastic changes in the
sensorimotor cortex in the motor representations of the jaw-opening (LAD, RAD) and
tongue protrusive (GG) muscles at days 1, 7, and 28 of continuous orthodontic force
application. One of the primary roles of the oral cavity, teeth in particular, is to provide
effective chewing function through a well-maintained occlusion of the teeth in dental
arches those are stable. The regulatory function of the PMRs and other orofacial receptors
on orofacial muscle activities has important orthodontic implications. Their activation can
result in excitatory and inhibitory reflexes and afferent inputs to the face sensorimotor
cortex that can influence complex integrative mechanisms involving CNS centres and may
strengthen and/or normalize the forces of masticatory and facial muscles (Trulsson and
Essix, 2004; Miles, 2004; for review, see, Turker, 2002; Turker et al, 2007).
Neurophysiological studies further suggest that in malocclusions of teeth, the abnormal
occlusal relationships of teeth may not provide the appropriate PDL sensory input to the
cerebral cortex and other CNS regions, resulting in dysfunction of sensorimotor integration
and thereby impairment of oral perception and motor behaviour (Ahlgren, 1966; Subtelny,
1970, Bakke et al., 1992; Yashiro and Takada, 1999; Trovato et al., 2009). For a more
effective treatment orthodontists therefore need to understand the nature of the oral sensory
changes that can produce neuroplastic alterations in mechanisms underlying sensorimotor
integrative functions that influence the development of oral motor behaviour, and include
adjunctive clinical rehabilitative approaches that target these mechanisms in humans
suffering from orofacial sensorimotor deficits causing skeletal and/or dental malocclusions.
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3.7. Figures and Tables
Fig. 3-1. Experiment timeline. The groups were designed to reflect the phases of OTM reported in the
literature. The E1, E7, and E28 groups were the experimental groups that had activated orthodontic springs
for 1, 7, and 28 days, respectively; the S1, S7, and S28 groups were the sham control groups that had
inactivated orthodontic springs for 1, 7, and 28 days, respectively; the N group was the control group with no
orthodontic spring.
Arrival 0 days 1 day 7 days 28 days
ICMS mapping for
the N group,
Radiographs taken
and orthodontic
spring attached in
the S1, E1, S7, E7,
S28 and E28
groups
Radiographs
taken and ICMS
mapping for the
S7 and E7 groups
Radiographs
taken and ICMS
mapping for the
S28 and E28
groups
Radiographs
taken and ICMS
mapping for the
S1 and E1 groups
Chapter 3
117
Fig. 3-2. A. Illustration of LAD, RAD, GG, Ma, and Bu muscles where EMG electrodes were inserted. B.
The intracortical microstimulation (ICMS) mapping area at AP2.5, AP3.0, AP3.5, and AP4.0 anterior to
Bregma. Each dot represents a microelectrode penetration site from which ICMS evoked EMG responses in
LAD, RAD, GG or in any combinations of these muscles (overlapped representations). C. Example of a set of
5 ICMS-evoked EMG responses recorded from LAD of a single rat. D. Example of data from C after being
rectified and averaged by a 4ms-moving average window. Any ICMS site from which at least 3 of the 5 EMG
evoked responses had an amplitude larger than the baseline (green line) by 2 SD (white line) was defined as a
“ positive ICMS site” and the onset latency (red line) of these responses was noted. Adapted from Avivi-
Arber, doctoral thesis submitted to University of Toronto, 2009.
Buccinator (Bu)
Chapter 3
118
Fig. 3-3. Nissl-stained coronal sections (100 µm) from a representative rat sensorimotor cortex at AP
planes 4.0 (A), 3.5 (B), 3.0 (C), and 2.5 (D) mm anterior to bregma. Arrowheads indicate borders between
granular (sensory) and agranular (motor) cortex. Electrolytic lesions were placed at the bottom of each
positive ICMS penetration. E: Higher magnification of the ventrolateral aspect of D, illustrating the border
between the granular and agranular insular cortex. F: Template adapted from “A rat brain atlas” (Swanson,
2004), corresponding to plane AP 2.8, illustrating the cortical layers within the primary somatosensory cortex
(SSp) and the primary motor cortex (MOp) and the gustatory (GU) insular cortex. AP, anteroposterior. Scale
bar = 1 mm. Reproduced from Avivi-Arber et al., 2010a; with permission.
Chapter 3
119
GG
AP4.0
AP3.5
RAD
LAD
No response
mm
AP2.5
AP3.0
Motor maps in face-M1 and face-S1(60 µA ICMS)
S1 E1
Left Right Left Right
S1
S1
S1
M1
M1
M1
Fig. 3-4A. Representative motor maps of LAD, RAD, and GG in a rat face-M1 and S1 from the S1 and
the E1 groups. The cortical sites where ICMS evoked LAD, RAD, or GG EMG activity are plotted on the
corresponding cortical coronal histological section at AP2.5, AP3, AP3.5, and AP4 planes with LAD in blue,
RAD in green, and GG in red markings, whereas no responses in sites for LAD, RAD, and GG were marked
in black. Extensive bilateral representations of LAD, RAD, and GG were found in face-M1 and face-S1.
ICMS simultaneously evoked LAD, RAD, and GG EMG activity from multiple sites. MMRM ANOVA
analysis revealed that in the E1 group, in the left face-M1, there was a decrease in number of positive ICMS
sites compared to the S1 group for both RAD and LAD (P = 0.007, = 0.034), whereas in the right face-M1 for
LAD only (P < 0.001)., while in the left face-S1, for RAD and GG (P < 0.001, < 0.001), whereas in the right
face-S1 for LAD and GG (P < 0.001, <0.001). LAD – left anterior digastric, RAD – right anterior digastric,
GG – genioglossus, AP – Anteroposterior, S1 – Sham 1 day group, E1 – Experimental 1 day group.
For Fig. 3-4A, B, C, a representative histological template depicting the AP planes that were mapped was
provided by Dr. Avivi-Arber L. and has been used to display the changes in the motor maps for the various E
and S groups, and the template for AP2.5 (right cortical side for the E1, E7, and E28) has been adapted from
“A rat brain atlas” (Swanson, 2004).
Chapter 3
120
GG
AP4.0
AP3.5
RAD
LAD
No response
mm
AP2.5
AP3.0
Motor maps in face-M1 and face-S1(60 µA ICMS)
S7 E7
Left Right Left Right
S1
S1
S1
M1
M1
M1
Fig. 3-4B. Representative motor maps of LAD, RAD, and GG in a rat face-M1 and S1 from the S7 and
the E7 groups. Multivariate analysis revealed that in the E7 group, in the left face-M1, there was a significant
increase in the number of positive ICMS sites compared to the S7 group for RAD and GG (P < 0.001, =
0.026, respectively), whereas in the right face-M1 for RAD only (P < 0.001). Further, in the left face-M1 in
the E7 group, the increase for RAD was larger compared to GG (P < 0.001). S7 – Sham 7 day group, E7 –
Experimental 7 day group.
Chapter 3
121
GG
AP4.0
AP3.5
RAD
LAD
No response
mm
AP2.5
AP3.0
Motor maps in face-M1 and face-S1(60 µA ICMS)
S28 E28
Left Right Left Right
S1
S1
S1
M1
M1
M1
Fig. 3-4C. Representative motor maps of LAD, RAD, and GG in a rat face-M1 and S1 from the S28
and the E28 groups. Multivariate analysis revealed that in the E28 group, in the left and right face-M1, there
was a significant decrease in the number of positive ICMS sites compared to the S28 group for RAD only (P
< 0.001, = 0.045, respectively), and for RAD + GG (P < 0.001, = 0.005, respectively), whereas only in right
face-M1 for RAD + LAD (P = 0.013), in the left face-S1, for RAD and GG (P = 0.048, < 0.001, respectively),
and the number of LAD positive ICMS sites in the left face-S1 was significantly reduced compared to right
face-S1 (P = 0.011). S28 – Sham 28 day group, E28 – Experimental 28 day group.
Chapter 3
122
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
20
40
60
80
100
120
LAD Left Face-M1 sites
LAD Right Face-M1 sites
**
*
** ^
!
!
Animal Groups
N S1 E1 S7 E7 S28 E28
Nu
mber
of
posi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
20
40
60
80
100
120
GG Left Face-M1 sites
GG Right Face-M1 sites
*
*
*
^^
!
Animal Groups
N S1 E1 S7 E7 S28 E28
Nu
mber
of
posi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
20
40
60
80
100
120
RAD Left Face-M1 sites
RAD Right Face-M1 sites
*
*
*
*
*
^^ ^
^^ ^
!
#
#
#
#
Muscles
LAD RAD GG
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
20
40
60
80
100
120
Left Face-M1 sites for E7 group
Right Face-M1 sites for E7 group
*
#
*
#
A
Fig. 3-5. The number of positive ICMS sites in left and right face-M1 at ICMS intensity of 60 µA for LAD (A), RAD (B), and
GG (C) in all the groups, and for LAD, RAD, and GG in the E7 group (D). MMRM ANOVA Sidak comparison revealed that in
the left face-M1 there was a significant decrease in the number of positive LAD sites (A) in the E1 group compared to S1 and E7
groups (* P = 0.034, 0.011, respectively), an increase in the E7 group compared to the E28 group (^ P = 0.011); whereas in the right
face-M1, a decrease in the E1 group compared to the S1, N, and E7 groups (* P < 0.001, < 0.001, < 0.001, respectively). In addition,
in the E28 group, there was a significant decrease in the number of LAD positive ICMS sites in left face-M1 compared to right face-
M1 (! P = 0.001). In the left face-M1 there was a significant decrease in the number of positive RAD sites (B) in the E1 group
compared to the S1, N and E7 groups (* P = 0.007, < 0.001, < 0.001, respectively), an increase in the E7 group compared to the S7, N
and E28 groups (^ P < 0.001, = 0.004, < 0.001, respectively), and a decrease in the E28 group compared to the S28 and N groups (# P
< 0.001, = 0.002, respectively); whereas in the right face-M1, a decrease in the E1 group compared to the N and the E7 groups (* P =
0.004, < 0.001, respectively), an increase in the E7 group compared to the S7, N and E28 groups (^ P < 0.001, = 0.004, < 0.001,
respectively), and a decrease in the E28 group compared to the S28 and N groups (# P = 0.045, 0.038, respectively). In the left face-
M1 there was a significant decrease in the number of positive GG sites (C) in the E1 group compared to the N and E7 groups (* P =
0.024, < 0.001, respectively), an increase in the E7 group compared to the S7 and E28 groups (^ P = 0.026, < 0.001, respectively);
whereas in the right face-M1, a decrease in the E1 group compared to the N group (* P = 0.011). In the E7 group, a significant
increase in number of positive ICMS sites in left face-M1 was revealed for RAD (B), and GG (C) compared to right face-M1 (! P =
0.001, < 0.001, respectively) and in right face-M1 for LAD (A) compared to left face-M1 (! P = 0.024). Also, in the E7 group (D)
only, in the left face-M1, there was a significant increase in number of positive ICMS RAD sites compared to LAD and GG (* P <
0.001, < 0.001, respectively), while in the right face-M1, for RAD compared to GG (# P < 0.001), and for LAD compared to GG (# P
= 0.003).
C D
B
Chapter 3
123
Animal Groups
N S1 E1 S7 E7 S28 E28
Nu
mber
of
posi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
10
20
30
40
50
60
LAD+GG Left Face-M1 sites
LAD+GG Right Face-M1 sites
* ^
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
10
20
30
40
50
60
RAD+LAD Left Face-M1 sites
RAD+LAD Right Face-M1 sites
*
*
*
^^^
#
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
10
20
30
40
50
60
RAD+GG Left Face-M1 sites
RAD+GG Right Face-M1 sites
*
*
^
!
####
*
Animal Groups
N S1 E1 S7 E7 S28 E28
Nu
mb
er o
f P
osi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
10
20
30
40
50
60
RAD+LAD+GG Left Face-M1 sites
RAD+LAD+GG Right Face-M1 sites
^
A
Fig. 3-6. The number of positive ICMS sites in left and right face-M1 at ICMS intensity of 60 µA for LAD + GG (A), RAD +
GG (B), RAD + LAD (C) and RAD + LAD + GG (D). MMRM ANOVA Sidak comparison revealed that in the left face-M1
there was a significant increase in the number of positive LAD + GG sites (A) in the E7 group compared to the E28 group (^ P =
0.005); whereas in the right face-M1, a decrease in the E1 group compared to the N group (* P = 0.007). In the left face-M1 there
was a significant decrease in the number of positive RAD +GG sites (B) in the E1 group compared to the E7 group (* P < 0.001),
an increase in the E7 group compared to the E28 group (^ P < 0.001), and a decrease in the E28 group compared to the S28 and N
groups (# P < 0.001, 0.006, respectively); whereas in the right face-M1, a decrease in the E1 group compared to the N group (* P
= 0.009), and a decrease in the E28 group compared to the S28 and N groups (# P = 0.005, 0.022, respectively). In the left face-M1
there was a significant decrease in the number of positive RAD + LAD sites (C) in the E1 group compared to the E7 group (* P <
0.001), an increase in the E7 group compared to the S7 and E28 groups (^ P = 0.004, < 0.001, respectively); whereas in the right
face-M1, a decrease in the E1 group compared to the E7 and N groups (* P < 0.001, P = 0.049, respectively), an increase in the E7
group compared to the S7 group (^ P = 0.013), and a decrease in the E28 group compared to S28 group (# P = 0.013). In the left
face-M1 there was a significant increase in the number of positive RAD + LAD + GG sites (D) in the E7 group compared to the
E28 group (^ P = 0.009). There was a significant increase in the number of positive ICMS RAD + GG sites in the left-face M1 in
the E7 group compared to the right face-M1 (! P = 0.004). Also, in the E7 group, in the left face-M1, there was a significant
increase in number of positive ICMS sites for RAD + GG compared to LAD + GG and RAD + LAD + GG (P = 0.04, 0.006,
respectively), for RAD + LAD compared to LAD + GG and RAD + LAD + GG (P = 0.007, < 0.001, respectively); in the right
face-M1 there was a significant increase in number of positive ICMS sites for RAD + LAD compared to LAD + GG , RAD + GG ,
and RAD + LAD + GG (P < 0.001, < 0.001, < 0.001, respectively). In the E28 group, in the right face-M1, there was a significant
decrease in the number of positive ICMS sites for RAD + GG compared to LAD +GG (P = 0.018), and RAD + LAD + GG
compared to LAD + GG (P = 0.003).
C D
B
Chapter 3
124
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
2
4
6
8
10
12
14
16
LAD Left Face-S1 sites
LAD Right Face-S1 sites
* *
!
*
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
2
4
6
8
10
12
14
16
GG Left Face-S1 sites
GG Right Face-S1 sites
*
*
*
**
^^
!
**
#
##
Animal Groups
N S1 E1 S7 E7 S28 E28
Num
ber
of
po
siti
ve
ICM
S s
ites
(M
ean +
SE
M)
0
2
4
6
8
10
12
14
16
RAD Left Face-S1 sites
RAD Right Face-S1 sites
*
*
*
*
*^
^ ^
^ ^
#
Fig. 3-7. The number of positive ICMS sites in left and right face-S1 at ICMS intensity of 60 µA for LAD (A), RAD (B), and
GG (C). MMRM ANOVA Sidak comparison revealed that in the right face-S1 there was a significant decrease in the number of
positive LAD sites (A) in the E1 group compared to the S1, N, and E7 groups (* P < 0.001, = 0.040, < 0.001, respectively), a
significant decrease in the left face-S1 compared to the right face-S1 in the E28 group (! P = 0.011). In the left face-S1 there was a
significant decrease in the number of positive RAD sites (B) in the E1 group compared to the S1, N, and E7 groups (* P < 0.001, =
0.048, < 0.001, respectively), an increase compared to the S7, N and E28 groups (^ P < 0.001, = 0.016, < 0.001, respectively), and
a decrease compared to the S28 group for RAD (# P = 0.048); whereas in the right face-S1, a decrease in the E1 group compared
to the E7 group (* P < 0.001), an increase in the E7 group compared to the S7 and E28 groups (^ P = 0.006, < 0.001, respectively).
In the left face-S1 there was a significant decrease in the number of positive GG sites (C) in the E1 group compared to the S1, N
and E7 groups (* P < 0.001, < 0.001, < 0.001, respectively), an increase in the E7 group compared to the S7 and E28 groups (^ P =
0.006, < 0.001, respectively), and a decrease in the E28 group compared to the S28 and N groups for GG (# P < 0.001, < 0.001);
whereas in the right face-S1, a decrease in the E1 group compared to the S1, N, E7, and E28 groups (* P < 0.001, < 0.001, <
0.001, = 0.018, respectively), a decrease in the E28 group compared to the N and E7 groups (# P = 0.022, * P < 0.001,
respectively). Also, in the left face-S1, there was a significant increase in number of positive ICMS GG sites compared to right
face-S1 (! P < 0.001).
C
A B
Chapter 3
125
Muscles
LAD RAD GG
Nu
mb
er o
f p
osi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
2
4
6
8
10
12
14
16
Left Face-S1 sites for E1 group
Right Face-S1 sites for E1 group
*
*
Muscles
LAD RAD GG
Nu
mb
er o
f p
osi
tiv
e IC
MS
sit
es (
Mea
n +
SE
M)
0
2
4
6
8
10
12
14
16
Left Face-S1 sites for E28 group
Right Face-S1 sites for E28 group
#
##
#
Muscles
LAD RAD GG
Num
ber
of
posi
tive
ICM
S s
ites
(M
ean +
SE
M)
0
2
4
6
8
10
12
14
16
Left Face-S1 sites for E7 group
Right Face-S1 sites for E7 group
^
^
^^
C
A
Fig. 3-8. The number of positive ICMS sites in left and right face-S1 at ICMS intensity of 60 µA for LAD, RAD, and GG in
the E1 (A), E7 (B), and E28 (C) groups. In the E1 group, in the left face-S1, number of positive ICMS sites for LAD was
significantly decreased compared to GG (* P = 0.003); in right face-S1, RAD was significantly decreased compared to GG (* P =
0.038). In the E7 group, in the left face-S1, number of positive ICMS sites for RAD was significantly increased compared to LAD
(^ P < 0.001), GG was significantly increased compared to LAD and RAD (^ P < 0.001, <0.001, respectively); in right face-S1, GG
was significantly increased compared to LAD (^ P = 0.006). In the E28 group, in the left face-S1, number of positive ICMS sites
for LAD and RAD was significantly decreased compared to GG (# P < 0.001, = 0.021, respectively); in right face-S1, LAD and
RAD was significantly reduced compared to GG (# P = 0.021, < 0.001, respectively).
B
Chapter 3
126
Fig. 3-9. AP distribution of the positive ICMS penetrations in the left (-ve coordinates on x-axis) and the right
(+ve coordinates on x-axis) face-M1 and face-S1 irrespective of the muscle and the ML position. The number
of positive penetrations in the AP coordinates was the highest in the AP2.5 - AP3 mm coordinates from the
landmark bregma in all the groups. In the left face-M1 and face-S1, multivariate analyses indicated that in the
coordinate AP2.5 (most posterior coordinate), the number of positive penetrations significantly decreased in the E1
group compared to the S1 and the N groups (* P < 0.001, P < 0.001, respectively), and decreased in the E28 group
compared to the S28 group (# P = 0.001) to suggest a decrease in the posterior extent in the number of positive
ICMS penetrations in the E1 group compared to the S1 and N groups, and the E28 group compared to the S28
group; in the coordinate AP4 (most anterior coordinate), there was no significant difference between the E groups
compared to their analogous S groups and the other E groups. In the right face-M1 and face-S1, in the coordinate
AP2.5, the number of positive penetrations significantly decreased in the E28 group compared to the S28 group (* P
= 0.017) to suggest a decrease in the posterior extent in the number of positive ICMS penetrations in the E28 group
compared to the S28 group, increased in the E7 group compared to the E1 (^ P < 0.001), and E28 (^ P < 0.001)
groups; in coordinate AP4, decreased in the E28 group compared to the S28 group (* P < 0.001) to suggest a
decrease in the anterior extent in the number of positive ICMS penetrations for the E28 group compared to the S28
group, increased in the E7 group compared to the E1 (^ P < 0.001), and E28 (^ P < 0.001) groups.
AP coordinates in face-M1 and face-S1
-8 -6 -4 -2 0 2 4 6 8
Num
ber
of p
osit
ive
ICM
S p
enet
rati
ons
(mea
n)
-2
0
2
4
6
8
S1
E1
S7
E7
S28
E28
N
*^
*
*
#^
#
^
*^
*
^
^
^#
#
*
*
^
^ *
**
*
*
*
#
#
#
^
^
^
*^
*^
^
^
Chapter 3
127
Fig. 3-10. ML distribution of the positive penetrations in the left (-ve coordinates on x-axis) and the right (+ve coordinates
on x-axis) face-M1 and face-S1 irrespective of the muscle and the AP position. In the left face-M1 and face-S1, multivariate
analyses indicated that in the coordinate ML2 (most medial coordinate) the number of positive penetrations significantly
increased in the E7 group compared to the E1 (^ P < 0.001) and E28 (^ P = 0.014) groups; in the coordinate ML2.5, decreased in
the E1 group compared to the N group (* P = 0.021) to suggest a decrease in the medial extent in the number of positive ICMS
penetrations in the E1 group compared to the N group, increased in the E7 group compared to the E1 group (^ P = 0.002). In the
coordinate ML3, the number of positive penetrations significantly decreased in the E1 group compared to the S1 group (* P =
0.014), and decreased in the E28 group compared to the S28 (# P = 0.002) and N (# P = 0.014) groups to suggest a decrease in
the medial extent in the number of positive ICMS penetrations in the E28 group compared to the S28 and N groups. In the right
face-M1 and face-S1, the number of positive ICMS penetrations in the coordinate ML3 decreased in the E28 group compared to
the S28 group (* P = 0.014) to suggest a decrease in the medial extent in the number of positive ICMS penetrations in the E28
group compared to the S28 group; in the coordinate ML4.5, the number of positive ICMS penetrations decreased in the E1 group
compared to S1 (* P = 0.001) E28 (* P = 0.014), and N (* P < 0.001) groups to suggest a decrease in the lateral extent in the
number of positive ICMS penetrations in the E1 group compared to the S1, E28 and N groups, and also decreased in the E28
group compared to the S28 group (# P = 0.014), increased in the E7 group compared to the E1 (^ P < 0.001), and E28 (^ P <
0.001) groups; in the coordinate ML5, decreased in the E28 group compared to the S28 group (* P < 0.001) to suggest a decrease
in the lateral extent in the number of positive ICMS penetrations in the E28 group compared to the S28 group, increased in the E7
group compared to E1(^ P = 0.001), E28 (^ P = 0.001), and N (^ P < 0.001) groups.
ML coordinates in face-M1 and face-S1
-8 -6 -4 -2 0 2 4 6 8
Num
ber
of p
osit
ve I
CM
S p
enet
rati
ons
(mea
n)
-2
0
2
4
6
8
S1
E1
S7
E7
S28
E28
N
^
^^
*
^
^^
*#
*##
*
*
^
^
^
#
#
*
*
*
*
#
#
^
^
*
**#
#
^
^^
*
**
*
#
#^
^
^
*
*
^
^
^^
Chapter 3
128
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
2.5
3.0
3.5
4.0
4.52.0
2.53.0
3.54.0
4.55.0
5.5
DE
(m
m)
AP
(mm
)
ML (mm)
*
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
2.5
3.0
3.5
4.0
4.52.0
2.53.0
3.54.0
4.55.0
5.5
DE
(m
m)
AP
(mm
)
ML (mm)
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
2.5
3.0
3.5
4.0
4.52.0
2.53.0
3.54.0
4.55.0
5.5
DE
(m
m)
AP
(mm
)
ML (mm)
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.03.2
3.43.63.84.04.24.4
3.0
3.5
4.0
4.5
2.02.5
3.03.5
4.04.5
5.05.5
DE
(m
m)
AP
(mm
)
ML (mm)
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.03.2
3.43.63.84.04.24.4
3.0
3.5
4.0
4.5
2.02.5
3.03.5
4.04.5
5.05.5
DE
(m
m)
AP
(mm
)
ML (mm)
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.03.2
3.43.63.84.04.24.4
3.0
3.5
4.0
4.5
2.02.5
3.03.5
4.04.5
5.05.5
DE
(m
m)
AP
(mm
)
ML (mm)S1
E1
S7
E7
S28
E28
N
A
C
LAD
RAD
E GG
D
B
F
Chapter 3
129
Fig. 3-11. CoG of the positive ICMS sites in face-M1 and face-S1 for LAD (A – right face-M1 and face S1, B –
left), RAD (C – right, D – left), and GG (E – right, F – left). In the AP plane, in the left and the right face-M1 and
face-S1, the CoG for LAD, RAD, and GG were located between AP3 and AP3.5 coordinates, with no significant
differences across the groups or between the left and the right face-M1 and face-S1. In the ML plane, in the left and
the right face-M1 and face-S1, the CoG for LAD, RAD, and GG were located between ML2 and ML3.5 coordinates.
However, for LAD, in the right face-M1 and face-S1, the ML position of the CoG shifted medially significantly in
the E1 group compared to the S1 group (* P = 0.022). Although, for the CoG for LAD in the other groups, and in all
the groups for RAD and GG, there were no statistically significant differences in the ML positions, however a trend
was noticed for the E1 group compared to the S1 and N groups to shift medially, the E7 group compared to the S7
and N groups to shift laterally, and for the E28 group compared to the S28 and N groups to shift medially. In the De
plane, in the left and the right face-M1 and face-S1, the CoG for LAD, RAD, and GG were located between De2
and De3.2 coordinates. Although, there were no statistically significant differences in the De positions for the CoG
in all the groups for LAD, RAD and GG, however a trend was noticed for the E1 group compared to the S1 and N
groups to shift superficially, the E7 group compared to the S7 and N groups to shift deeper, and for the E28 group
compared to the S28 and N groups to shift superficially.
Chapter 3
130
Group Muscle Left face-M1 Right face-M1 Left face-S1 Right face-S1
N
LAD 10.56 ± 0.71 09.43 ± 0.81* 10.83 ± 0.56 08.96 ± 0.24*
RAD 08.70 ± 0.17* 11.26 ± 0.84 09.06 ±
0.25* 11.20 ± 0.48
GG 11.00 ± 1.21 10.46 ± 0.88 11.30 ± 0.46 10.33 ± 0.47
S1
LAD 10.93 ± 0.56 08.51 ± 0.12* 10.30 ± 0.35 08.50 ± 0.20*
RAD 09.46 ± 0.41* 11.40 ± 1.03 09.13 ±
0.22*
11.45 ± 0.83
GG 12.80 ± 0.60 12.60 ± 0.36 12.00 ± 0.37 11.96 ± 0.24
E1
LAD 11.70 ± 1.00 09.06 ± 0.20* 10.76 ± 0.50 08.60 ± 0.22*
RAD 08.93 ± 0.31* 10.70 ± 0.59 08.86 ±
0.19*
10.06 ± 0.24
GG 11.43 ± 0.56 12.90 ± 0.78 10.9 ± 0.40 11.70 ± 0.49
S7
LAD 10.03 ± 0.21 08.50 ± 0.12* 10.26 ± 0.50 08.46 ± 0.13*
RAD 09.26 ± 0.71* 10.33 ± 0.40 08.96 ±
0.26*
10.50 ± 0.48
GG 09.66 ± 0.57 10.03 ± 0.19 10.30 ± 0.42 10.33 ± 0.23
E7
LAD 10.63 ± 0.69 09.26 ± 0.70* 10.00 ± 0.36 08.80 ± 0.29*
RAD 08.76 ± 0.29* 10.76 ± 1.20 08.60 ±
0.14*
10.20 ± 0.28
GG 11.36 ± 0.53 12.26 ± 0.95 11.36 ± 0.32 11.66 ± 0.67
S28
LAD 10.46 ± 0.68 08.66 ± 0.12* 10.85 ± 0.49 08.73 ± 0.16*
RAD 08.46 ± 0.08* 10.00 ± 0.54 08.56 ±
0.16*
10.23 ± 0.38
GG 10.20 ± 0.29 10.10 ± 0.24 10.56 ± 0.46 10.50 ± 0.37
E28
LAD 11.00 ± 0.61 08.93 ± 0.18* 10.76 ± 0.46 08.93 ±0.18*
RAD 09.06 ± 0.23* 11.53 ± 1.17 08.70 ±
0.14*
11.53 ± 0.43
GG 12.46 ± 0.66 11.96 ± 1.26 11.76 ± 0.36 11.46 ± 0.86
Table. 3-1. Onset latencies (ms) of ICMS (60 µA) –evoked EMG activites in LAD, RAD, and GG in
face-M1 and face-S1 (mean ± SEM). There were no significant differences across the groups in the mean
shortest onset latency for ICMS-evoked EMG activities in LAD, RAD, and GG in the left or right face-M1
and face-S1. In each group LAD and RAD had significantly shorter onset latency in the contralateral face-M1
and face-S1 (* P < 0.001). Also, there were no significant differences in the mean shortest onset latencies of
evoked LAD, RAD, or GG activities in the face-S1 when compared to face-M1.
Chapter 3
131
Group LMa RMa LBu RBu 20 µA
N 2.50 ± 0.42 2.00 ± 0.36 1.83 ± 0.47 1.83 ± 0.30 4.16 ± 0.60
S1 1.50 ± 0.42 1.33 ± 0.42 1.50 ± 0.34 1.83 ± 0.30 3.66 ± 0.55
E1 1.00 ± 0.25 1.33 ± 0.21 1.00 ± 0.36 1.33 ± 0.42 2.80 ± 0.37
S7 1.33 ± 0.49 1.16 ± 0.30 1.16 ± 0.30 1.50 ± 0.42 3.16 ± 0.30
E7 1.33 ± 0.42 1.33 ± 0.42 1.16 ± 0.47 1.50 ± 0.34 4.16 ± 0.74
S28 1.33 ± 0.42 1.50 ± 0.22 1.33 ± 0.42 1.00 ± 0.36 2.60 ± 0.81
E28 1.50 ± 0.42 1.66 ± 0.49 1.16 ± 0.30 1.00 ± 0.25 2.33 ± 0.42
Table. 3-2. Number of positive ICMS sites in the left and right face-M1 and face-S1 for LMa, RMa,
LBu, RBu, and at ICMS intensity of 20 µA for LAD, RAD, and GG. Since the number of positive sites for
LMa, RMa, LBu, RBu, and for 20 µA intensity ICMS for LAD, RAD, and GG in the face-M1 and face-S1
was minimal, this data was not included in the extensive data analysis.
Chapter 3
132
Muscle Predictor F value, DF, Statistical
Significance
LAD
Study Group 07.94, 6/35, < 0.0001
Cortical Side 59.73, 1/35, < 0.0001
Study Group * Cortical side 01.55, 6/35, = 0.1911
RAD
Study Group 11.23, 6/35, < 0.0001
Cortical Side 50.02, 1/35, < 0.0001
Study Group * Cortical side 00.73, 6/35, = 0.6253
GG
Study Group 12.04, 6/35, < 0.0001
Cortical Side 11.60, 1/35, = 0.0017
Study Group * Cortical side 03.49, 6/35, = 0.0083
RAD + LAD
Study Group 08.63, 6/35, < 0.0001
Cortical Side 00.02, 1/35, = 0.8809
Study Group * Cortical side 01.06, 6/35, = 0.4062
LAD + GG
Study Group 03.79, 6/35, = 0.0052
Cortical Side 10.47, 1/35, = 0.0027
Study Group * Cortical side 02.52, 6/35, = 0.0392
RAD + GG
Study Group 06.46, 6/35, < 0.0001
Cortical Side 09.32, 1/35, = 0.0043
Study Group * Cortical side 01.55, 6/35, = 0.1897
LAD + RAD + GG
Study Group 03.33, 6/35, = 0.0107
Cortical Side 00.04, 1/35, = 0.8453
Study Group * Cortical side 01.34, 6/35, = 0.2667
Table 3-3. Mixed model repeated measures ANOVA, followed by post-hoc Sidak-adjusted pairwise
comparisons where applicable were used to determine whether study group, cortical side, or any interactions
of these effects significantly affected the number of positive ICMS sites. These tests were performed
separately for each muscle and each combination of muscles in face-M1.
Chapter 3
133
Muscle Predictor F value, DF, Statistical
Significance
LAD
Study Group 14.46, 6/35, < 0.0001
Cortical Side 50.71, 1/35, < 0.0001
Study Group * Cortical side 01.14, 6/35, = 0.3618
RAD
Study Group 07.74, 6/35, < 0.0001
Cortical Side 75.07, 1/35, < 0.0001
Study Group * Cortical side 03.68, 6/35, = 0.0061
GG
Study Group 13.35, 6/35, < 0.0001
Cortical Side 07.82, 1/35, = 0.0083
Study Group * Cortical side 02.48, 6/35, = 0.0418
Table 3-4. Mixed model repeated measures ANOVA, followed by post-hoc Sidak-adjusted pairwise
comparisons where applicable were used to determine whether study group, cortical side, or any interactions
of these effects significantly affected the number of positive ICMS sites. These tests were performed
separately for each muscle in face-S1.
CHAPTER 4
MECHANICAL AND THERMAL HYPERSENSITIVITIES
ASSOCIATED WITH ORTHODONTIC TOOTH
MOVEMENT (OTM): A BEHAVIOURAL RAT MODEL FOR
OTM-INDUCED PAIN
Chapter 4
135
4.1. Abstract
Objectives: To test if orofacial mechanical and thermal hypersensitivities occur in rats
during orthodontic tooth movement (OTM).
Material and Methods: Sprague-Dawley rats (140-160 g), 6 weeks old, were divided into
an experimental (E) group (n=7) that had an active orthodontic spring placed in the right
side of their mouth, and a sham (S) group (n=7) with an inactive orthodontic spring.
Mechanical sensitivity preoperatively (1 day before attaching the orthodontic spring) and
postoperatively (1 hr, 3 hrs, 6 hrs, days 1 – 7, day 14, day 21, and day 28 after orthodontic
spring attachment) was tested bilaterally on the cheek, upper lip, and maxillary incisor
labial gingiva by recording the threshold for a head withdrawal response evoked by von
Frey filaments. Thermal sensitivity at these times was also tested bilaterally on the cheek
by applying a noxious thermal stimulus and measuring head withdrawal response duration,
response score, and response percentile rate. Statistical analyses involved a mixed model
repeated measures ANOVA (MMRM ANOVA).
Results: The mechanical and thermal sensitivities at all ipsilateral and contralateral sites
were significantly increased (P <0.01) in the E group in the early postoperative period (1 –
5 days) with peaks reached on day 1, and then returned to preoperative levels until
postoperative day 28. However, there was no significant change in mechanical and thermal
sensitivities for the S group from the preoperative level for all the tested sites for the entire
testing period. The significant mechanical hypersensitivity in the E group was indicated by
a decrease in the threshold for head withdrawal response, whereas the significant thermal
hypersensitivity was indicated by an increase in all the 3 thermal response parameters, the
head withdrawal response duration, the response score, and the response percentile rate.
Conclusions: This is the first study to document bilateral orofacial mechanical and thermal
hypersensitivities that last for up to 5 days in a rat OTM-induced pain model. This
correlates with the time course of OTM-induced neuroplastic changes in the face-M1 and
face-S1 that were evident at postoperative days 1, 7, and 28, suggesting that the latter
neuroplastic changes in the face-M1 and face-S1 are unlikely related to maintained OTM-
Chapter 4
136
induced pain since orofacial mechanical and thermal sensitivities had returned to
preoperative levels almost 3 weeks before day 28.
4.2. Introduction
We have documented neuroplastic changes in the sensorimotor cortex associated
with OTM in a time-dependent manner (see chapter 3). Earlier studies have reported that
acute post-operative pain may induce sensorimotor cortex neuroplasticity in rodents and
humans (Ridding and Rothwell, 1997; Monfils et al., 2005; Boudreau et al., 2007; Adachi
et al., 2008). Therefore, it is possible that the OTM-induced face sensorimotor cortex
neuroplasticity could have been the consequence of OTM-evoked pain. Orthodontic
treatment is a common clinical procedure and most of the patients experience pain and this
has been suggested to affect treatment outcomes and compliance (Jones and Chan, 1992a;
Patel, 1992; Sergl et al., 2000; Kluemper et al., 2002; Asham, 2004, Keim, 2004; Fleming
et al., 2009; Hammad et al., 2012). A majority of the patients undergoing orthodontic
treatment first experience pain approximately 4 hours after orthodontic appliance
placement, and pain reaches its peak levels at 24 hours, usually lasts for 2-3 days and then
gradually dissipates completely by 5-6 days (Jones, 1984; Ngan et al., 1989, 1994; Jones
and Chan, 1992a, b; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and Dincer, 2004;
Polat et al., 2005; Polat, 2007). An immediate and delayed painful response after
orthodontic force application has also been reported in the initial few days of OTM
(Burstone, 1962; Jones and Chan, 1992b; Krishnan, 2007). The immediate pain has been
attributed to compression of the PDL and the resulting input from activated nociceptors,
and the slightly delayed pain to a hyperalgesic state. However, the etiology and
pathophysiology of OTM-induced pain are still largely unknown and no animal model has
been developed to simulate clinical OTM-induced pain in humans.
Experiments on pain in human subjects are practically challenging, fundamentally
subjective, and ethically self-limiting, and thus laboratory animal models of pain are a more
practical substitute to study mechanisms of pain. A number of animal models have been
developed to study various types of pain-related behaviours and mechanisms (for review,
see Ren and Dubner, 1999; Siddall and Munglani, 2003; Ma, 2007; Mogil, 2009). In the
Chapter 4
137
orofacial region, the infraorbital nerve ligation (ION-CCI) model (Vos et al., 1994, 1998),
the inferior alveolar nerve injury (IANX) model (Iwata et al., 2001; Piao et al., 2006;
Okada-Ogawa et al., 2009), and the cervical nerve transection (CNX) model (Kobayashi et
al., 2011) have been used (for review, see Iwata et al., 2011). Other behavioural models to
study TMJ, masticatory muscle and pulp pain mechanisms have been developed by
characterizing the nociceptive behavioural responses induced by the injection of algesic
chemicals into these tissues or by using other approaches to disrupt these tissues (Yu et al.
1995, 1996; Bakke et al., 1998a; Chiang et al., 1998, 2005, 2007, 2011; Cairns et al., 1998,
2007; Roveroni et al., 2001; Hu et al., 2002; Lam et al., 2005a, b; Ro, 2005; Xie et al.,
2007; Adachi et al., 2008; Zhang et al., 2008; Itoh et al., 2011; Tsuboi et al., 2011; Cao et
al., 2013; for review, see Khan and Hargreaves, 2010, Sessle, 2011a). These models have
led to the elucidation of a variety of central and peripheral mechanisms in TMJ, muscle,
and tooth pulp pain, including central sensitization in brainstem and thalamic nociceptive
neurons. In addition, there is evidence in some of these models that noxious orofacial
stimulation may induce face-M1 neuroplasticity, consistent with findings in humans that
acute orofacial pain produces a reversible face-M1 neuroplasticity (Boudreau et al., 2007,
2010).
The majority of behavioural studies using orofacial pain models in animals have
typically assessed pain behaviour in terms of evoked withdrawal responses and
hypersensitivity. These include behavioural hyperalgesia, defined as an exaggerated
nociceptive response to a noxious stimulus, and allodynia, pain induced by a normally
nonnoxious stimulus (Vos et al., 1994, 1998; Le Bars et al., 2001; Iwata et al., 2011).
Quantification of behavioural responses to cutaneous hyperalgesia in the orofacial region in
animals has relied upon mechanical testing (Rosenfeld et al., 1978; Morris et al., 1982;
Clavelou et al., 1989; Vos et al., 1994, 1998) and thermal testing procedures (Mor and
Carmon, 1975; Carmon and Frostig, 1981; Hargreaves et al., 1988, Hargreaves, 2011).
Since there is a need for animal models of clinical OTM-induced pain in humans and we
have documented that OTM induces sensorimotor cortical neuroplasticity that conceivably
could be a result of OTM-induced pain (chapter 3), the objective of this study was to test if
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138
orofacial mechanical and thermal hypersensitivities occur in rats during OTM. Some
findings of this study have been published briefly in abstract form (Sood et al., 2012, 2013).
4.3. Materials and Methods
4.3.1. Animal preparation
Experiments were performed on 14 6-week-old adult male Sprague-Dawley rats
(140-160 g) that were housed in cages (27cm X 45 cm X 20 cm) in a temperature (21±1°C)
- and humidity (50±5%) - controlled environment under a 12 h light/dark cycle (lights on at
07:00 a.m.) and that received water and mashed diet (Rodent diet #2018M, Harlan Teklad)
ad libitum. The rats were acclimatized to the environment for 1 week before the initiation
of the study. The animals were monitored on a daily basis to assess body weight and food
consumption, general behaviour and any post-operative complications that could ensue
from tissue injury as outlined in chapter 2. All experimental procedures were approved by
the University of Toronto Animal Care Committee, in accordance with the Canadian
Council on Animal Care Guidelines and the regulations of the Ontario Animals for
Research Act (R.S.O. 1990), and the guidelines of the International Association for the
Study of Pain. All experimental procedures were carried out by one investigator to ensure
consistency in the experimental procedures and the experimenter was blinded to the animal
groups, and the data analyses were performed by another experimenter who was also
blinded to the animal groups.
4.3.2. Study groups and mechanical and thermal testing procedures
The rats were separated into 2 groups, an experimental (E) group (n = 7) that
received a NiTi closed-coil orthodontic spring (coil diameter 0.22 mm, eyelet diameter 0.56
mm, force on activation 10 cN, GAC, NY, U.S.A.) that was activated to induce OTM, and
a sham (S) group (n = 7) that received the orthodontic spring but in an inactive state. The
orthodontic springs were attached under general anaesthesia (inhalation isoflurane 5%
induction, 2~2.5% maintenance) (see chapter 2).
Chapter 4
139
4.3.2.1. Testing procedure
For observation of responses to mechanical and thermal stimulation, rats were
placed in a cylindrical restrainer (10 cm diameter, 20 cm length) with an open posterior end
and a small opening in the anterior end through which the rats could place their snouts. This
restrainer permitted the investigator access to the face of the rat, but covered the eyes so
that the rat could not visualize the approach of the investigator to perform the stimulation
test. Further, the restrainer was large enough to allow the rat to rotate to the posterior end to
escape the stimulus. Multiple grooves cut into the side of the restrainer posterior to the
initial 1 cm from the anterior end permitted the investigator to visualize the rat after the
stimulus induced a withdrawal response of the head into the restrainer. Before the
stimulation session, rats were adapted to the restrainer for 15 min daily, and then the series
of mechanical and thermal stimulations were initiated. Stimulations were administered
when the rat was neither moving nor freezing and were applied perpendicular to the sagittal
plane of the head. The next stimulus was applied only when the rat resumed this position
and at least 30 s after the preceding stimulation. Three consecutive days before the
placement of the orthodontic spring, the rats were habituated to the restrainer. One day
prior to the placement of the orthodontic spring, the preoperative values for the mechanical
and thermal tests were obtained. Rats were then tested for mechanical and thermal
sensitivities at 3 hrs, 6 hrs, days 1-7, day 14 and day 28 after the placement of the
orthodontic spring. Testing was conducted at times between 0700 and 1500 hr. of the day.
Rats were tested in the test room with a constant background noise (50 dB) that was used to
decrease any interference of sudden auditory stimulation.
4.3.2.2. Nature of stimuli and the stimulation sites
For mechanical stimulation, von Frey filaments (Pressure Aesthesiometer, Stoelting
Co, Chicago, IL) of varying diameters for which the force required to bend each filament
was 1 g, 1.4 g, 2 g, 4 g, 6 g, 8 g, 10 g, 16 g in an ascending series were used. During one
session, the complete series of von Frey hair intensities was presented in an ascending
series until a response was obtained. Each testing site was stimulated with each filament 5
consecutive times in an ascending order starting with 1 g. When 3 out of 5 stimulations for
Chapter 4
140
a filament resulted in a head withdrawal response (see below), the force level of that
filament was considered the threshold mechanical response level. Once the threshold
response was obtained, the threshold value was confirmed by dropping down one smaller
size in the series of von Frey filament stimulation intensities and observing if the
withdrawal response could no longer be evoked by that stimulus intensity (Vos et al., 1994;
1998; Imamura et al., 1997; Ren, 1999; Iwata et al., 2001, Suzuki et al., 2013). The sites
were tested in a random order with at least 1 m time lag between the different test sites.
Further, a time lag of 30 s was maintained between each filament contact in each testing
site. Mechanical stimuli were applied to the cheek, the upper lip, and the maxillary incisor
labial gingiva. Radiographic images and anatomical landmarks of the head of the rat were
used to select the stimulation sites. The site of the cheek was the midpoint vertically
between the second and the third row of whiskers and horizontally the most posterior extent
of the vibrissal pad where it meets the furry skin of the cheek. This point was midway on
the cheek between the maxillary incisor and the maxillary molar teeth, and although being
on the hairy vibrissal pad, was covered with only a small amount of hair and thereby
provided an unobstructed area for mechanical testing. Secondly, the anterior opening of the
restrainer permitted this area of the rat’s face to be easily accessible for testing. For the
upper lip, the testing was done at a spot midway along the length and the width of the upper
lip. In testing for the maxillary incisor labial gingiva, caution was taken to prevent making
any contact with the upper lip. These areas were stimulated on both sides of the face, i.e.
ipsilateral and contralateral to the side where the orthodontic spring was attached.
For thermal testing, a beam of noxious radiant heat was aimed at the cheek. The
stimulation was limited to the ipslilateral and the contralateral cheek site only, and the site
was similar to the one used for mechanical testing on the cheek. The light source was made
from the lamp housing of a fiber optic microscope illuminator (Lype Laser, China). The
radiant heat stimulus was a focused beam of light from a modified microscope illuminator,
the aperture of which was 10 cm from the stimulation site. Thermal testing was done at the
strength of 22 A and 200 ms duration to generate a noxious radiant heat, as documented in
another study in our laboratory (Wang et al., 2012). Guideline marks on the laboratory table
helped maintain a constant distance between the skin and the heat source. Head withdrawal
Chapter 4
141
latency along with the response duration was measured by using a stopwatch to give the
total response duration (TRD), and was determined 5 times on each side of the face with 2
min intervals between each stimulus; any response that occurred in at least 3 out of 5
thermal stimuli was considered as a positive response. Since the values for head withdrawal
latency and response duration could not be separated because the response was very rapid,
the TRD value was measured and used in the analysis. A 6s of thermal stimulation cut-off
was used to prevent tissue damage.
4.3.2.3. Response scoring
For mechanical testing, the response threshold was noted for each of the testing
sites when there was a head withdrawal response, i.e. the rat pulled its head briskly
backward when the stimulus was applied (Hargreaves et al., 1988; Vos et al., 1994;
Imamura et al., 1997; Ren, 1999; Iwata et al., 2001, Suzuki et al., 2013). Further, to
compare if the change in mechanical response threshold affected one of the tested site more
than the other in the E group, the change in postoperative response threshold from the
preoperative response threshold was compared amongst the different tested sites on
analogous days. For thermal testing, in addition to measuring the TRD (see above), each
response was scored (Mor and Carmon, 1975; Carmon and Frostig, 1981; Hargreaves et al.,
1988; Schouenborg et al., 1992; Fan et al., 1995) by grading the response from 0 to 3 [0=
no response, 1 = slight twitch (approx. 0.5s), 2 = distinct movement away from the
stimulus/ brief stroke of the face by the rat’s paw (range of 0.7-1.2s), 3 = very strong
movement/turn around]. Also, the frequency of a positive response during the thermal
testing procedure was calculated as the response percentile rate.
4.3.3. Statistical analyses
Statistical differences between groups at different time points were determined by
using multivariate [mixed model repeated-measures (MMRM) ANOVA] analyses,
followed by post-hoc Sidak-adjusted pairwise comparisons as appropriate. A probability
level of P < 0.05 was considered statistically significant. Data was analyzed by a statistician
using the SAS statistical software program (version 9.3). All values are expressed as mean
± SEM.
Chapter 4
142
4.4. Results
The condition of the rats was monitored on a daily basis during the course of the
experiment and no abnormal behavior and orthodontic spring related complications were
apparent, e.g., standing in a corner of the cage for prolonged periods, strange eating or
chewing behaviour, swaying their heads back and forth, vibrating their tails, vocalizing, or
tissue laceration and infection. As described in chapter 2, before the orthodontic spring was
placed, E and S group animals had a similar daily gain in body weight. However, after the
placement of the orthodontic spring, the E group had a small but significant loss of weight
for day 1 as compared to the S group, but thereafter gained weight again at a similar rate to
that of the S group (P < 0.001) (see chapter 2).
Significant changes in mechanical and thermal sensitivities following OTM were
documented during the testing period that extended from 1 day before (preoperative) and
28 days after (postoperative) the placement of the orthodontic spring on the maxillary
molars and the maxillary incisors.
4.4.1. Response threshold related to mechanical stimulation
In the S group, the postoperative response threshold at any of the tested sites (cheek,
upper lip, and maxillary incisor gingiva) did not differ significantly from the preoperative
response threshold value at that same site (P > 0.05). However, MMRM ANOVA revealed
significant differences in treatment (P = 0.036). In the E group, a significant difference was
revealed in time (P < 0.0001), and also in treatment * time interaction effects (P < 0.0001).
There was no significant difference between the mean values of the ipsilateral and the
contralateral sides of the tested sites (P = 0.69), indicating that the contralateral response
threshold decrease reached statistical significance at the same time as the ipsilateral side (at
day 1 for all sites, except at 6 h for the maxillary incisor gingival site). The significant
change was reflected in a decrease in response threshold that reached its peak on
postoperative day 1, but none of the significant changes in response thresholds to
mechanical stimulation of the orofacial areas lasted longer than postoperative day 4 (Fig. 4-
1A, B, C).
Chapter 4
143
At the cheek site, post-hoc analysis revealed a significant decrease in the response
thresholds in the E group on each of the days 1 – 4 compared to the preoperative value (P <
0.0001) and compared to analogous days in the S group (P < 0.0001) (Fig. 4-1A). A
significant decrease in postoperative response threshold was also observed at the upper lip
site in the E group on each of the days 1 - 4 compared to the preoperative value (P <
0.0001) and compared to analogous days in the S group (P < 0.01) (Fig. 4-1B). At the
maxillary incisor gingival site, a significant decrease in the response thresholds in the E
group was revealed at 6 h and each of the days 1 – 4 compared to the preoperative value (P
< 0.0001), and compared to the analogous time period in the S group (P < 0.01) (Fig. 4-
1C). The postoperative response threshold at each of these sites returned to the preoperative
levels by day 5, and thereafter remained at those levels until day 28. Amongst the three
tested sites, the lowest response thresholds were detected at the maxillary incisor gingival
site (Fig. 4-1A, B, C). Further comparison of the early postoperative decrease in
mechanical response threshold to the preoperative value between the 3 sites for the E group
on the analogous days revealed that there was no significant difference in the level of
decrease in response thresholds compared to their preoperative value at all the sites.
4.4.2. Responses related to thermal stimulation
The responses evoked by noxious thermal stimulation applied bilaterally to the
cheek site were measured in terms of three parameters described below. In the S group, the
postoperative value at the tested site did not differ significantly from the preoperative test
value for that site, but multivariate analysis revealed significant differences in treatment (P <
0.05). In the E group, a significant difference was also revealed in time (P < 0.0001), and also
in treatment * time interaction effects (P < 0.0001). There was no significant difference in any
of the measured thermal response parameters between the sides of the tested sites (P > 0.05),
indicating that the contralateral thermal response values reached statistical significance
around the same time as that for the ipsilateral side (at day 1), but none of the significant
changes to thermal stimulation of the cheek site lasted longer than postoperative day 5 (Fig.
4-2A, B, C).
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Post-hoc analysis revealed a significant increase in TRD for each side in the E
group on each of the days 1 – 5 compared to the preoperative value (P < 0.0001) and
compared to that for the analogous days in the S group (P < 0.0001) (Fig. 4-2A). Further, a
significant increase for each side in the response score (0 - 3) in the E group on each of the
days 1 – 5 compared to the preoperative value (P < 0.0001) and compared to that for the
analogous days in the S group (P < 0.0001) was revealed (Fig. 4-2B). Also, a significant
increase occurred for each side in the response percentile rate in the E group on each of the
days 1 – 5 compared to the preoperative value (P < 0.0001) and compared to that for the
analogous days in the S group (P < 0.001) (Fig. 4-2C).
4.5. Discussion
This is the first study to document that orofacial mechanical and thermal
sensitivities change in a rat OTM-induced pain model. The mechanical and thermal
sensitivities at all ipsilateral and contralateral sites tested were significantly increased (P
<0.01) in the E group in the early postoperative period (1 – 5 days) with peaks reached on
day 1, and then returned to preoperative levels until postoperative day 28. The mechanical
hypersensitivities reflected a decrease in the mechanical response thresholds, and the
thermal hypersensitivities consisted of an increase in the response duration, response score,
and response percentile rate. Between postoperative days 4 and 6, the sensitivities returned
to preoperative control level and were maintained at this level for the rest of the 28 days
testing period. Further, both mechanical and thermal hypersensitivities occurred in
ipsilateral as well as contralateral tested sites. However, there was no significant change in
mechanical and thermal sensitivities for the S group from the preoperative control level for
all the tested sites for the entire testing period.
In a study in the same OTM rat model (see chapter 3), we have documented a
significant decrease in the number of positive ICMS sites for anterior digastric and
genioglossus motor representations in the face-M1 and face-S1 at postoperative day 1,
followed by a significant increase in motor representations at postoperative day 7, and
another significant decrease in motor representations at postoperative day 28. Thus,
neuroplastic changes in the face-M1 and face-S1 at postoperative day 1 could conceivably
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be related to OTM-induced nociceptive inputs associated with the orofacial
hypersensitivities, and the increase in the number of sites at postoperative day 7 could be
related to the absence of the nociceptive inputs by postoperative day 6. Indeed, short-term
neuroplastic changes in the face-M1 have been documented with the application of noxious
orofacial stimuli; these are expressed as a decreased cortical excitability (Adachi et al.,
2008), consistent with our E1 findings (chapter 3). However, the latter neuroplastic changes
in the face-M1 and face-S1 at postoperative day 28 are unlikely related to maintained
OTM-induced pain as mechanical and thermal sensitivities had returned to preoperative
levels well before postoperative day 28, and are more likely related to the altered occlusion
that is evident at postoperative day 28 (see chapter 2).
4.5.1. OTM pain - Peripheral mechanisms
It has been suggested that OTM results from an inflammatory process in the PDL
(Vandevska-Radunovic, 1999; Wise and King, 2008; Krishnan and Davidovitch, 2009) and
dental pulp (Kvinnsland and Kvinnsland, 1990; Norevall et al., 1998; Leavitt et al., 2002;
Yamaguchi et al., 2004). It has also been suggested that the nociceptive impulses generated
by OTM are initiated in the PDL and gradually diminish with every passing day, reflecting
the adaptation of PDL neural elements to environmental changes caused by orthodontic
forces (Krishnan and Davidovitch, 2006). An immediate pain response in the first few
hours of OTM results from compression of the PDL by the movement of the tooth and the
resulting inputs from nociceptors; whereas the slightly delayed gradual painful response is
reflected in a hyperalgesic state attributed to peripheral sensitization of nociceptive
afferents to released algogens such as histamine, bradykinin, PGEs, serotonin, and
substance P (Burstone, 1962; Jones and Chan, 1992b; Fujiyoshi et al., 2000; for review, see
Vandevska-Radunovic, 1999; Krishnan, 2007; Wise and King, 2008). Hence the
inflammatory process and peripheral sensitization in the PDL and associated structures may
have contributed to the documented short-term hypersensitivity in the early period of OTM
in the present study. Central mechanisms of nociception likely have contributed also to the
hypersensitivity state, as noted below.
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OTM is also associated with an impairment of chewing and biting ability, decrease
in biting force (Miyamoto et al., 1996; Alomari and Alhaija, 2012), a decrease in the
frequency of tooth contacts during the early stages of OTM and a decrease in pressure pain
threshold (PPT) for masseter and anterior temporalis muscles has been reported (Goldreich
et al., 1994; Miyamoto et al., 1996; Michelotti et al., 1999; Tanaka et al., 2003). Thus,
occlusal changes during the early period of OTM may also have contributed through
intraoral and muscle hyperalgesia to the short term hypersensitivity state documented in the
present study.
The present study demonstrated a return in the mechanical and thermal sensitivities
to the preoperative level by postoperative day 6 and this level was maintained for the rest of
the 28-day testing period, suggesting that long-term nociceptive mechanisms were not
present beyond the early period of OTM. Also, studies have demonstrated that peripheral
receptor mechanisms exist (e.g., opioid, GABA) that mediate analgesic effects that are
prominent in painful orofacial inflammatory conditions (Yu et al., 1994; Bakke et al.,
1998b; Hargreaves 2006; Sessle, 2011a), and thus these peripheral inhibitory mechanisms
may have been involved in abolition of OTM-induced nociceptive behaviour by day 6
documented in the present study (see below).
4.5.2. OTM pain - Central mechanisms
As a result of the ensuing inflammatory process during OTM, the process of
peripheral sensitization of the nociceptive endings can result in an augmented afferent
barrage being conducted along the nociceptive afferents into the CNS and the production of
trigeminal central sensitization that can contribute to orofacial hyperalgesia (Sessle, 2006;
Henry and Hargreaves, 2007; Sessle, 2011a). Thus, central sensitization could have
contributed to the orofacial hypersensitivities in the early period of OTM, but since the
present study demonstrated a return in the mechanical and thermal sensitivities to the
preoperative control level by postoperative day 6 and this level was maintained for the rest
of the 28 day testing period, long-term central sensitization mechanisms were unlikely
present beyond the early period of OTM. Central mechanisms may also have contributed to
shortening the duration of OTM-induced pain. OTM-induced nociceptive afferent inputs
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have been suggested to activate the descending serotonergic inhibitory system in the
periaqueductal grey and its projections to the trigeminal subnucleus caudalis at day 1 (Kato
et al., 1995; Yamashiro et al., 2000. 2001; Hasegawa et al., 2012). Also, it has been
demonstrated that inflammatory process that activate nociceptive afferent inputs also
enhanced CNS inhibitory pathways using endogenous opioids and other neurochemicals
(e.g., GABA, noradrenalin) that act on nociceptive transmission (Chiang et al., 1994; Yu et
al., 1994; Mason, 2005; Sessle, 2011a), and upregulation of the endogenous opioid gene in
trigeminal subnucleus suboralis (Vo) during OTM has been documented (Balam et al.,
2005). Therefore, these antinociceptive mechanisms may have contributed to the
dissipation of the OTM-induced orofacial hypersensitivities by day 6.
4.5.3. Mechanical and thermal hypersensitivities of the orofacial region as
an index of OTM-induced pain
There is evidence that intraoral noxious stimulation results in sensitization of
nociceptive neurons in trigeminal subnucleus caudalis and other parts of the brainstem
complex and in components of the trigeminal ascending pain pathways to the thalamus,
limbic system, the sensorimotor cortex and other cortical regions (Park et al., 2006; Zhang
et al., 2006; Magdalena et al., 2004; Chen et al., 2007; Adachi et al., 2008; Stabile et al.,
2009; Novaes et al., 2010; Iwata et al., 2011). As noted above, trigeminal central
sensitization also occurs with the time course that suggests it may contribute to the short-
term hypersensitivities documented in the present study. The time-dependent
hypersensitivity changes in the present study also correlated well with the early period
time-related pain incidence reported in human studies (Jones, 1984; Ngan et al., 1989,
1994; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and Dincer, 2004; Polat et al.,
2005; Polat, 2007). Further, the mechanical and thermal sensitivities for the S group were
maintained at the preoperative control level for all the tested sites for the entire testing
period. This finding addresses the concern that response parameters to repeated application
of the same series of mechanical and thermal stimuli might be influenced by factors such as
tissue damage, learning effect, animal fatigue, and increased animal irritability introduced
by the testing procedures (Le Bars et al., 2001). Hence, these observations in this rat OTM-
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induced pain model provide strong evidence for the validity of using mechanical and
thermal hypersensitivities as a measure of OTM-induced pain.
4.5.4. Orofacial mechanical and thermal hypersensitivities contralateral
to the side of orthodontic spring application
In the present study, mechanical and thermal hypersensitivities were present
bilaterally although the active orthodontic spring was attached on the right side intraorally.
This is in line with hyperalgesia contralateral to spinal nerve ligation and injury reported by
several groups (Seltzer et al., 1990; Kim and Chung, 1992; DeLeo et al., 1994). Trigeminal
primary afferent projections to the contralateral subnucleus caudalis (Capra, 1995; Sessle,
2000, 2011a; Waite, 2004), and also projections from subnucleus caudalis to the
contralateral side (Capra, 1995; Sessle, 2000; Waite, 2004) have been reported. In addition,
both ipsilateral and contralateral orofacial hypersensitivities have been demonstrated in
acute and chronic orofacial pain models (Vos et al. 1994, 1998; Chiang et al., 1998, 2007;
Iwata et al., 2011; Sessle, 2011a; Tsuboi et al., 2011; Cao et al., 2013) that are suggestive
of neuroplastic changes reflecting central sensitization in the subnucleus caudalis (Iwata et
al., 2001, 2004; Okada-Ogawa et al., 2009; Saito et al., 2008; Shibuta et al., 2012).
4.5.5. Strengths, limitations and future directions
Most of the OTM pain studies, both clinical on human subjects and in animals, have
used the Waldo’s method (1954) to induce OTM associated pain. However, unlike the
present study, these studies have not tested if orofacial nociceptive behaviour occurs in
these models. The orthodontically related disadvantages in the use of Waldo's method are
that the intensity of the initial force that the elastic generates was calculated to be
approximately 80 - 200 g (Azuma et al., 1970; Ren, 2004). This value is equivalent to 1.6 -
4 kg for humans because the surface of roots of man’s first molar has an area of about 20 -
50 times larger than that of rat molar (Sato et al., 1984, Ren, 2004). Firstly, this heavy force
applied to rat molars may not simulate the clinical condition of OTM in humans. Secondly,
when this heavy a force is applied to molars in a rat it may lead to an immediate forced
eruption of the tooth causing hyperocclusion. Studies of occlusal trauma have documented
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hyperocclusion based hypersensitivity (Cao et al., 2009; Cao et al, 2013). Thirdly, the
elastic used in the Waldo’s method suffers from a rapid dissipation of force (Ren, 2004).
Therefore, the use of an orthodontic appliance that generates a mild constant force that
better simulates clinical conditions, like a Ni-Ti orthodontic spring used in the present
study, is a more appropriate means of inducing OTM for experimental pain studies.
In comparison of thermal and mechanical methods, thermal methods may have a
higher sensitivity to detect hyperalgesia and allodynia (Vinegar et al., 1978; Hargreaves et
al., 1988, Hargreaves, 2011) and the development of hyperalgesia may differ in
responsiveness to thermal and mechanical stimuli (Handwerker et al., 1987, Le Bars et al.,
2001). Hence, both mechanical and thermal methods were employed in the present study.
Previous studies on chronic constriction injury (CCI) pain models and other pain
models, including orofacial models (e.g. inferior alveolar nerve transection, infraorbital
nerve transection, occlusal interference) have demonstrated that mechanical allodynia
depends primarily on the alteration of Aδ fibre properties whereas production of heat
hyperalgesia depends mainly on the alteration of C fibre properties (Le Bars et al., 2001;
Nakanishi et al., 2004; Toda et al., 2004). The present study revealed both mechanical and
thermal hypersensitivities produced by OTM, and further electrophysiological and
histological studies are needed to delineate the impact of OTM on different neural
structures of the PDL and their influence on mechanical and thermal sensitivities. Also, the
present study design limited its aim to study OTM-induced nociception that was related to
the evoked behavioural changes, and future studies to study spontaneous behavioural
changes in response to pain are suggested to clarify mechanisms related to spontaneous
pain related to OTM. Furthermore, future studies are warranted that test for the presence of
any secondary allodynia and hyperalgesia beyond the trigeminal innervation domain
(Hargreaves et al., 1988, Le Bars et al., 2001; Henry and Hargreaves, 2007; Sessle, 2011a)
to complement the OTM-induced rat pain model used in the present study.
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4.6. Conclusions
This project has introduced an OTM rat model that uses orthodontic force
parameters that are well defined and within the physiological limits when applied to the
teeth of rats and correlates well with the orthodontic forces applied in a clinical scenario in
humans. The present study has extended this model to characterize mechanical and thermal
hypersensitivities that are associated with OTM. Orofacial mechanical and thermal
hypersensitivities during OTM were apparent bilaterally and were significantly increased in
the E group in the early postoperative period (1 – 5 days) with peaks reached on day 1, and
then returned to preoperative levels until postoperative day 28. Also, hypersensitivities
measured in the present study correlated well with the time-dependent pain reported in
clinical OTM pain studies. This animal model provides easy implementation of mechanical
and thermal hypersensitivities as a behavioural model of allodynia/hyperalgesia related to
OTM and can serve as an important experimental tool to assess OTM-induced nocifensive
behaviours and to study the underlying mechanisms of OTM-induced pain.
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4.7. Figures
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
Res
po
nse
thre
sho
lds
(g)
2
4
6
8
10
* *
* *
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
Res
po
nse
thre
sho
lds
(g)
2
4
6
8
10
^
^ ^^
^
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
Res
po
nse
thre
sho
lds
(g)
2
4
6
8
10
Response thresholds on ipsilateral side for E (n=7)
Response thresholds on ipsilateral side for S (n=7)
Response thresholds on contralateral side for E (n=7)
Response thresholds on contralateral side for S (n=7)
##
# #
Fig. 4-1. Response threshold evoked by mechanical stimulation at the bilateral cheek (A), upper lip (B), and
maxillary incisor gingival (C) sites. A. At the cheek site, post-hoc analysis revealed a significant decrease in the
response thresholds in the E group on each of the days 1 – 4 compared to the preoperative value (P < 0.0001) and
compared to analogous days in the S group (*P < 0.0001). B. A similar trend of decrease in postoperative response
threshold was observed at the upper lip site in the E group on each of the days 1 - 4 compared to the preoperative value
(P < 0.0001) and compared to analogous days in the S group (#P < 0.01). C. At the maxillary incisor gingival site, a
significant decrease in the response thresholds in the E group was revealed on 6 h and each of the days 1 – 4 compared
to the preoperative value (P < 0.0001), and compared to the analogous time period in the S group (^P < 0.01).
C
A B
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152
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
TR
D (
s)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
TRD on ipsilateral side for E (n=7)
TRD on ipsilateral side for S (n=7)
TRD on contralateral side for E (n=7)
TRD on contralateral side for S (n=7)
* * *
*
*
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
Res
po
nse
Per
centi
le R
ate
0
20
40
60
80
100
Response percentile rate on ipsilateral side for E (n=7)
Response percentile rate on ipsilateral side for S (n=7)
Response percentile rate on contralateral side for E (n=7)
Response percentile rate on contralateral side for S (n=7)
^ ^
^^
^
Preoperative and Postoperative time
Preop. 1 h 3 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 14 d 21 d 28 d
Res
po
nse
sco
res
(0 -
3)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Response score on ipsilateral side for E (n=7)
Response score on ipsilateral side for S (n=7)
Response score on contralateral side for E (n=7)
Response score on contralateral side for S (n=7)
##
#
##
B
C
A
Fig. 4-2. Responses evoked by noxious thermal stimulation on the cheek site tested using the total response
duration (TRD) (A), response score (B), and response percentile rate (C). A. There was a significant increase in
the TRD in the E group on each of the days 1 – 5 compared to the preoperative value (P < 0.0001) and compared to the
analogous days in the S group (*P < 0.0001). B. There was a significant increase in the response score in the E group
on each of the days 1 – 5 compared to the preoperative value (P < 0.0001) and compared to the analogous days in the
S group (#P < 0.0001). C. There was a significant increase in the response score in the E group on each of the days 1 –
5 compared to the preoperative value (P < 0.0001) and compared to the analogous days in the S group (^P < 0.001).
CHAPTER 5
GENERAL DISCUSSION AND CONCLUSIONS
Chapter 5
154
This doctoral thesis is the first to address the hypothesis that OTM is associated
with neuroplasticity of the rat’s face-M1 and face-S1 motor representations and with
mechanical and thermal hypersensitivities in the orofacial region. To address this
hypothesis, the objectives of the study were:
1. To develop an OTM rat model, and design and manufacture a cephalostat for
standardized radiographic measurement of the amount and the rate of OTM.
2. To use ICMS and EMG recordings to test if neuroplastic changes occur in the ICMS-
defined motor representations of anterior digastric (LAD, RAD), masseter (LMa, RMa),
buccinator (LBu, RBu), and genioglossus (GG) muscles within the rat’s face-M1 and face-
S1 during OTM; the analyses to include any alterations in the number of ICMS sites
representing these muscles and in the onset latencies of ICMS-evoked responses in the
muscles.
3. To test if orofacial mechanical and thermal hypersensitivities occur in rats during OTM.
The OTM rat model that was developed reflects the advantages of using orthodontic
force parameters that are well defined and within the physiological limits when applied to
the teeth of rats and correlate well with the orthodontic forces applied in a clinic scenario in
humans. This study documented neuroplastic changes induced in the face-M1 and face-S1
by OTM. These changes may reflect adaptive sensorimotor changes in response to the
altered environment in the oral cavity induced by OTM. OTM resulted in significant
changes in motor representations of the LAD, RAD, and GG muscles in the ICMS-defined
face-M1 and face-S1 at time points of days 1, 7, and 28 of continuous orthodontic force
application and in the number of positive ICMS sites in face-M1 that could simultaneously
activate muscle activity in different combinations of LAD, RAD, and GG. The study also
discovered orofacial mechanical and thermal sensitivities changes in a rat OTM-induced
pain model. Changes in both mechanical and thermal sensitivities at all the sites tested after
orthodontic spring attachment followed a time course that could divide the OTM testing
period into an early and a late period. In the early period (1 - 5 days postoperative), there
was a significant increase in the mechanical and thermal sensitivities which returned to the
preoperative control level before the late period (6 - 28 days postoperative) of OTM and
remained at the preoperative level thereafter. Thus, the hypersensitivity at day 1 was
Chapter 5
155
associated with a decrease in the number of positive ICMS sites for RAD, LAD and GG
motor representations in the face-M1 and face-S1, and the return of sensitivity to
preoperative levels at day 7 with an increase in the number of positive ICMS sites of the
motor representations. However, the decrease in the number of ICMS sites observed at day
28 cannot be attributed to the maintenance of nociceptive afferent inputs related to OTM-
induced pain since mechanical and thermal sensitivities had returned to preoperative levels
well before day 28. Since alteration of the occlusion at day 28 was documented by our
radiographic measurements, it more likely that the neuroplastic change documented at day
28 may be more a result of the occlusal change, although OTM-related nociceptive inputs
from the molars cannot be ruled out as it was not possible in the present study to measure
behavioural responses to mechanical and thermal molar stimulation in awake rats.
5.1. Findings related to specific objectives of the study
5.1.1. OTM rat model
This study has introduced a new OTM rat model for studying tooth movement and
associated neuroplastic changes induced by OTM. Analyses of radiographs taken using the
custom-designed cephalostat to standardize the x-ray tube, rat head, and film distance
revealed that the adjusted OTM of the three maxillary molars and maxillary incisors, that
included a component of distal drift (DD) that is normally evident in the maxillary molars
of rats, was 1.75 ± 0.23 mm in a span of 28 days. The rate of OTM was uniform throughout
the 28 days of the experiment. The force-delivery method of attaching an orthodontic
spring on the rat’s teeth had defined and measurable force parameters with force levels that
were of constant magnitude and in a direction that was maintained parallel to the occlusal
plane for the duration of the experiment. The use of the NiTi closed-coil orthodontic spring
in this study produced a constant level of 10 ± 4 cN force throughout the experimental
period. Further, in relation to the force parameters, since the molar teeth of rats are
approximately 20 - 50 times smaller than that of human molars, the force magnitude
applied to the rat teeth was adjusted to this difference in tooth size to maintain the force
applied to the rat molars proportional to orthodontic forces applied to human teeth (Sato et
al., 1984; Ren et al. 2004; Tan et al., 2009).
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The amount and rate of OTM recorded in the present study compared well with
values reported by other studies that have used a similar NiTi closed-coil orthodontic spring
on the maxillary teeth in rats (Ren et al., 2003., 2004; Rozman et al., 2010), and the amount
and rate were equivalent to the distal movement of 1.22 to 1.91 mm reported for the
maxillary canine in human studies for 28 days (Daskalogiannakis and McLachlan, 1996;
Iwasaki et al., 2000, 2009; Hayashi et al., 2004). The optimal level of force required to
induce the maximum rate of OTM in the shortest term has eluded the orthodontic
profession. Studies in rats indicate that forces of less than 10 cN may be the ideal force
magnitude in terms of inducing an optimal rate of OTM (Kohno et al., 2002; Ren et al.,
2004; Gonzales et al., 2008). This level of force correlates well with studies on the activity
in the microvasculature of the PDL during OTM (Oppenheim, 1944; Kondo, 1969,
Vandevska-Radunovic et al., 1994; Noda et al. , 2009) that have suggested that a light force
maintains the patency of the blood vessels of the PDL and thereby promotes biological
processes of OTM, while a heavier force causes partial or total occlusion of the blood
vessels that results in degeneration or necrosis of the PDL (Schwarz, 1932; Storey, 1952,
1973; Reitan, 1957, 1964, 1967; Gianelly, 1969; Gaengler and Merte, 1983; for review, see
Meikle, 2006; Wise and King, 2008; Krishnan and Davidovitch, 2009).
A non-linear rate of OTM due to the different phases of OTM has been reported in
humans and rats (Reitan, 1967; Storey, 1973; King et al., 1991; Pilon et al., 1996; van
Leeuwen et al., 1999), with an initial lag phase of OTM that has been explained on the
basis of the time taken to recruit cells in the PDL to eliminate the hyalinized tissue that
develops in the PDL due to orthodontic force (Burstone, 1984; King et al., 1991). A
constant rate of OTM was however documented in the present study in the rat until the end
of day 28. A similar trend of a constant rate of OTM without a lag phase has been reported
in other studies on rats (Kohno et al., 2002; Ren et al., 2004; Gonzales et al., 2008; Rozman
et al., 2010) and humans (Iwasaki et al., 2000, 2009). This constant rate of OTM may be
attributed to a lower magnitude of forces applied in the present study that promoted
physiological OTM without undermining resorption as a distinct separate phase and thereby
circumventing the lag phase of tooth movement described in the literature (Reitan, 1967;
Storey, 1973; Burstone, 1984; King et al., 1991; Pilon et al., 1996; van Leeuwen et al.,
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157
1999). Further, recent studies that have used low levels of orthodontic force have reported
traces of hyalinized tissue not only in the initial but also in the linear phase of experimental
OTM (Kohno et al., 2002; von Bohl et al., 2004a, b; Iino et al., 2007), suggesting that the
development and removal of hyalinized tissue is a continuous process instead of a exclusive
single phase event during OTM (von Bohl and Jagtman, 2009).
Inherent DD of the molar teeth in rats has been documented in relation to the
posterior lengthening of the jaw (Kraw and Enlow, 1967; Vignery and Baron, 1980; King
et al., 1991; Tsuchiya et al., 2013). The amount of DD of the maxillary molars in the
present study progressed at a constant rate and correlated with the levels described in the
studies reported by King et al. (1991) and Ren et al. (2004). It seems reasonable to adjust
the decrease of the I-M distance to calculate the amount of OTM by factoring-in the
inherent DD of the maxillary molars that the orthodontic spring has to overcome to induce
OTM. The requirements of an ideal OTM Animal Model are listed in Table 5-1 (see
below).
5.1.2. A behavioural rat model that measures mechanical and thermal
hypersensitivities associated with OTM
Pain during OTM is a common and a complex experience and is amongst the most
cited negative experiences of orthodontic treatment (Kluemper et al., 2002; Asham, 2004,
Keim, 2004; Hammad et al., 2012). Every year more than 6 million patients seek
orthodontic treatment in the United States and Canada, a number that has grown by 45%
over the past 10 years (AAO bulletin, 2005). Orthodontic treatment was reported painful by
more than 90% of the orthodontic patients and this can negatively influence the desire to
initiate treatment, and almost 30% of the orthodontic patients confessed of considering
ceasing treatment prematurely because of the pain they experienced during treatment
(Oliver and Knapman, 1985; Bergius et al., 2000; Otasevic et al., 2006; Pringle et al.,
2009). The incidence and severity of OTM pain reported are higher when compared with
the pain associated with extraction of teeth, and thus can challenge compliance in the
treatment and the treatment outcome (Jones and Chan, 1992a; Patel, 1992; Sergl et al.,
2000; Fleming et al., 2009).
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158
This study documented that orofacial mechanical and thermal hypersensitivities
occur in a rat OTM-induced pain model. The mechanical and thermal sensitivities at all
ipsilateral and contralateral sites tested were significantly increased in the E group in the
early postoperative period (1 – 5 days) with peaks reached on day 1, and then returned to
preoperative control levels that were maintained until postoperative day 28. However, there
was no significant change in mechanical and thermal sensitivities for the S group from the
preoperative level for all the tested sites for the entire testing period. Further, the
mechanical and thermal hypersensitivity states were also detected in the contralateral tested
sites and thereby suggest that intrasubject experimental designs that use the contralateral
side as the control side for testing pain might result in erroneous comparative conclusions.
It has been suggested that OTM-induced nociceptive impulses are initiated in the
PDL and gradually diminish with every passing day due to the adaptation of PDL neural
elements to environmental changes caused by orthodontic forces (Krishnan and
Davidovitch, 2006). The PDL is abundantly supplied by two kinds of sensory receptors,
Ruffini-like endings that are activated by stretch and sustained pressure, and free nerve
endings that are activated by noxious stimuli (Maeda et al., 1999; Toda et al., 2004;
Trulsson, 2007). One possible explanation for the changes in sensitivity ratings in the early
period of OTM is that the nociceptive endings in the PDL are undergoing peripheral
sensitization that accounts for the hypersensitivity. Further, OTM also leads to alteration of
the dental occlusion. The immediate tooth movement across the width of the PDL may
create a condition of transient occlusal trauma due to hyperocclusion that is of acute nature
and very painful condition (Miles, 2004). This could have lead to masticatory muscle
hyperalgesia induced by occlusal interference (Cao et al., 2009; 2013). A decrease in
pressure pain threshold (PPT) for Ma and anterior temporalis muscles along with a decrease
in the frequency of tooth contacts have been reported during the early stages of OTM
(Michelotti et al., 1999). Further, the time-dependent hypersensitivity changes in the
present study correlated well with time-related pain reported in human studies (Jones, 1984;
Ngan et al., 1989, 1994; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and Dincer,
2004; Polat et al., 2005; Polat, 2007). Hence, these observations in the rat OTM-induced
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pain model suggest that OTM-induced mechanical and thermal hypersensitivities may be
useful measure of OTM-induced pain.
5.1.3. Neuroplasticity in the face-M1 and face-S1 associated with OTM
This study documented neuroplastic changes induced in the sensorimotor cortex by
OTM. OTM resulted in significant changes in the motor representations of the jaw-opening
(LAD, RAD) and tongue protrusive (GG) muscles in the ICMS-defined face-M1 at days 1,
7, and 28 of continuous orthodontic force application. Analogous neuroplastic changes
were also revealed in the ICMS-defined motor representations within face-S1. These
neuroplastic changes in the face-M1 and face-S1 may reflect adaptive sensorimotor
changes in response to the altered environment in the oral cavity induced by OTM.
5.1.3.1 Features of jaw and tongue motor representations in the face-M1and face-S1
Some of the characteristic features of face-M1 found in other studies were also
documented in this study (Donoghue and Wise, 1982; Neafsey et al., 1986; Satoh et al.,
2006, Adachi et al., 2007, Avivi-Arber et al., 2010a, b). This included sites from which
LAD, RAD and GG could be activated individually with significant contralateral face-M1
predominance for RAD and LAD, and overlapping muscle representations in face-M1 of
LAD, RAD, and GG. It has been proposed that such overlapping of motor representations
in face-M1, along with the extensive bilateral representations of LAD, RAD and GG
muscles, may form an essential substrate for the dynamic coordination of bilateral orofacial
movements involving the complex synergistic activities of these muscles, and for face-M1
neuroplastic mechanisms manifested as reorganization of motor representations within
face-M1 (for review, see Sanes and Donoghue, 2000; Sanes and Schieber, 2001; Avivi-
Arber et al., 2011).
The study also revealed that ICMS applied to the face-S1 evoked LAD, RAD and
GG EMG activity. These findings are consistent with previous findings in rats (Avivi-Arber
et al., 2010a) and support the view that face-S1 as well as face-M1 may play a role in the
generation and control of orofacial movements (Murray et al., 2001; Yao et al., 2002). The
observed ICMS-evoked EMG activities could conceivably have been the result of spread of
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ICMS currents from face-S1 to face-M1 either directly (Cheney, 2002) or indirectly
through axon collaterals (Henry and Catania, 2006; Chakrabarti and Alloway, 2006;
Iyengar et al., 2007). However, this is unlikely for several reasons. Firstly, as the distance
between face-M1 and many of the positive ICMS sites within the face-S1 was much larger
than the estimated extent of ICMS current spread of less than 0.5mm at 60 μA ICMS
intensity (Asanuma, 1989; Cheney, 2002; Schieber, 2001). Secondly, consistent with
Avivi-Arber et al. (2010a), many of the positive ICMS sites within the face-S1 had short
onset latencies (8-12 ms), comparable to those of face-M1, thus suggesting relatively direct
projections to brainstem motoneurons rather than projections via face-M1. Thirdly, efferent
projections from rat face-S1 to motoneurons have been documented (Rathelot and Strick,
2006; Chang et al., 2009; Yoshida et al., 2009; Tomita et al., 2012; Haque et al., 2012).
Although ICMS within the mapped area evoked LAD, RAD and GG EMG activity,
jaw-closing (Ma) and cheek muscle (Bu) EMG activity was usually not observed. The very
few sites from which Ma EMG activity could be initiated is consistent with face-M1 studies
in rats and monkeys that document minimal jaw-closing motor representation (Neafsey et
al., 1986; Huang et al., 1988; Murray et al. 2001; Avivi-Arber et al., 2010a). Therefore, this
suggests that face-M1 and face-S1 play an important role in the generation of some but not
all orofacial movements. However, since cold block inactivation of face- M1 results in a
significantly increased spontaneous EMG activity of the Ma muscles in monkeys it has
been suggested that while ICMS of face-M1 facilitates the spontaneous and reflex-induced
activity of the motoneurons supplying the RAD and LAD, it may inhibit the activity of the
motoneurons supplying the Ma muscle (Yamamura et al., 2002). Thus, it seems plausible
that face-M1 neurons may have a predominantly inhibitory effect on Ma motoneurons, and
also on Bu motoneurons, or the Ma and Bu motor representations are masked by an
inhibitory effect of intracortical interneurons such as documented in limb-M1 (Chase et al.,
1973; Ethier et al., 2007).
5.1.3.2. Effects of OTM on face-M1 and face-S1
M1 and S1 neuroplasticity has been associated with motor function recovery
following central or peripheral injury, changes in muscle use or disuse, learning of novel
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motor skills, and adaptive processes (Kaas, 2007; Martin, 2009; Avivi-Arber et al., 2011,
Kleim, 2011; Nudo, 2011). Earlier studies have shown that alterations in somatosensory
inputs induced by deafferentation (Franchi, 2001; Yildiz et al., 2004; Halkjaer et al., 2006;
Adachi et al., 2007) or sustained somatosensory stimulation (Hamdy et al., 1998; Adachi et
al., 2008) may result in neuroplastic changes within face-M1 (Toldi et al., 1996; Farkas et
al., 2000; Sanes and Donoghue, 2000; Adachi et al., 2008, Avivi-Arber et al., 2010a) and
face-S1 (for review, see Feldman and Brecht, 2005; Petersen, 2007; Fox, 2009; Barnes and
Finnerty, 2010), as reflected in an altered cortical excitability or motor representations.
However, until now no study had addressed the question if neuroplastic changes occur in
face-M1 and face-S1 during OTM. Hence a hypothesis tested in the present project was
related to whether neuroplastic changes occur in the ICMS-defined motor representations
of GG, AD, Ma, and Bu muscles within the rat’s face-M1 and face-S1 during OTM. The
present study found that OTM resulted in significant changes in motor representations of
the LAD, RAD, and GG muscles in the ICMS-defined face-M1 and face-S1 at time points
of days 1, 7, and 28 of continuous orthodontic force application. This was reflected in a
change in the number of positive ICMS AD and GG sites and in the number of positive
ICMS sites in face-M1 from which ICMS could simultaneously activate muscle activity in
different combinations of LAD, RAD, and GG. Further, there was no significant difference
in the onset latency for any of the muscles across the groups, and between face-M1 and
face S1 within each group. Also, the extent of the face-M1 and face-S1 territory for LAD,
RAD, and GG representations during OTM revealed significant changes in the number of
positive penetrations in the anteroposterior (AP) and the mediolateral (ML) planes and
suggested a trend in the shift of the centre of gravity (CoG).These findings provide the
documentation that OTM can be associated with neuroplastic changes within the face-M1
and that face-S1 motor outputs also have the capacity to undergo neuroplastic changes
following OTM. The findings of the present study are consistent with the findings of other
neuroplasticity studies of the sensorimotor cortex in our laboratory following manipulation
of the orofacial region (Adachi et al., 2007; Sessle et al., 2007; Avivi-Arber et al., 2010a;
Veeraiyan et al., 2011). These findings are consistent with studies in humans showing face-
M1 neuroplasticity in association with motor skill acquisition, chewing and swallowing
patterns, and peripheral manipulations including dental implants and occlusal splints
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(Tamura et al., 2002, 2003; Shinagawa et al., 2003; Svensson et al., 2003b, 2006; Boudreau
et al., 2007; Kordass et al., 2007; Yan et al., 2008; Baad-Hansen et al., 2009; Byrd et al.,
2009; Martin, 2009; Shibusawa et al., 2009, 2010; Arima et al., 2011, Trulsson et al.,
2012).
5.2. Possible mechanisms of OTM-induced neuroplasticity in the
sensorimotor cortex
Mechanisms underlying face-M1 and face-S1 neuroplasticity following
manipulations in the orofacial region are not well understood. Understanding the
anatomical substrates and physiological mechanisms that underlie the neuroplastic changes
in the sensorimotor cortex could inform new or improved approaches to target the
sensorimotor cortical neuroplasticity for desired orthodontic outcomes.
OTM is associated with altered PMR responses, changes in occlusal contacts and
pain and may thus result in altered afferent inputs to face-M1 and face-S1. The orofacial
hypersensitivity at day 1 of OTM was associated with a decrease in the number of positive
ICMS sites for AD and GG motor representations in the face-M1 and face-S1, and the
return of sensitivity to preoperative control levels by day 7 was associated with the increase
in the number of positive ICMS sites of the motor representations. However, the decrease
in the number of ICMS sites observed at day 28 likely cannot be attributed to the
maintenance of nociceptive afferent inputs related to OTM-induced pain since mechanical
and thermal sensitivities had returned to preoperative levels well before day 28. Since
alteration of the occlusion at day 28 was documented by our radiographic measurements, it
seems logical to assume that the neuroplastic change documented by day 28 may be more a
result of the occlusal change, although OTM-related intraoral nociceptive inputs from the
molars cannot be ruled out as it was not possible in the present project to test for the
presence of intraoral nociceptive inputs from the molars in awake rats. One of the
mechanisms suggested for M1 neuroplasticity is that novel or repeated peripheral inputs
activate a set of neural signaling pathways which induce gene transcription within the M1
and S1 to translate proteins to orchestrate changes in synaptic strength. The change in
synaptic strength then results in a reorganization of cortical microcircuitry that is
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manifested as a redistribution of ICMS-evoked motor representations (Hess and Donoghue,
1994; Kleim et al., 1996, 1998, 2003a, 2004; Nudo et al., 1996; Monfils et al., 2005; Nudo,
2011). Also, changes in sensory inputs have been suggested to alter the balance between
sensory inputs and motor outputs and lead to LTP or LTD of synaptic efficacy
(Buonomano and Merzenich, 1998; Rioult-Pedotti and Donoghue, 2003; Nudo, 2008).
Consequently, ICMS can either excite neighbouring neurons directly or indirectly through
horizontal interconnections that previously were non-responsive, thereby accounting for
alterations in the ICMS-defined motor representations (Jacobs and Donoghue, 1991;
Schieber, 2001; Monfils et al., 2005).
Further, changes in sensory inputs to the CNS, such as those associated with OTM
may result from altered oral motor behaviour. Face-M1 and face-S1 receive somatosensory
inputs from oral tissues involved in orofacial movements (for review, see Murray et al.,
2001; Sessle, 2011a). Adapting to an altered pattern of mastication during OTM may
require repetition of the novel motor movements which may be analogous to learning a
novel motor skill (Adams, 1987; Sessle, 2011b). Motor maps reflect a level of synaptic
connectivity within the sensorimotor cortex that is required for the performance of skilled
movement. Thus, repeated jaw or tongue movements may result in sustained
somatosensory inputs to face-S1 and face-M1 that may induce enhanced synaptic efficacy
(Asanuma, 1989; Monfils et al., 2005; Nudo et al., 2008). Enhanced synaptic efficacy can
facilitate the ability for ICMS of face-M1 and face-S1 areas to evoke movements that
previously could not be evoked by a similar stimulus to these areas, and thereby for
example lead to an increased motor representation. Similarly, a decreased motor
representation may reflect decreased synaptic efficacy associated with decreased
somatosensory inputs and decreased motor function (Monfils et al., 2005; Nudo, 2011).
The change of the motor map is thought to be the result of disruption of cortical circuitry,
as there is a loss of synapses but not neurons within these regions. Motor rehabilitation
restores skilled movement, reinstates motor maps, and increases synapse number (Nudo et
al., 1996; Frost et al., 2003; Kleim et al., 2003b). These mechanisms could be involved in
the present project’s documented decrease of RAD, LAD, and GG representations within
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face-M1 and face-S1 at day 1 and day 28 and an increase of these muscle representations at
day 7 during OTM.
Sensorimotor neuroplasticity is thought to arise during learning or skill acquisition
and is not simply use-dependent. Thus, if there is no component of skill learning involved,
motor representations do not increase in size and this state of sensorimotor neuroplasticity
suggests a consolidation of motor skill (Plautz et al., 2000; Remple et al., 2001; Kleim et
al., 2004; Monfils et al., 2005; Nudo, 2008). Further, current research suggests both
implicit and explicit learning stages in motor learning in humans employ different neural
structures (Ashe et al., 2006; Nudo, 2008; Orban et al., 2010, 2011; Steenbergen et al., 2010).
Much of motor sequence learning is implicit, as the different elements of the movements
are made in sequential order without specific awareness about the sequence, and the
learning sequence originates in M1 and then propagates to premotor areas. However, with
repeated practice, learning may become explicit, as the subject is aware of the instructional
set (Nudo, 2008). Studies have reported modification of M1 representations during both the
implicit and explicit phases of the task, e.g. TMS-induced M1 representations of finger or
tongue muscles increase along with decreased reaction times during the implicit learning
phase (Pascual-Leone et al., 1994, 1995; Zhuang et al., 1997; Boudreau et al., 2007, 2010).
Further, some of these studies reported that the expansion of M1 representations began to
retract just before explicit knowledge occurred and continued to retract below that of
baseline levels with continued learning. It has also been suggested that specific neural
signals may also be required on a continued basis for not only motor map reorganization
but also for map maintenance (Kleim et al., 2003a). Therefore, it is possible that these
mechanisms could explain the changes observed in the present study at day 28 of OTM, as
temporally the jaw and tongue motor learning sequence due to changes in occlusal contacts
may have translated into the explicit phase of learning. Future studies are needed to address
this possibility (see section 5.5.2).
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5.3. Are OTM-induced neuroplastic changes adaptive or
maladaptive in nature?
Neuroplasticity can be adaptive when associated with a gain in function (Cohen et
al., 1997) or maladaptive when associated with negative consequences, such as function
loss or injury potentiation (Nudo, 2006). In the present study, the significant decrease in the
number of positive sites at day 1 could conceivably be attributed to the nociceptive inputs
induced by OTM that alter synaptic connectivity in the face sensorimotor cortex and also at
different levels of the ascending nociceptive pathways and thereby modify sensorimotor
functions. This could also be linked to conscious reactions by the animal to adapt its
orofacial sensorimotor functions in view of its altered intraoral state. These possibilities are
consistent with clinical findings of decreased chewing force, increased number of chewing
cycles to form a bolus, decreased activity of masseter and temporalis, and decreased pain
pressure threshold of the masseter in the early period of OTM (Goldreich et al., 1994;
Miyamoto et al., 1996; Miyawaki and Takada, 1997; Ferrario et al., 1999a, 1999b, 2002;
Michelotti et al., 1999). Therefore, neuroplasticity reported at day 1 could be an adaptive
protective change in response to the initial hyperalgesic state induced by OTM. On the
other hand, at day 7 of OTM, the number of sites in the face sensorimotor cortex
significantly increased. Based on the concept of use-dependent cortical neuroplasticity
(Nudo et al., 1992; 1996a, b; Nudo, 2003; Monfils and Teskey, 2004; Cramer et al., 2011),
it is possible that neuroplastic changes evident at day 7 were related to the animal’s
acquiring novel sensorimotor behaviours to adapt to the changes in the dental occlusion
induced by OTM. Thus, based on the nature of the adaptation of the jaw and tongue
muscles to accommodate the changes induced in the oral cavity by OTM, the neuroplastic
changes in the sensorimotor cortex could be considered as adaptive during days 1 and 7.
Further, as noted above, the significant decrease in the number of positive sites documented
at day 28 was unlikely related to sustained nociceptive inputs. Rather the OTM-induced
decrease could reflect the consolidation of the animal’s acquisition of the new sensorimotor
behaviours, along the lines of the stages in motor learning reported in the literature
(Pascual-Leone et al., 1994, 1995; Kleim et al., 2003a; Ashe et al., 2006; Nudo, 2008).
Although OTM is reported to cause a transient impairment of the number, functional
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properties and distribution of the PMRs (Loescher et al., 1993; Okazaki, 1994; Long et al.,
1996; Ogawa et al., 2002), once the teeth are in their desired position and the tissues have
healed, the response of the PMRs is reported to improve and return to normal levels
(Nakanishi et al., 2004). But whether this return of PMR normal features is associated with
a regaining of normal orofacial sensorimotor behaviours or instead with sustained
modifications of these behaviours in association with the neuroplastic changes in the
sensorimotor cortex at day 28 is unknown. Therefore, as noted below, future behavioural
studies in association with investigations of cortical neuroplasticity are indicated.
5.4. Targeting neuroplasticity mechanisms in orthodontics
The orofacial region may be considered as a region involved in sensorimotor
learning where repetitive stimulation of the sensory receptors of the skin, oral mucosa, and
neighbouring structures results in a patterned neural input and processing of information in
the cerebral cortex and other brain regions. This results in sensory perception, learning, and
memory of the experience, and thereby regulates motor control of muscles of the orofacial
region (Trulsson and Essix, 2004; Turker et al., 2007; Sessle, 2011b). To understand the
mechanisms of CNS function and its link to developmental pathology, it would help to
understand the nature of the environmental stimulation and the input that the stimulus
imparts to the CNS.
The integration of orofacial sensorimotor functions and behaviours depends on the
development of neural circuits connecting the sensory receptors through the CNS to
muscles. On the other hand, the motor output also has an influence on the sensory input,
i.e., the movements of the orofacial muscles stimulate the sensory receptor inputs that then
may be used to guide oral function (Huang et al., 1989b; Lin and Sessle, 1994; Martin et
al., 1997, 1999; Lin et al., 1998; Hiraba and Sato, 2005a, b). Thus, the receptors of the skin
of the orofacial region, PDL, and the tongue are functionally integrated with muscles of the
jaw, facial expression and tongue. These sensory and motor orofacial functions may also
contribute to changes in growth and development of the craniofacial region, as noted
below.
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5.4.1. The classic concepts of growth and development of the face vis-à-vis
new insights in neuroscience
The traditional orthodontic viewpoint of growth and development of the face has
been that the sensory and motor functions of the orofacial region are under genetic control
and this encoding then serves as a template leading to the development of the craniofacial
region (Moyers, 1988; Linder-Aronson and Woodside, 2000). However, the current
concepts of neuroplasticity, including the evidence that sensory experiences may modify
the structure and function of the brain and thereby influence the sensorimotor function of
the orofacial region, make it relevant to reconsider the traditional concepts of growth and
development of the face.
5.4.1.1. Functional matrix theory
Moss theorized that the major determinant of the growth of the maxilla and
mandible is the enlargement of the nasal and oral cavities, which grow in response to
functional needs (Moss and Salentijn, 1968; Moss, 1997a, b, c, d). However, the theory
does not clarify the mechanisms by which the functional needs are translated into the
growth of the tissues around the mouth and nose - could sensorimotor cortical
neuroplasticity mechanisms be the missing link?
The receptors of the PDL, alveolar bone, muscles, tendons, and the TMJs are
involved in the mechanosensory process (see chapter 1). Further, it has been documented
that bone cells (except osteoclasts) are linked by gap junctions to form an osseous
connected cellular network (CCN) that permits the intercellular transmission of ions and
small molecules and permits electrical transmission in bone cells (Moss, 1997a, b). Skeletal
muscle contraction is a typical mechanotransduction process causing deformation of bone
(Atchley, 1993; Reddi, 1994; Moss, 1997a; Ozcivici et al., 2010), and can initiate
membrane action potentials in the entire osseous CCN and osteocytes in the periosteum
(van't Veen et al., 1995). According to the functional matrix theory, the muscles, glands,
neurovascular bundles and teeth are termed the periosteal matrices that are involved in
epigenetic regulation of bone morphogenesis (Moss, 1969). The functional matrix concept
of growth and development of the orofacial region suggests that orofacial postnatal growth
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and development is accomplished by adaptive modifications of orofacial sensorimotor
functions that influence primarily the growth and attachment of the musculature, and
secondarily the skeletal structures of the orofacial region (Moss, 1997d). Thus, an extension
of this concept is that orofacial sensory and motor activity may lead to neuroplastic changes
in the cerebral cortex and may indirectly influence bone remodelling dynamics through the
motor output to the muscles of the jaw, face, and tongue.
5.4.1.2. Petrovic’s servosystem theory
The concept that the PMRs of the teeth and the TMJs are involved in the guidance
of jaw movement to occlude in maximum intercuspal position could be consistent with the
cybernetic model proposed by Petrovic and Stutzmann (1977) describing the physiological
influence of the occlusal relationship of the teeth on growth of the maxilla and mandible.
This servosystem theory of craniofacial growth states that anterior growth of the maxilla
creates a slight occlusal deviation between the maxillary and mandibular dentitions.
Perception of this occlusal deviation by PMRs and TMJ triggers the lateral pterygoid
muscle to become more active tonically to position the mandible anteriorly. This increased
muscle activity along with the protrusion of the mandible, and in the presence of
appropriate hormonal factors, stimulate growth at the mandibular condyle (Carlson, 2005)
(Fig. 5-1). Clinical investigations have suggested that any deviation of the normal occlusal
relationship results in disruption of the physiological growth association of the maxilla and
the mandible leading to an abnormal relationship of the jaws (Lavergne and Petrovic,
1983). Conflicting evidence exists however whether the lateral pterygoid muscle activity is
involved in mandibular growth (McNamara, 1973; Sessle et al., 1990). Thus, orofacial
sensory and motor activity that may lead to neuroplastic changes in the sensorimotor cortex
reported in the thesis could be the link in the CNS suggested by the servosystem theory to
influence the growth of the maxilla and the mandible.
5.4.2. Equilibrium theory revisited yet again
Factors that could influence the equilibrium of tooth position include the light but
long-lasting pressures from the intrinsic factors such as the tongue, lips, cheeks at rest, and
extrinsic factors such as behaviours (e.g. thumb sucking) and orthodontic appliance forces.
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In addition, significant equilibrium effects can be expected from occlusal forces, the
elasticity of gingival fibres and from the metabolic activity within the PDL. The major
equilibrium influences reported on the jaws are functional processes affecting the positional
changes, e.g. respiratory needs influence head, jaw and tongue posture (Proffit, 1978).
These concepts maintain their scientific validity to this day.
Fast adapting mechanoreceptors in the tongue tip respond to tongue movements
only if the receptive field of the sensory afferents at the tip of the tongue is brought in
contact with other intraoral structures and objects, such as the lower incisor teeth (Trulsson
and Essick, 1997; Trulsson and Johansson, 2002). This highlights the concept of
multisensory integration of function that associates various sensory modalities evoked by
the different structures in the same regional source (Maravita et al., 2003), e.g. in the
context of the orofacial region, this concept would suggest that the ability of the
sensorimotor cortex to discriminate the position of the tongue is increased in the presence
of its reference to the anterior teeth. Therefore, the position of lower incisor teeth may
influence the anterior boundary of the postural position of the tongue (Tolu et al., 1993,
1994a, b; Yagi et al., 2008). Taken together, the tongue posture is therefore secondarily
dependent on the normal arrangement of the teeth in the dental arch for multisensory
experience inputs and the activation of PMRs through the activity of the tongue muscles.
Since very similar neurophysiological properties have been reported for Ruffini-like PMRs
and the Ruffini mechanoreceptors of the perioral skin, namely slow adaptation, presence of
spontaneous activity, a hyperbolic stimulus-response relationship to maintained loads, and
directional sensitivity to loads (Trulsson and Johansson, 2002), the action potentials of their
afferents provide similar discriminative information of their spatial and temporal elements
to the sensorimotor cortex. These similarities in the nature of the inputs may generate motor
outputs to muscles associated with these receptors, namely the Ruffini-like ending PMRs
that are activated by the tongue muscles (Tolu et al., 1993, 1994a, b; Yagi et al., 2008) and
the Ruffini stretch receptors of perioral skin that are activated by deformation of the skin
through contraction of the muscles of facial expression (Trulsson and Essick, 2004). Thus,
it may be hypothesized that the tongue, jaw and perioral muscles of facial expression may
interact actively to maintain equilibrium of the position of the teeth through sensorimotor
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cortical control of their respective receptors regulating the tension and the proprioceptive
function of their respective muscles. The property of sensorimotor cortical neuroplasticity
may be the substrate that permits the modification of the tissue force balance on the teeth
and jaws to accommodate changes in teeth and jaw positions during OTM.
5.4.3. Retention mechanisms and other orthodontic implications
It is a common trend to utilize retention appliances after an active phase of
orthodontic treatment to maintain the teeth in their corrected positions and to prevent their
relapse to their original state of occlusion. One of the reasons cited for relapse of
orthodontic treatment is the positioning of the teeth in an unstable position after treatment
so that the enveloping soft tissues are constantly applying pressures on the teeth and the
supporting dentoalveolar bone resulting in a relapse of the treatment outcome (Blake and
Bibby, 1998; Littlewood et al., 2006; Bondemark et al., 2007). Thus, if teeth are positioned
in the unstable zone of the soft-tissue equilibrium, a gradual withdrawal of the retention
appliances after treatment would not suffice to maintain the treatment result on a long-term
basis. However, in clinical practice a good level of stability of the achieved orthodontic
result is reported for upper intercanine and upper and lower intermolar widths, except
mandibular intercanine width (Glenn et al. 1987; Moussa et al. 1995; Blake and Bibby,
1998). This result may be attributed to the fact that the inputs generated by PMRs during
function of the anterior and the posterior teeth that have directional sensitivity (Trulsson,
2007; Svensson and Trulsson, 2009) may indirectly influence perioral, jaw and tongue
muscle behaviour through neuroplastic changes of the sensorimotor cortex and thus modify
its output to brainstem motoneurons supplying these muscles and thereby harmonize the
soft tissue function to the corrected tooth position.
Understanding the specific behavioural and neural signals that drive sensorimotor
cortex neuroplasticity will help guide the development of novel orthodontic interventions
for desired therapeutic outcomes. The significance of neuroplasticity for rehabilitation is
that it provides a mechanistic basis for understanding therapeutic interventions. Thus, it
may be possible to develop more effective treatment protocols if the effects of such
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interventions on neuroplasticity in the sensorimotor cortex, and the underlying
mechanisms, can be elucidated.
The implications of the recent findings in the physiology of the neural systems are
profound. Since the physiology of the neuron may not be regulated by its genes
exclusively, but operates in harmony with environmental factors and experience, the
growth and functioning of the nervous system that forms the substrate of the oral
sensorimotor functions can be considered as an epigenetic mechanism that responds to
environmental experiences. Comprehensive orthodontic management should include
analysis of the orofacial sensorimotor system and evaluation of its
physiological/pathological effects. Orthodontic biomechanical therapy by using motor
training/habit breaking appliances may influence oral neuromuscular behaviour in a more
conducive way and these adjunctive treatment modalities may significantly enhance
achievement of the orthodontic treatment goals.
5.5. Study limitations and future directions
5.5.1. Validity of the use of ICMS technique to study sensorimotor
cortical changes
The ICMS technique was the approach used in this thesis project to document that
OTM induces sensorimotor cortical neuroplasticity. This technique is considered to be
appropriate and precise for mapping the functional properties of motor outputs within the
sensorimotor cortex and reveals the extent of motor representations through the
measurement of the ICMS-evoked EMG responses in the target muscles (for reviews, see
Asanuma, 1989; Cheney, 2002). However, the overall extent of motor representations can
be influenced by the cortical depth of ICMS application, any previous ICMS stimulation,
depth of anaesthesia, muscle posture, as well as individual variations of the subjects
(Asanuma, 1989; Donoghue and Wise, 1982; Sessle and Wiesendanger, 1982; Nudo et al.,
1992; Schieber, 2001; Cheney, 2002; Tehovnik et al., 2006; Tandon et al., 2008).
Therefore, precautions were taken in the present study to control for the possible sources of
variability by ensuring comparable anaesthetic, experimental conditions, procedural
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protocols, and stimulation parameters were applied to all the study groups. Further, the
animals were maintained at a similar narrow range of anaesthetic state. The general features
of the motor maps in control animals obtained in the present study were comparable to
those documented in other studies (Donoghue and Wise, 1982; Neafsey et al., 1986; Huang
et al., 1989b; Adachi et al., 2007; Tandon et al., 2008; Avivi-Arber et al., 2010a).
The ICMS technique also has some limitations in the reliability and precision to
delineate functional boundaries for analyzing motor representations within the mapping
area (Huntley and Jones, 1991; Nudo et al., 1992). It has been demonstrated that at 30 µA
threshold ICMS intensities, the motor responses can be evoked within ~100 μm of the
microelectrode position in M1 (Asanuma, 1989); however, the horizontal spatial resolution
in the present study was 500 μm, i.e. inter-microelectrode distances. Therefore, the
mapping procedure used in the present study may have been sensitive to only large changes
in motor representations and could have missed smaller changes. The study also used
suprathreshold ICMS intensity of 60 μA that may have activated additional distant
pyramidal neurons through direct current spread or indirectly through activation of axon
collaterals thereby possibly resulting in overestimated motor representations that could
have masked changes that could have been revealed by mapping at threshold ICMS
intensities (Sessle and Wiesendanger, 1982; for reviews, see Asanuma, 1989; Schieber,
2001; Cheney, 2002; Tehovnik et al., 2006). Also, the ICMS technique is an artificial
means of activating S1 as well as M1 outputs and may not reflect exactly the mechanisms
that are at work during natural sensorimotor function, where extensive facilitatory and
inhibitory interactions are at play. Other emerging techniques, such as optical imaging,
voltage-sensitive dye imaging and optogenetic stimulation (Petersen, 2007; Han et al.,
2009; Feldmeyer et al., 2013), could be used in animals and methods such as ƒMRI applied
in humans to test for neuroplastic changes in motor representations in the face sensorimotor
cortex.
Chapter 5
173
5.5.2. Time course and mechanisms involved in OTM-induced
sensorimotor cortical neuroplasticity
Since only 3 time points were assessed in the present study, OTM-induced
neuroplasticity could be studied at various extended time points to gain insights into the
time course of the OTM effects on the sensorimotor cortex. The time points between day 7
and 28 would be of special interest to determine the exact time point at which the
neuroplastic change reflected in a decrease in the motor representations begins, and if it is
linked to any sensorimotor changes in the animals behaviour that is simultaneously
monitored. Also, it would be of interest to study time points beyond the day 28 of OTM to
provide details into whether the shift in neuroplasticity pattern of the sensorimotor cortex is
maintained or motor representations return to normal levels to attain a state of homeostasis
akin to which has been reported in the neuroplasticity literature (Turrigiano and Nelson,
2004; Turrigiano, 2008; Feldman, 2009). The mechanisms underlying the neuroplasticity
should also be studied in the future by employing electrophysiological and
immunohistochemical techniques to determine, for example, the cellular processes and
chemical mediators involved, the role of sensory inputs and their source, other CNS regions
involved in relaying these sensory influences to the sensorimotor cortex, and the relative
role of face-M1 versus face-S1 in behavioural changes that may be associated with the
sensorimotor cortical neuroplasticity (e.g., by monitoring EMG, tongue and jaw
movements in awake rats).
5.5.3. Sex differences in sensorimotor cortex neuroplasticity, mechanical
and thermal hypersensitivity states, and amount and rate of OTM
It was beyond the scope of the present studies to investigate possible sex-related
differences in sensorimotor cortex neuroplasticity, mechanical and thermal hypersensitivity
states, and the amount and rate of OTM, but these need to be considered in future studies of
OTM-induced neuroplasticity for the following reasons. Estrogen receptors have been
reported in muscle tissue (Dahlberg, 1982; Meyer and Raap, 1985), TMJ (Aufdemorte et
al., 1986; Milam et al., 1987), and dorsal root ganglion neurons (Sohrabji et al., 1994),
suggesting that deep tissues as well as peripheral ganglia are potential targets for sex
Chapter 5
174
steroid modulation of sensory functions. Further, the presence of trigeminal subnucleus
caudalis (Vc) estrogen receptors has been documented (Amandusson et al., 1996; Bereiter
et al., 2005), and actions of estrogen on central trigeminal nociceptive processing has been
reported (Tashiro et al., 2009a, b). In acute pain assays female rats and mice have been
found to be more sensitive to nociceptive stimulation than males (Cairns et al., 2001; Mogil
and Chanda, 2005, Mogil, 2009), although in human studies, conflicting results have been
reported in relation to gender differences in OTM-induced pain perception (Scheurer et al.,
1996; Fernandes et al., 1998; Erdinc and Dincer, 2004; Alomari and Alhaija, 2012).
Further, since the rate of OTM strongly depends on the activity of osteoclasts that resorb
bone (Igarashi et al., 1994; Wise and King, 2008; Krishnan and Davidovitch, 2009), cyclic
changes in serum estrogen may be associated with the estrous-cycle-dependent variation in
tooth movement through its effects on bone resorption (Haruyama et al., 2002).
5.5.4. Translation of experimental animal models to human OTM
sensorimotor neuroplasticity and pain states
The findings in the present rat OTM model imply that peripheral orofacial
manipulation contributes to sensorimotor changes and orofacial pain states. However, the
direct translation of all results of experimental animal models to human neuroplasticity and
pain states is somewhat limited by genetic and species differences. For example, genetics
influence responses to stress, acute and chronic pain, susceptibility to inflammatory
conditions (Cizza and Sternberg 1994; Vendruscolo et al., 2006), and the effects of
analgesic drugs such as morphine and the response of rodents to nociceptive tests (Elmer et
al. 1998; Mogil, 2005, 2012; Seltzer and Dorfman. 2004; Sorge et al., 2012). And while the
organization of the sensorimotor system of the rat is generally analogous to that of other
animals including humans (Paxinos 2004; Kaas, 2008; Nudo , 2008; Hedegaard et al.,
2012), there are some differences for higher-order CNS structures, e.g., representation in
the M1 of the face and vibrissae in the rat occupies a disproportionately larger area than in
many higher mammals that have larger representations of the digits (Welker, 1971; Nudo et
al., 1992; Brett-Green et al., 2001; Monfils et al., 2005; Frostig, 2006), and unlike
carnivores and primates, no interneurons or GABAergic cells are present in the VPM of
Chapter 5
175
rats (Barbaresi et al., 1986; Williams and Faull, 1987; Amadeo et al., 2001). Nonetheless,
as noted above, the OTM-induced orofacial hypersensitivity and face sensorimotor cortical
neuroplasticity are consistent with previous findings of OTM-induced clinical pain (Jones,
1984; Ngan et al., 1989, 1994; Scheurer et al., 1996; Firestone et al., 1999; Erdinc and
Dincer, 2004; Polat et al., 2005; Polat, 2007) and of face sensorimotor cortex
neuroplasticity (Tamura et al., 2002, 2003; Shinagawa et al., 2003; Svensson et al., 2003b,
2006; Boudreau et al., 2007; Kordass et al., 2007; Yan et al., 2008; Baad-Hansen et al.,
2009; Byrd et al., 2009; Martin, 2009; Shibusawa et al., 2009, 2010; Arima et al., 2011,
Trulsson et al., 2012). However, orofacial mechanical and thermal sensitivities as measured
in the present study reflect only indirect measures of evoked nociceptive behaviour and
further studies are needed to test for other types of nociceptive behaviour, e.g., spontaneous
or ongoing pain or dental pain that may have relevance to OTM-related pain in humans.
Although the amount and rate of OTM documented in the present study was similar
to those reported in humans (Daskalogiannakis and McLachlan, 1996; Iwasaki et al., 2000,
2009; Hayashi et al., 2004), the teeth were moved into a state of malocclusion from normal
occlusion, whereas in clinical practice the goal of the orthodontic treatment is to resolve an
existing dental malocclusion. Hence methods such as gene manipulation or jaw growth
modification need to be explored that could first create a state of malocclusion in the rat
and then an active orthodontic spring could be placed to treat that malocclusion to simulate
a clinical scenario.
Sensorimotor cortical neuroplasticity also has clinical significance in humans since
it has been suggested that peripheral interventions can lead to either adaptive M1
neuroplasticity, e.g. motor recovery after stroke, or maladaptive sensorimotor cortical
neuroplasticity, e.g. chronic pain following peripheral tissue injury (Cohen et al., 1997;
Nudo, 2006). Therefore, as noted above, OTM studies in humans to map motor
representations that use TMS, fMRI, etc. and to test approaches aimed at modifying face
sensorimotor cortex neuroplasticity would provide further important information about
sensorimotor cortex neuroplasticity and its clinical relevance.
Chapter 5
176
Fig. 5-1. Petrovic’s servosystem theory on maxillomandibular growth that suggests that CNS mechanisms
play a critical role in the regulation the growth of the mandible. Reproduced from Lavergne and Petrovic,
1983; with permission.
Chapter 5
177
1) Animal occlusion
i. Normal occlusion*
ii. Malocclusion
2) Force application
i. Magnitude*
ii. Intermittent or constant (constant*)
iii. Direction maintained parallel to the occlusal plane*
3) Tooth movement
i. Amount*
ii. Rate*
iii. Accurate and Sensitive measurement method*
iv. Ease of measurement method*
v. Repeatability of the measurement method*
4) Cellular changes in the PDL
5) Types of sensory inputs induced by OTM
i. Mechanosensory
ii. Nociceptive
6) Nervous system changes induced by OTM
i. Changes in sensorimotor cortex*
ii. Changes in other CNS regions
7) Behavioural changes induced by OTM
i. Sensory
a. Mechanical sensitivity*
b. Thermal sensitivity*
c. Spontaneous nociceptive behaviour
ii. Motor
a. Masticatory
b. Other motor behaviours
iii. Other (e.g. emotional, motivational, cognitive)
Table 5-1. Requirements of an ideal OTM Animal Model. * Parameters investigated in the present study.
178
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APPENDIX
Appendix 1. Data from individual animals
Expt. 1
Tooth movement
RAT DATE RT DIF LT DIF(DD) TM
E34 1 day -0.04
4.00E-03 -0.044
E35 1 day -0.01
2.00E-03 -0.012
E36 1 day -0.06
8.00E-03 -0.068
E37 1 day -0.03
9.00E-03 -0.039
E38 1 day -0.01
8.00E-03 -0.018
E39 1 day -0.05
9.00E-03 -0.059
E310 1 day -0.07
6.00E-03 -0.076
E311 1 day -0.01
2.00E-03 -0.02
RAT DATE RT DIF
LT DIF(DD) TM
E34 1 wk -0.25 0.03 -0.28 E35 1 wk -0.28 0.01 -0.29 E36 1 wk -0.29 0.13 -0.42 E37 1 wk -0.27 0.11 -0.38 E38 1 wk -0.37 0.06 -0.43 E39 1 wk -0.37 0.02 -0.39 E310 1 wk -0.45 0.09 -0.54 E311 1 wk -0.25 0.03 -0.28
RAT DATE RT DIF
LT DIF(DD) TM
E34 2 wks -0.62 0.05 -0.67 E35 2 wks -0.32 0.04 -0.36 E36 2 wks -1.39 0.19 -1.58 E37 2 wks -0.82 0.18 -1 E38 2 wks -1.46 0.12 -1.58
241
E39 2 wks -0.55 0.04 -0.59 E310 2 wks -0.9 0.12 -1.02 E311 2 wks -0.32 0.08 -0.4
RAT DATE RT DIF
LT DIF(DD) TM
E34 3 wks -0.95 0.08 -1.03 E35 3 wks -0.83 0.07 -0.9 E36 3 wks -1.82 0.23 -2.05 E37 3 wks -1.36 0.25 -1.81 E38 3 wks -1.97 0.15 -2.22 E39 3 wks -0.78 0.06 -0.84 E310 3 wks -1.72 0.14 -1.86 E311 3 wks -0.86 0.11 -0.97
RAT DATE RT DIF
LT DIF(DD) TM
E34 4 wks -1.22 0.11 -1.33 E35 4 wks -1.11 0.09 -1.2 E36 4 wks -2.16 0.29 -2.45 E37 4 wks -1.72 0.33 -2.05 E38 4 wks -2.41 0.21 -2.62 E39 4 wks -0.86 0.08 -0.94 E310 4 wks -2.02 0.2 -2.22 E311 4 wks -1.06 0.14 -1.2
RAT DATE RT DIF LT DIF
S31 1 day 0.01 2.00E-
03
S32 1 day 0.07 3.00E-
03
S34 1 day 0.01 3.00E-
03
S35 1 day 0.02 5.00E-
03
S36 1 day 0.06 9.00E-
03
S37 1 day 0.02 5.00E-
03
S45 1 day 0.01 5.00E-
03
S46 1 day 0.06 9.00E-
03
242
RAT DATE RT DIF LT DIF S31 1 wk 0.01 0.01 S32 1 wk 0.01 0.01 S34 1 wk 0.06 0.04 S35 1 wk 0.1 0.075 S36 1 wk 0.08 0.13 S37 1 wk 0.02 0.08 S45 1 wk 0.06 0.08 S46 1 wk 0.08 0.11
RAT DATE RT DIF LT DIF S31 2 wks 0.04 0.05 S32 2 wks 0.11 0.12 S34 2 wks 0.13 0.13 S35 2 wks 0.13 0.19 S36 2 wks 0.12 0.14 S37 2 wks 0.06 0.09 S45 2 wks 0.11 0.18 S46 2 wks 0.17 0.19
RAT DATE RT DIF LT DIF S31 3 wks 0.12 0.21 S32 3 wks 0.16 0.16 S34 3 wks 0.15 0.2 S35 3 wks 0.18 0.26 S36 3 wks 0.12 0.15 S37 3 wks 0.09 0.11 S45 3 wks 0.13 0.23 S46 3 wks 0.19 0.25
RAT DATE RT DIF LT DIF S31 4 wks 0.11 0.24 S32 4 wks 0.18 0.18 S34 4 wks 0.14 0.29 S35 4 wks 0.21 0.31 S36 4 wks 0.13 0.26 S37 4 wks 0.16 0.18 S45 4 wks 0.14 0.28 S46 4 wks 0.22 0.33
243
Rate of tooth movement
Rat 0 to 1wk.
1 to 2wks.
2 to 3wks
3 to 4wks
E34 0.28 0.39 0.36 0.3 E35 0.29 0.07 0.54 0.3 E36 0.42 1.16 0.47 0.4 E37 0.38 0.62 0.81 0.24 E38 0.43 1.15 0.64 0.4 E39 0.39 0.2 0.25 0.1 E310 0.54 0.48 0.84 0.36 E311 0.28 0.12 0.57 0.23
Weight changes
RAT -1 day
0 day
1 day
2 days
3 days
4 days
5 days
6 days
1 wk
2 wks 3 wks 4 wks
E1 -6 0 -2 7 15 24 33 39 48 108 171 233 E2 -10 0 -6 3 10 16 23 28 34 89 138 187 E3 -4 0 -1 7 16 25 36 44 55 119 187 248 E4 -6 0 -3 5 11 20 28 35 43 99 155 211 E5 -9 0 -6 4 9 15 20 25 32 79 129 178 E6 -7 0 -5 5 10 16 22 28 34 84 133 184
E7 -5 0 -3 4 9 17 23 30 38 95 147 204 E8 -5 0 -4 4 12 18 26 36 44 94 149 210
RAT
-1 day
0 day
1 day
2 days
3 days
4 days
5 days
6 days
1 wk
2 wks 3 wks 4 wks
S1 -5 0 4 9 15 22 29 34 41 90 139 192 S2 -9 0 8 17 26 35 44 53 62 124 181 239 S3 -7 0 6 15 23 30 39 48 57 111 163 221 S4 -9 0 6 14 24 33 42 52 60 121 175 234 S5 -5 0 4 12 17 23 30 37 42 94 145 194 S6 -8 0 5 13 22 29 37 45 54 115 169 220 S7 -9 0 7 18 27 35 44 54 63 127 185 247
S8 -5 0 5 11 15 22 31 37 44 99 152 198
244
Expt. 2
Positive face-M1 sites
Rat LAD LT LAD RT RAD LT RAD RT GG LT GG RT N1 21 65 61 37 34 38 N2 46 51 59 42 47 39 N3 31 53 37 36 37 41 N4 42 58 55 39 48 35 N5 22 22 53 49 38 29 N6 14 33 43 22 10 19
LAD LT LAD RT RAD LT RAD RT GG LT GG RT
S11 25 38 50 32 25 43
S12 39 27 39 26 28 26 S13 65 60 59 31 34 28 S14 42 58 60 58 29 36 S15 28 57 50 24 38 13 S16 31 52 43 29 41 18
LAD LT LAD RT RAD LT RAD RT GG LT GG RT E11 4 26 16 21 4 11 E12 22 22 30 18 19 12 E13 16 18 26 15 18 10
E14 23 19 29 11 16 10 E15 14 21 20 9 17 16 E16 13 11 27 13 20 12
LAD LT LAD RT RAD LT RAD RT GG LT GG RT S71 32 49 57 31 37 21 S72 26 57 47 67 20 31 S73 24 36 23 15 34 26 S74 20 44 34 26 25 22 S75 34 35 37 30 32 29 S76 32 43 65 23 40 19
LAD LT LAD RT RAD LT RAD RT GG LT GG RT E71 50 53 113 73 47 9 E72 38 69 106 76 50 17 E73 58 55 64 60 65 50 E74 35 67 91 85 50 35 E75 24 30 29 20 71 40 E76 38 69 114 76 50 35
245
LAD LT LAD RT RAD LT RAD RT GG LT GG RT S281 42 63 60 55 52 45 S282 26 55 38 16 21 36 S283 14 42 63 49 42 38 S284 20 59 54 40 45 33 S285 36 58 46 41 39 45 S286 27 31 69 38 38 33
LAD LT LAD RT RAD LT RAD RT GG LT GG RT E281 9 16 23 1 13 18
E282 12 50 22 18 33 16 E284 16 49 20 23 17 40 E285 13 25 39 33 26 26 E286 18 40 21 9 11 11 E287 22 42 27 21 20 20
Positive face-S1 sites
Rat LAD LT LAD RT RAD LT RAD RT GG LT GG RT N1 2 7 7 4 11 13 N2 5 6 7 5 16 13 N3 4 6 4 4 13 12
N4 5 7 6 4 9 11 N5 2 3 6 5 13 10 N6 2 4 5 2 4 6
S11 3 5 7 4 9 15 S12 4 4 5 3 9 6 S13 7 8 8 4 11 10 S14 5 8 8 7 10 11 S15 3 7 7 3 13 5 S16 3 4 5 3 11 8
E11 1 3 2 2 1 4
E12 3 2 3 2 7 4 E13 1 1 3 1 7 4 E14 3 2 3 1 5 3 E15 2 2 2 1 5 6 E16 1 2 2 1 6 4
S71 4 5 7 3 13 7
246
S72 3 6 5 7 6 10 S73 4 4 3 2 12 9 S74 3 5 4 4 8 7 S75 5 4 4 4 11 10 S76 4 5 7 3 13 7
E71 6 6 13 8 13 7 E72 4 8 12 9 14 8 E73 6 6 7 7 15 12 E74 4 7 10 10 13 10 E75 3 3 6 2 16 11
E76 4 8 12 9 14 10
S281 5 8 7 6 12 13 S282 3 7 4 2 10 10 S283 2 6 7 6 12 11 S284 2 6 6 5 12 9 S285 4 8 5 5 10 11 S286 3 5 8 4 10 8
E281 1 2 3 1 5 7 E282 1 6 3 2 8 7 E284 2 6 2 3 6 10
E285 1 3 5 4 7 8 E286 2 5 3 1 5 6 E287 2 5 3 2 6 7
Positive sites for combination of muscles
RAD+LAD LT
RAD+LAD RT
LAD+GG LT
LAD+GG RT
RAD+GG LT
RAD+GG RT
RAD+LAD+GG LT
RAD+LAD+GG RT
N1 23 41 18 44 32 34 18 34 N2 47 45 47 43 52 42 44 40 N3 35 21 41 45 31 31 15 21 N4 43 40 27 34 27 32 11 22
N5 22 7 17 7 35 27 16 6 N6 16 22 3 18 9 12 3 12
S1 28 29 28 27 34 36 19 14 S1 31 26 24 24 29 19 25 14 S1 37 23 27 27 31 17 27 16 S1 28 29 28 28 34 36 19 20
247
S1 29 26 19 29 31 10 19 15 S1 29 27 20 24 29 12 16 14
E1 5 23 1 11 3 10 1 10 E1 22 18 19 15 25 14 18 14 E1 14 10 12 9 16 11 12 9 E1 26 8 18 8 20 6 18 5 E1 12 10 14 19 15 9 12 9 E1 14 16 10 11 12 13 11 9
S7 28 23 29 19 39 15 24 15
17 43 12 48 12 54 7 37
2 10 7 24 21 13 2 10
16 23 18 29 19 21 13 21
33 31 28 29 26 21 4 0
28 23 29 19 39 15 24 15
E7 53 45 28 10 44 9 27 9
36 38 20 15 30 13 17 12
58 58 56 44 59 47 52 42
38 65 21 30 38 35 21 29
26 22 27 28 35 21 26 21
36 38 20 15 30 13 17 12
S28 20 22 18 30 45 46 17 24
26 13 19 43 15 8 19 8
16 36 14 37 38 31 14 27
10 36 9 31 34 26 8 24
36 38 35 46 31 37 27 29
30 35 26 20 33 28 26 17
E28 10 3 6 17 11 3 6 3
13 19 6 20 1 12 6 12
26 23 10 26 9 19 8 17
12 17 11 18 20 19 11 12
12 1 6 24 6 1 6 1
15 3 12 20 1 1 6 1
ML spread of penetrations
248
rat
-6
-5.
5 -5
-4.5
-4
-3.5
-3
-2.
5 -2
-1.
5 -1
-0.
5 0
0.
5 1
1.
5 2
2.
5 3
3.
5 4
4.
5 5
5.
5 6
N 0 0 0 4 4 4 4 3 0 0 0 0 0 0 0 0 1 3 4 3 4 2 0 0 0
0 0 0 3 4 4 5 4 2 0 0 0 0 0 0 0 0 2 3 4 4 3 1 0 0
0 1 1 2 3 3 2 1 0 0 0 0 0 0 0 0 0 0 2 3 3 3 0 0 0
0 1 1 2 2 3 2 2 0 0 0 0 0 0 0 0 1 1 3 3 4 3 1 0 0
0 0 0 1 2 3 3 3 2 0 0 0 0 0 0 0 0 1 4 4 4 2 1 0 0
0 0 0 2 4 4 2 0 0 0 0 0 0 0 0 0 0 1 4 4 4 2 0 0 0
S1 0 0 1 3 4 4 2 2 0 0 0 0 0 0 0 0 0 1 3 4 3 2 0 0 0
0 0 0 2 3 4 4 3 1 0 0 0 0 0 0 0 0 0 1 3 3 3 1 0 0
0 0 0 0 2 4 4 3 1 0 0 0 0 0 0 0 1 2 4 4 3 2 1 0 0
0 0 0 0 1 4 4 4 1 0 0 0 0 0 0 0 1 3 3 3 4 1 0 0 0
0 0 0 2 3 4 4 3 2 1 0 0 0 0 0 0 0 0 2 4 4 3 3 3 0
0 0 1 3 4 4 2 2 0 0 0 0 0 0 0 0 0 1 3 4 3 2 0 0 0
E1 0 0 0 0 1 2 4 1 0 0 0 0 0 0 0 0 1 0 4 2 3 1 0 0 0
0 0 0 1 2 3 3 0 0 0 0 0 0 0 0 0 0 1 2 2 2 0 0 0 0
0 0 0 1 3 3 2 1 0 0 0 0 0 0 0 0 0 1 3 3 1 0 0 0 0
0 0 0 2 2 4 2 2 0 0 0 0 0 0 0 0 0 2 2 2 0 1 0 0 0
0 0 0 0 0 1 3 2 1 0 0 0 0 0 0 0 0 1 2 2 3 1 0 0 0
0 0 0 1 3 3 2 1 0 0 0 0 0 0 0 0 0 1 3 3 1 0 0 0 0
S7 0 0 0 3 3 4 3 2 1 0 0 0 0 0 0 0 1 2 4 4 3 3 1 0 0
0 0 0 3 4 4 4 2 0 0 0 0 0 0 0 0 0 2 4 4 4 3 3 0 0
0 0 1 2 3 3 3 1 0 0 0 0 0 0 0 0 1 1 2 3 2 2 2 0 0
0 0 1 3 3 4 3 3 0 0 0 0 0 0 0 0 0 3 3 4 3 3 1 0 0
0 0 1 3 2 4 4 0 0 0 0 0 0 0 0 0 0 1 3 4 4 4 2 0 0
0 0 0 3 3 4 3 2 1 0 0 0 0 0 0 0 1 2 4 4 3 3 1 0 0
E7 0 0 1 2 2 3 4 3 1 0 0 0 0 0 0 0 0 2 3 4 3 2 2 0 0
1 1 2 4 4 4 3 3 0 0 0 0 0 0 0 0 1 4 4 4 4 4 3 0 0
0 0 2 2 3 2 4 3 2 1 0 0 0 0 0 0 1 2 4 4 4 4 2 1 0
0 0 0 3 4 4 4 2 0 0 0 0 0 0 0 0 0 1 3 3 4 4 2 1 0
0 0 2 3 4 4 4 4 2 1 0 0 0 0 0 0 1 2 4 4 4 4 4 1 0
0 0 0 3 3 3 3 2 0 0 0 0 0 0 0 0 0 0 3 3 3 3 3 0 0
249
S28 0 0 2 3 3 4 4 3 2 0 0 0 0 0 0 0 0 3 4 3 4 4 2 0 0
0 0 2 3 3 3 2 1 0 0 0 0 0 0 0 0 0 0 3 4 4 3 3 0 0
0 0 2 4 4 4 3 2 0 0 0 0 0 0 0 0 1 2 4 4 4 3 3 0 0
0 0 0 4 4 3 3 1 0 0 0 0 0 0 0 0 0 0 3 4 4 3 1 0 0
0 0 0 3 3 4 4 1 0 0 0 0 0 0 0 0 0 2 4 4 4 3 2 0 0
0 0 0 2 3 4 4 3 0 0 0 0 0 0 0 0 0 1 4 4 4 3 1 0 0
E28 0 0 0 0 2 1 3 2 0 0 0 0 0 0 0 0 0 0 2 3 4 4 0 0 0
0 0 0 1 3 3 4 3 1 0 0 0 0 0 0 0 1 1 1 3 2 1 1 0 0
0 0 1 2 2 1 1 0 0 0 0 0 0 0 0 0 0 2 3 2 3 2 0 0 0
0 0 0 3 3 3 2 2 1 0 0 0 0 0 0 0 0 1 2 3 3 2 1 0 0
0 0 1 1 2 2 3 1 0 0 0 0 0 0 0 0 0 1 3 2 2 2 0 0 0
0 0 0 2 3 3 3 1 0 0 0 0 0 0 0 0 0 0 3 2 0 0 0 0 0
AP spread of penetration
rat
-5
-4.
5 -4
-3.
5 -3
-2.
5 -2
-1.
5 -1
-0.
5 0 0.5 1
1.5 2
2.5 3
3.5 4
4.5 5
N 0 0 5 4 5 5 0 0 0 0 0 0 0 0 0 5 6 4 2 0 0
0 2 5 4 6 5 0 0 0 0 0 0 0 0 0 6 5 4 2 0 0
0 0 0 3 5 5 0 0 0 0 0 0 0 0 0 3 4 4 0 0 0
0 0 0 1 5 7 0 0 0 0 0 0 0 0 0 5 6 4 1 0 0
0 0 1 3 5 5 0 0 0 0 0 0 0 0 0 4 4 5 3 0 0
0 0 3 3 3 3 0 0 0 0 0 0 0 0 0 3 4 4 3 1 0
S1 0 0 4 3 6 3 0 0 0 0 0 0 0 0 0 2 4 4 3 0 0
0 0 3 3 6 5 0 0 0 0 0 0 0 0 0 4 4 3 0 0 0
0 0 2 3 5 4 0 0 0 0 0 0 0 0 0 4 7 3 3 0 0
0 0 3 3 4 4 0 0 0 0 0 0 0 0 6 4 3 2 0 0 0
0 0 3 7 5 4 0 0 0 0 0 0 0 0 0 5 5 6 3 0 0
0 0 3 3 6 5 0 0 0 0 0 0 0 0 0 4 4 3 0 0 0
E 0 0 1 1 1 3 2 0 0 0 0 0 0 0 1 4 4 1 1 0 0
250
1
0 0 0 2 3 4 0 0 0 0 0 0 0 0 0 2 4 1 0 0 0
0 0 2 1 2 5 0 0 0 0 0 0 0 0 0 4 2 1 1 0 0
0 0 1 5 5 1 0 0 0 0 0 0 0 0 0 2 2 2 1 0 0
0 0 0 3 2 2 0 0 0 0 0 0 0 0 0 2 4 3 0 0 0
0 0 2 1 2 5 0 0 0 0 0 0 0 0 0 4 2 1 1 0 0
S7 0 0 1 5 6 4 0 0 0 0 0 0 0 0 0 5 6 5 2 0 0
0 0 4 4 5 4 0 0 0 0 0 0 0 0 0 5 6 6 3 0 0
0 0 1 3 6 3 0 0 0 0 0 0 0 0 0 5 7 1 0 0 0
0 0 3 5 6 3 0 0 0 0 0 0 0 0 0 3 6 5 3 0 0
0 0 2 3 4 5 0 0 0 0 0 0 0 0 0 5 3 5 5 0 0
0 0 1 3 6 3 0 0 0 0 0 0 0 0 0 5 7 1 0 0 0
E7 0 0 2 2 6 6 0 0 0 0 0 0 0 0 0 6 6 2 2 0 0
0 0 3 5 6 8 0 0 0 0 0 0 0 0 0 6 6 7 5 0 0
0 0 1 3 8 7 0 0 0 0 0 0 0 0 0 8 5 5 4 0 0
0 0 4 4 5 4 0 0 0 0 0 0 0 0 0 5 5 6 2 0 0
0 0 4 6 8 6 0 0 0 0 0 0 0 0 0 5 6 8 5 0 0
0 0 0 5 5 4 0 0 0 0 0 0 0 0 0 4 5 5 1 0 0
S28 0 0 4 4 7 6 0 0 0 0 0 0 0 0 0 6 5 6 3 0 0
0 0 1 4 6 3 0 0 0 0 0 0 0 0 0 4 5 5 3 0 0
0 0 4 4 6 5 0 0 0 0 0 0 0 0 0 5 7 6 3 0 0
0 0 4 4 6 5 0 0 0 0 0 0 0 0 0 4 5 3 3 0 0
0 0 2 5 4 4 0 0 0 0 0 0 0 0 0 5 5 6 3 0 0
0 0 3 4 5 4 0 0 0 0 0 0 0 0 0 5 5 4 3 0 0
E28 0 0 0 3 2 2 0 0 0 0 0 0 0 0 0 2 4 1 0 0 0
0 0 0 1 3 4 0 0 0 0 0 0 0 0 0 4 4 3 2 0 0
0 3 3 5 4 0 0 0 0 0 0 0 0 0 0 0 7 2 1 0 0
0 0 0 0 3 4 0 0 0 0 0 0 0 0 0 5 5 2 0 0 0
0 0 0 4 5 5 0 0 0 0 0 0 0 0 0 5 4 3 0 0 0
0 0 2 2 4 4 0 0 0 0 0 0 0 0 0 2 2 1 0 0 0
Centre of gravity
251
LAD
Rat Muscle AP Left ML Left Depth Left AP Right
ML Right
Depth Right
N1 LAD 2.5 0 0
2.5 3.37 3.7 N1 LAD 3 4.08 4.1
3 3.31 3.40833
N1 LAD 3.5 0 0
3.5 3.37 3.05128 N1 LAD 4 3.86 2.98571
4 3.14 2.88571
N1 LAD 4.5 3.5 3
4.5 3 2.3 N2 LAD 2.5 3.9 4.3
2.5 4.09 3.975
N2 LAD 3 3.49 3.75
3 3.92 3.74054 N2 LAD 3.5 3.05 3.26667
3.5 3.43 3.33913
N2 LAD 4 3.97 3.22667
4 3.46 2.88 N2 LAD 4.5 3 2.6
4.5 3.78 3.06667
N3 LAD 2.5 0 0
2.5 0 0 N3 LAD 3 4.08 5.03333
3 3.84 4.675
N3 LAD 3.5 3.67 3.08889
3.5 3.57 3.71818 N3 LAD 4 0 0
4 3.36 4.14545
N3 LAD 4.5 0 0
4.5 0 0 N4 LAD 2.5 3.63 3.93333
2.5 3.68 4.608
N4 LAD 3 3.62 3.03
3 3.2 3.16522 N4 LAD 3.5 3.06 2.96667
3.5 3.4 3.17333
N4 LAD 4 3.97 3.22667
4 4 3.7 N4 LAD 4.5 0 0
4.5 0 0
N5 LAD 2.5 0 0
2.5 4.25 4.5 N5 LAD 3 3.5 3.9
3 3.5 3.3
N5 LAD 3.5 3.06 2.96667
3.5 3.25 3.15 N5 LAD 4 2.25 1.7
4 0 0
N5 LAD 4.5 0 0
4.5 3.17 2.33333 N6 LAD 2.5 0 0
2.5 4.25 4.8
N6 LAD 3 4 3.8
3 3.87 3.88333 N6 LAD 3.5 4 4.1
3.5 3.25 3.225
N6 LAD 4 3.86 2.98571
4 3.25 2.7 N6 LAD 4.5 0 0
4.5 3 2.2
S11 LAD 2.5 0 0
2.5 4.08 3.98333 S11 LAD 3 4.5 4.4
3 3.97 4.17895
S11 LAD 3.5 3.7 3.24
3.5 3.52 3.27273 S11 LAD 4 3.5 2.6
4 3.25 3.03333
S11 LAD 4.5 3.5 3
4.5 2.83 2.46667 S12 LAD 2.5 4 3.6
2.5 4.3 4.33333
S12 LAD 3 3.68 3.55294
3 4.21 4.14286 S12 LAD 3.5 3.54 3.46154
3.5 3.5 3.62105
252
S12 LAD 4 3.07 3.14286
4 3.56 3.025 S12 LAD 4.5 3.17 2.63333
4.5 0 0
S13 LAD 2.5 3.04 3.1
2.5 3.73 3.69 S13 LAD 3 2.5 2.8
3 3.65 3.02963
S13 LAD 3.5 2.93 2.71429
3.5 3.5 2.84444 S13 LAD 4 2.5 1.8
4 3.1 2.6
S13 LAD 4.5 3.5 2.15
4.5 3.28 2.35556 S14 LAD 2.5 3.25 2.7
2.5 3.47 3.44211
S14 LAD 3 3 2.7
3 3.39 2.55556 S14 LAD 3.5 0 0
3.5 3.64 2.2
S14 LAD 4 0 0
4 4 2.33333
S14 LAD 4.5 0 0
2.5 3.62 2.25 S15 LAD 2.5 3.5 4.2
3 4.25 4.24
S15 LAD 3 3.5 3.66667
3.5 4.54 4.42857 S15 LAD 3.5 3.5 4.2
4 4.64 3.888
S15 LAD 4 2.65 3.12
4.5 4.54 3.77692 S15 LAD 4.5 2.72 2.73333
2.5 3.7 3.14
S16 LAD 2.5 4 3.6
3 4.3 4.33333 S16 LAD 3 3.68 3.55294
3.5 4.21 4.14286
S16 LAD 3.5 3.54 3.46154
4 3.5 3.62105 S16 LAD 4 3.07 3.14286
4.5 3.56 3.025
S16 LAD 4.5 3.17 2.63333
4 0 0
E11 LAD 2.5 2.5 2.9
2.5 3.42 3.83333 E11 LAD 3 3 3.2
3 3.46 3.55
E11 LAD 3.5 3 2.6
3.5 3 2.8 E11 LAD 4 3 2.6
4 3 2.3
E11 LAD 4.5 0 0
4.5 0 0 E12 LAD 2.5 3.95 4.11
2.5 3.83 4.50667
E12 LAD 3 3.68 3.32857
3 3.19 3.14444 E12 LAD 3.5 3.25 3.1
3.5 3.08 2.93333
E12 LAD 4 0 0
4 0 0 E12 LAD 4.5 0 0
4.5 0 0
E13 LAD 2.5 4.12 3.95
2.5 3.36 3.68571
E13 LAD 3 3.45 3.48
3 3 3.53333 E13 LAD 3.5 3.12 2.85
3.5 3.17 3.2
E13 LAD 4 0 0
4 0 0 E13 LAD 4.5 0 0
4.5 0 0
E14 LAD 2.5 3.67 4.13333
2.5 0 0 E14 LAD 3 3.95 3.90667
3 3.37 3.37037
E14 LAD 3.5 3.54 3.8
3.5 3.27 3.13846 E14 LAD 4 0 0
4 3.17 3.03333
253
E14 LAD 4.5 0 0
4.5 3 2.6 E15 LAD 2.5 3 3.5
2.5 4.37 4.09333
E15 LAD 3 2 2
3 3.63 3.32 E15 LAD 3.5 2 2
3.5 4.1 3.5
E15 LAD 4 0 0
4 0 0 E15 LAD 4.5 0 0
4.5 0 0
E16 LAD 2.5 2.5 2.9
2.5 3.42 3.83333 E16 LAD 3 3 3.2
3 3.46 3.55
E16 LAD 3.5 3 2.6
3.5 3 2.8 E16 LAD 4 3 2.6
4 3 2.3
E16 LAD 4.5 0 0
4.5 0 0
S71 LAD 2.5 0 0
2.5 0 4.2 S71 LAD 3 4.21 4.2
3 0.11 3.6
S71 LAD 3.5 3.84 3.51818
3.5 0.12 3.19286 S71 LAD 4 3.5 2.9
4 0.06 3.05
S71 LAD 4.5 0 0
4.5 0 2.3 S72 LAD 2.5 4.25 3.98
2.5 0.08 3.57391
S72 LAD 3 4 4.48
3 0.08 3.925 S72 LAD 3.5 3.75 3.58
3.5 0.11 3.36522
S72 LAD 4 3.5 3.33333
4 0.15 3.57333 S72 LAD 4.5 3.92 3.2
4.5 0 2.6
S73 LAD 2.5 0 0
2.5 0.25 4.4
S73 LAD 3 0 0
3 0.12 3.48 S73 LAD 3.5 3.5 2.7
3.5 0.13 3.21429
S73 LAD 4 3.5 2.7
4 0.13 2.66667 S73 LAD 4.5 0 0
4.5 0 0
S74 LAD 2.5 0 0
2.5 0.07 4.16842 S74 LAD 3 0 0
3 0.13 3.38333
S74 LAD 3.5 3 2
3.5 0 2.4 S74 LAD 4 3 2
4 0 2.4
S74 LAD 4.5 3 2
4.5 0 0 S75 LAD 2.5 3.5 3.2
2.5 0.08 3.82222
S75 LAD 3 3.57 3.4
3 0.09 3.08649
S75 LAD 3.5 3.77 3.71667
3.5 0.12 2.77333 S75 LAD 4 3.4 2.76
4 0.11 2.525
S75 LAD 4.5 0 0
4.5 0 0 S76 LAD 2.5 0 0
2.5 0 4.2
S76 LAD 3 4.21 4.2
3 0.11 3.6 S76 LAD 3.5 3.84 3.51818
3.5 0.12 3.19286
S76 LAD 4 3.5 2.9
4 0.06 3.05 S76 LAD 4.5 0 0
4.5 0 2.3
254
E71 LAD 2.5 3.52 3.208
2.5 3.5 3.22857 E71 LAD 3 3.72 3.08148
3 3.58 3.19355
E71 LAD 3.5 2.75 3.15
3.5 3.57 3.01429 E71 LAD 4 0 0
4 3.4 2.84
E71 LAD 4.5 0 0
4.5 3.5 2.5 E72 LAD 2.5 4.43 5.11429
2.5 0 0
E72 LAD 3 4.46 4.29231
3 3.47 3.33125 E72 LAD 3.5 4.03 3.74118
3.5 3.53 3.13684
E72 LAD 4 3.6 3.64
4 3.09 3.28696 E72 LAD 4.5 0 0
4.5 3.67 3.2
E73 LAD 2.5 3.42 3.4973
2.5 3.77 3.79556 E73 LAD 3 3.32 3.51034
3 3.43 3.36429
E73 LAD 3.5 3.29 3
3.5 3.61 3.09697 E73 LAD 4 3 2.46667
4 3.5 2.93333
E73 LAD 4.5 3.5 2.4
4.5 0 0 E74 LAD 2.5 3.87 4.35
2.5 3.35 4.025
E74 LAD 3 3.37 3.55
3 3.5 3.5 E74 LAD 3.5 3.5 3.7
3.5 3.44 3.325
E74 LAD 4 3.25 2.675
4 4 4.4 E74 LAD 4.5 0 0
4.5 3.42 3.1
E75 LAD 2.5 4 3.5
2.5 4.1 4.16 E75 LAD 3 4.27 3.65455
3 4.15 3.6
E75 LAD 3.5 3.3 3.02
3.5 3.69 3.21667 E75 LAD 4 3.14 2.41818
4 3.55 2.62105
E75 LAD 4.5 3.5 2.4
4.5 3.72 2.91111 E76 LAD 2.5 0 0
2.5 4.5 5.06667
E76 LAD 3 3.83 3.43333
3 4.28 4.15556 E76 LAD 3.5 3.42 2.95385
3.5 3.97 3.68
E76 LAD 4 3.25 2.675
4 3.42 3.1 E76 LAD 4.5 0 0
4.5 0 0
S281 LAD 2.5 4.3 4.57391
2.5 3.4 3.78462 S281 LAD 3 3.78 3.57778
3 3.61 3.72727
S281 LAD 3.5 3.5 3.1
3.5 3.57 3.34286 S281 LAD 4 3.42 3.18462
4 4.11 3.75714
S281 LAD 4.5 0 0
4.5 0 0 S282 LAD 2.5 4.5 4.2
2.5 4.29 4.43158
S282 LAD 3 3.82 3.8
3 4.22 3.912 S282 LAD 3.5 4 3.86667
3.5 3.65 3.69412
S282 LAD 4 3.5 3.4
4 3.38 2.83077 S282 LAD 4.5 0 0
4.5 0 0
255
S283 LAD 2.5 0 0
2.5 0 0 S283 LAD 3 4.5 4
3 4.14 3.79091
S283 LAD 3.5 3.97 3.78667
3.5 3.6 3.25 S283 LAD 4 0 0
4 3.53 3.32632
S283 LAD 4.5 0 0
4.5 3.5 2.92 S284 LAD 2.5 0 0
2.5 4.42 5.02
S284 LAD 3 4.25 4.55
3 4.33 4.85556 S284 LAD 3.5 4 4
3.5 4.05 4.32727
S284 LAD 4 4 2.6
4 3.25 3.175 S284 LAD 4.5 0 0
4.5 0 0
S285 LAD 2.5 3.83 3.91667
2.5 4 3.76923
S285 LAD 3 3.77 3.63077
3 4.24 3.94545 S285 LAD 3.5 3.56 3.4125
3.5 3.55 3.07273
S285 LAD 4 2.95 2.96364
4 3.5 3.33333 S285 LAD 4.5 0 0
4.5 3.4 2.76
S286 LAD 2.5 3.87 3.81667
2.5 4.09 4.07273 S286 LAD 3 3.3 3.10667
3 3.5 3.13333
S286 LAD 3.5 3 2.2
3.5 3.33 2.7 S286 LAD 4 0 0
4 3.43 3.17143
S286 LAD 4.5 0 0
4.5 0 0
E281 LAD 2.5 0 0
2.5 0 0 E281 LAD 3 3.5 3.7
3 4.3 4.56
E281 LAD 3.5 3.5 3.92
3.5 4.23 3.90769 E281 LAD 4 3.17 2.86667
4 0 0
E281 LAD 4.5 0 0
4.5 0 0 E282 LAD 2.5 3 3.05714
2.5 4.03 4
E282 LAD 3 2.75 3.05
3 4 3.67143 E282 LAD 3.5 3 2.5
3.5 3.83 3.68
E282 LAD 4 0 0
4 4.23 3.50909 E282 LAD 4.5 0 0
4.5 3 3.5
E283 LAD 2.5 4.5 3.9
2.5 4 3.6 E283 LAD 3 4.15 3.94
3 3.43 3.37037
E283 LAD 3.5 3.94 3.3
3.5 3.42 3.016
E283 LAD 4 0 0
4 3 2.8 E283 LAD 4.5 0 0
4.5 0 0
E284 LAD 2.5 3.33 3.5
2.5 4.02 4.2 E284 LAD 3 3.19 3.35
3 3.63 3.32632
E284 LAD 3.5 3.4 3
3.5 3.39 2.62222 E284 LAD 4 3.5 2.2
4 0 0
E284 LAD 4.5 0 0
4.5 0 0 E285 LAD 2.5 0 0
2.5 4 4.8
256
E285 LAD 3 4.5 5
3 3.73 4.12 E285 LAD 3.5 3.41 3.3
3.5 3.38 3.60952
E285 LAD 4 3.5 2.6
4 2.93 2.97143 E285 LAD 4.5 3 2.6
4.5 3 2.9
E286 LAD 2.5 4.5 4
2.5 0 0 E286 LAD 3 4.5 4.4
3 3.36 3.68571
E286 LAD 3.5 3.25 3
3.5 3.24 3.03448 E286 LAD 4 0 0
4 2.9 3.08
E286 LAD 4.5 0 0
4.5 3.58 3.66667
RAD
Rat Muscle AP Left ML Left Depth Left AP Right
ML Right
Depth Right
N1 RAD 2 4 3.62
2 0 0 N1 RAD 2.5 3.97 3.64667
2.5 3.2 3.42
N1 RAD 3 3.85 2.96
3 2.85 3.08235 N1 RAD 3.5 3.95 3.14286
3.5 3 2.7
N1 RAD 4 3.71 2.94286
4 0 0 N2 RAD 2.5 3.65 3.78378
2.5 3.71 3.74118
N2 RAD 3 3.14 3.23721
3 3.53 3.27778 N2 RAD 3.5 3.74 3.21739
3.5 3.46 2.8
N2 RAD 4 3.14 2.62857
4 3.62 3.45
N2 RAD 4.5 3.17 2.8
4.5 0 0 N3 RAD 2 4.5 5.2
2 0 0
N3 RAD 2.5 4.15 5.23077
2.5 3.75 4.9 N3 RAD 3 3.77 3.30769
3 3.54 3.82857
N3 RAD 3.5 4 4.5
3.5 3.1 4.16 N3 RAD 4 0 0
4 0 0
N4 RAD 2.5 4.17 4.09756
2.5 3.66 4.8375 N4 RAD 3 3.58 2.94444
3 3.42 3.25
N4 RAD 3.5 3.5 2.7
3.5 3.07 3.02857 N4 RAD 4 0 0
4 0 0
N4 RAD 4.5 0 0
4.5 0 0 N5 RAD 2 3.72 3.87778
2 4.12 4.47059
N5 RAD 2.5 3.18 3.31429
2.5 3.85 3.73 N5 RAD 3 3.23 2.74545
3 3.24 2.71765
N5 RAD 3.5 2.32 2.12727
3.5 3.68 3 N5 RAD 4 3.5 2.6
4 3.33 2.43333
N6 RAD 2 4.5 4.3
2 4.25 4.75 N6 RAD 2.5 4 3.6
2.5 3.94 4.02222
N6 RAD 3 3.79 3.71429
3 3.45 3.21818
257
N6 RAD 3.5 4 2.6
3.5 3.25 2.7 N6 RAD 4 0 0
4 0 0
S11 RAD 2 4.28 3.3375
2 4 4.11111 S11 RAD 2.5 4.25 3.63333
2.5 3.83 4.04444
S11 RAD 3 3.79 3.00606
3 3.5 3.17647 S11 RAD 3.5 3.85 2.90769
3.5 3.25 3.03333
S11 RAD 4 3.5 2.46667
4 2.87 2.35 S12 RAD 2 4 3.6
2 4.35 4.5
S12 RAD 2.5 3.64 3.6381
2.5 4.18 4.36364 S12 RAD 3 3.18 3.14286
3 3.71 3.35714
S12 RAD 3.5 3.6 3.56
3.5 3.37 3.15 S12 RAD 4 3.17 2.68889
4 0 0
S13 RAD 2 3 2.9
2 0 0 S13 RAD 2.5 4 3.3
2.5 3 2.9
S13 RAD 3 2.5 2.8
3 3 2.6 S13 RAD 3.5 0 0
3.5 0 0
S13 RAD 4 0 0
4 0 0 S14 RAD 2 3.55 2.8
2 3.43 3.48
S14 RAD 2.5 3.67 2.91111
2.5 3.43 2.91429 S14 RAD 3 2.5 2.6
3 3.62 2.5
S14 RAD 3.5 4 2.8
3.5 0 0 S14 RAD 4 0 0
4 3.5 2.3
S15 RAD 2 3.5 3.8
2 4 4.3 S15 RAD 2.5 3.67 3.52
2.5 4.57 5.02857
S15 RAD 3 3.43 3.60909
3 4.67 4.46667 S15 RAD 3.5 2.52 2.92
3.5 4.58 4.53333
S15 RAD 4 2.8 2.72
4 3.9 3.48 S16 RAD 2 4 3.6
2 4.35 4.5
S16 RAD 2.5 3.64 3.6381
2.5 4.18 4.36364 S16 RAD 3 3.18 3.14286
3 3.71 3.35714
S16 RAD 3.5 3.6 3.56
3.5 3.37 3.15 S16 RAD 4 3.17 2.68889
4 0 0
E11 RAD 2.5 3 3.425
2.5 3.44 3.88889 E11 RAD 3 3 3.2
3 3.41 3.54545
E11 RAD 3.5 3 2.5
3.5 3 2.9 E11 RAD 4 3 2.6
4 3 2.4
E11 RAD 4.5 0 0
4.5 0 0 E12 RAD 2.5 4 4.2
2.5 3.57 3.74667
E12 RAD 3 3.72 3.176
3 3.35 3.22 E12 RAD 3.5 3.3 3.04
3.5 3 3.1
258
E12 RAD 4 0 0
4 0 0 E12 RAD 4.5 0 0
4.5 0 0
E13 RAD 2 4.12 3.95385
2 3.43 3.91429 E13 RAD 2.5 3.53 3.77778
2.5 3.08 3.63333
E13 RAD 3 3.12 3
3 3 3.2 E13 RAD 3.5 4 2.9
3.5 3 3
E13 RAD 4 3.5 2.5
4 0 0 E14 RAD 2.5 3.94 4.35556
2.5 3 2.8
E14 RAD 3 3.88 3.69655
3 3.42 3.57778 E14 RAD 3.5 3.63 3.81333
3.5 3.32 3.30909
E14 RAD 4 3.5 3.3
4 0 0
E14 RAD 4.5 0 0
4.5 0 0 E15 RAD 2.5 3 3.37143
2.5 4.31 4.125
E15 RAD 3 2.75 2.1
3 3.5 3.33333 E15 RAD 3.5 2 2
3.5 3.67 3.46667
E15 RAD 4 3 3.6
4 0 0 E15 RAD 4.5 0 0
4.5 0 0
E16 RAD 2.5 3 3.425
2.5 3.44 3.88889 E16 RAD 3 3 3.2
3 3.41 3.54545
E16 RAD 3.5 3 2.5
3.5 3 2.9 E16 RAD 4 3 2.6
4 3 2.4
E16 RAD 4.5 0 0
4.5 0 0
S71 RAD 2 4.21 4.65263
2 4 4.2 S71 RAD 2.5 4.12 3.93
2.5 3.5 3.5
S71 RAD 3 3.62 3.48333
3 3.07 3.24286 S71 RAD 3.5 3.39 3.25714
3.5 3.1 2.8
S71 RAD 4 3.5 3.8
4 0 0 S72 RAD 2 3.98 3.34167
2 4.27 3.73846
S72 RAD 2.5 3.98 3.88571
2.5 4.02 3.925 S72 RAD 3 3.72 3.6875
3 3.77 3.50909
S72 RAD 3.5 3.21 2.76667
3.5 4.1 3.6 S72 RAD 4 3.33 2.13333
4 3.17 2.6
S73 RAD 1.5 4.08 3.7
1.5 3.67 4
S73 RAD 2 3.97 3.2375
2 0 0 S73 RAD 2.5 3.5 2.6
2.5 3.62 3.3
S73 RAD 3 3.5 2.6
3 3.5 2.2 S73 RAD 3.5 0 0
3.5 0 0
S74 RAD 2.5 0 0
2.5 4.32 4.2 S74 RAD 3 4.21 4.65263
3 3.53 3.22667
S74 RAD 3.5 3.3 3.08
3.5 3.5 2.6 S74 RAD 4 0 0
4 0 0
259
S74 RAD 4.5 0 0
4.5 0 0 S75 RAD 2.5 3.5 3.2
2.5 0 0
S75 RAD 3 3.3 3.08
3 3.5 3.5 S75 RAD 3.5 4 4.1
3.5 3.53 3.22667
S75 RAD 4 0 0
4 0 0 S75 RAD 4.5 0 0
4.5 0 0
S76 RAD 2 0 0
2 4 4.2 S76 RAD 2.5 4.12 3.93
2.5 3.5 3.5
S76 RAD 3 3.62 3.48333
3 3.07 3.24286 S76 RAD 3.5 3.39 3.25714
3.5 3.1 2.8
S76 RAD 4 3.5 3.8
4 0 0
E71 RAD 2 3.35 3.26667
2 3.4 2.88571 E71 RAD 2.5 3.92 3.2623
2.5 3.52 3.22927
E71 RAD 3 3.29 2.75294
3 3.39 3.08889 E71 RAD 3.5 3.25 2.5
3.5 3.5 2.9
E71 RAD 4 2.75 1.95
4 3.67 3.43333 E72 RAD 1.5 4.5 4.95
1.5 3 3.2
E72 RAD 2 4.38 4.00769
2 3.42 3.2 E72 RAD 2.5 3.89 3.60571
2.5 3.39 3.15556
E72 RAD 3 3.75 3.51667
3 2.94 3.30769 E72 RAD 3.5 3.88 3.25882
3.5 3.25 2.84286
E73 RAD 2.5 3.63 3.62979
2.5 3.62 3.885
E73 RAD 3 3.46 3.63265
3 3.42 3.46452 E73 RAD 3.5 3.39 3.26667
3.5 3.57 3.22069
E73 RAD 4 2.87 2.55
4 3.58 2.83333 E73 RAD 4.5 2.75 1.95
4.5 3.67 3.43333
E74 RAD 2.5 4.07 4.23478
2.5 3.36 3.92727 E74 RAD 3 3.62 3.53846
3 3.41 3.70909
E74 RAD 3.5 3.33 3.6
3.5 3 3.5 E74 RAD 4 3.5 3.36
4 4.5 2.8
E74 RAD 4.5 0 0
4.5 0 0 E75 RAD 2 4.14 3.89091
2 4 4.26667
E75 RAD 2.5 4.15 3.50769
2.5 4.22 3.74444
E75 RAD 3 3.65 2.95556
3 3.68 3.31667 E75 RAD 3.5 3.14 2.52222
3.5 3.72 2.792
E75 RAD 4 3.45 2.2
4 3.8 2.88 E76 RAD 2.5 4 4.4
2.5 0 0
E76 RAD 3 3.77 3.36364
3 4.33 4.23333 E76 RAD 3.5 3.5 3.04615
3.5 3.91 3.69091
E76 RAD 4 3.4 2.72
4 3.4 3.08 E76 RAD 4.5 0 0
4.5 0 0
260
S281 RAD 2.5 4.29 3.92381
2.5 4 3.93333 S281 RAD 3 3.64 3.12222
3 3.86 3.50286
S281 RAD 3.5 3.2 2.82
3.5 3.4 3.92 S281 RAD 4 0 0
4 0 0
S281 RAD 4.5 0 0
4.5 0 0 S282 RAD 2.5 4.36 4.51429
2.5 4.5 4.8
S282 RAD 3 4.05 4.01818
3 4.12 4.175 S282 RAD 3.5 3.62 3.375
3.5 3.6 3.76
S282 RAD 4 3.5 3.4
4 3.12 3.1 S282 RAD 4.5 0 0
4.5 0 0
S283 RAD 2 0 0
2 0 0 S283 RAD 2.5 4.21 3.85263
2.5 4.17 3.94444
S283 RAD 3 3.94 3.69677
3 3.58 3.3 S283 RAD 3.5 3.69 2.95385
3.5 3.12 3.13333
S283 RAD 4 3.64 2.91429
4 3.2 3 S284 RAD 2.5 4.28 4.84444
2.5 4.37 4.9625
S284 RAD 3 4.35 4.28
3 4.44 4.9375 S284 RAD 3.5 3.89 3.71818
3.5 4 4.4
S284 RAD 4 3.75 4
4 3.5 3.05 S284 RAD 4.5 3.5 2.4
4.5 3.4 3.6
S285 RAD 2 3.79 4.12857
2 4 3.9 S285 RAD 2.5 3.73 3.48
2.5 3.85 3.9
S285 RAD 3 3.65 3.16471
3 3.64 3.26667 S285 RAD 3.5 3.28 3.125
3.5 3.53 3.2
S285 RAD 4 3.17 2.86667
4 3.5 3 S286 RAD 2.5 3.84 3.61429
2.5 4.08 4.33333
S286 RAD 3 3.35 3
3 3.53 3.12632 S286 RAD 3.5 3.13 2.46667
3.5 3.35 2.61538
S286 RAD 4 3.27 2.16364
4 3.5 3.5 S286 RAD 4.5 0 0
4.5 0 0
E281 RAD 2 0 0
2 0 0 E281 RAD 2.5 3.36 3.25714
2.5 3.71 4.02353
E281 RAD 3 3.3 3.54
3 4 3.8 E281 RAD 3.5 3.06 2.85
3.5 3.87 3.725
E281 RAD 4 3.25 3.08
4 3 3.24 E282 RAD 2.5 0 0
2.5 0 0
E282 RAD 3 3.06 3.08889
3 3.4 3.28 E282 RAD 3.5 2.83 2.53333
3.5 3.79 3.6
E282 RAD 4 2.75 3.56
4 3.71 4.02353 E282 RAD 4.5 0 0
4.5 3 3.4
261
E283 RAD 2.5 0 0
2.5 0 0 E283 RAD 3 2.75 3.56
3 3.71 4.02353
E283 RAD 3.5 3.4 3.18667
3.5 3.87 3.725 E283 RAD 4 3.25 3.08
4 3.5 3.6
E283 RAD 4.5 4 2.6
4.5 3.25 2.9 E284 RAD 2 3.06 2.85
2 0 0
E284 RAD 2.5 4.19 3.96923
2.5 3.27 3.26667 E284 RAD 3 3.78 3.17778
3 3.25 2.9
E284 RAD 3.5 3.25 3.08
3.5 3.5 3.6 E284 RAD 4 0 0
4 0 0
E285 RAD 2 4 2.6
2 0 0
E285 RAD 2.5 3.7 3.41429
2.5 3.56 3.6 E285 RAD 3 3.42 2.76923
3 3.5 2.8875
E285 RAD 3.5 3.67 3.66667
3.5 3 3.24 E285 RAD 4 0 0
4 0 0
E286 RAD 2 4.5 4.93333
2 4 4.6 E286 RAD 2.5 4.15 4.96923
2.5 3.5 3.6
E286 RAD 3 3.44 3.35556
3 3.5 3.6 E286 RAD 3.5 3.25 3.08
3.5 3.5 3.6
E286 RAD 4 0 0
4 0 0
GG
Rat Muscle AP ML Left Depth Left AP ML Right
Depth Right
N1 GG 2 4.1 3.75
2 3.8 4.12 N1 GG 2.5 3.93 3.80952
2.5 3.27 3.4
N1 GG 3 3.17 3.33333
3 3.3 3.392 N1 GG 3.5 3.93 3.04286
3.5 3.1 2.48
N1 GG 4 3.5 3.2
4 3.5 3.2 N2 GG 2 4.02 4.46667
2 4.22 4.17778
N2 GG 2.5 3.53 3.90909
2.5 4.12 4.17143 N2 GG 3 3.74 3.81538
3 3.59 3.43636
N2 GG 3.5 3.84 3.1375
3.5 3.53 2.83529 N2 GG 4 2.8 2.52
4 3.7 3.32
N3 GG 2 4.25 5
2 0 0 N3 GG 2.5 4.61 5.03478
2.5 3.85 4.71765
N3 GG 3 3.89 3.38261
3 3.55 3.67619 N3 GG 3.5 3.62 4.1
3.5 3.43 3.97333
N3 GG 4 0 0
4 0 0 N4 GG 2.5 4.53 4.30588
2.5 3.71 4.78333
N4 GG 3 3.75 2.9
3 3.55 3.47273
262
N4 GG 3.5 3.5 3.7
3.5 3 3.3 N4 GG 4 2.67 2.76667
4 3.93 3.11429
N4 GG 4.5 0 0
4.5 0 0 N5 GG 2 3.73 3.89091
2 4.19 4.15
N5 GG 2.5 3.5 3.7
2.5 3.92 3.76667 N5 GG 3 3.54 3.04324
3 3.45 3.08
N5 GG 3.5 2.67 2.76667
3.5 3.93 3.11429 N5 GG 4 0 0
4 3.62 3.3
N6 GG 2 0 0
2 4 4.4 N6 GG 2.5 4 3.8
2.5 3.73 3.4
N6 GG 3 4 3.4
3 3.5 3.3
N6 GG 3.5 3.5 3.7
3.5 3 2.7 N6 GG 4 0 0
4 3 2.6
S11 GG 2 0 0
2 4.5 4.3 S11 GG 2.5 4.11 3.49091
2.5 3.71 3.65714
S11 GG 3 4 3.26667
3 3.45 3.12 S11 GG 3.5 0 0
3.5 4.12 3
S11 GG 4 0 0
4 0 0 S12 GG 2 4.22 3.75556
2 4.3 4.38
S12 GG 2.5 3.81 3.68889
2.5 4.06 4.5 S12 GG 3 3.44 3.24444
3 3.77 3.58182
S12 GG 3.5 3.43 3.48571
3.5 3.5 3.13333
S12 GG 4 3.17 2.8
4 0 0 S13 GG 2 3.3 3.44
2 3.75 3.94
S13 GG 2.5 3.3 3.44
2.5 3.19 3.06154 S13 GG 3 3.21 3.17143
3 3.33 2.6
S13 GG 3.5 2.5 2.6
3.5 2.75 2.7 S13 GG 4 0 0
4 0 0
S14 GG 2 0 0
2 0 0 S14 GG 2.5 4.11 3.49091
2.5 4.75 4.75
S14 GG 3 3.44 3.24444
3 3.77 3.58182 S14 GG 3.5 4.22 3.75556
3.5 4.3 4.38
S14 GG 4 0 0
4 0 0
S15 GG 2 3.87 4.08333
2 4 4.6 S15 GG 2.5 4.11 3.49091
2.5 4.75 4.75
S15 GG 3 3.33 3.36667
3 4.56 4.175 S15 GG 3.5 3.25 3.19
3.5 4.5 4.4
S15 GG 4 2.5 2.6
4 3.67 3.13333 S16 GG 2 4.22 3.75556
2 4.3 4.38
S16 GG 2.5 3.81 3.68889
2.5 4.06 4.5 S16 GG 3 3.44 3.24444
3 3.77 3.58182
263
S16 GG 3.5 3.43 3.48571
3.5 3.5 3.13333 S16 GG 4 3.17 2.8
4 0 0
E11 GG 2 3.75 3.8
2 4 4.4 E11 GG 2.5 3 3.2
2.5 3.75 4
E11 GG 3 3 3.3
3 3.61 3.42222 E11 GG 3.5 0 0
3.5 0 0
E11 GG 4 0 0
4 0 0 E12 GG 2.5 4.03 4.2375
2.5 3.94 4.46667
E12 GG 3 3.62 3.48333
3 3.33 3.33333 E12 GG 3.5 2.75 2.5
3.5 3 3.2
E12 GG 4 0 0
4 0 0 E12 GG 4.5 0 0
4.5 0 0
E13 GG 2 3.9 3.6
2 3.37 3.5 E13 GG 2.5 3.73 3.42667
2.5 3.27 3.49231
E13 GG 3 3.08 3.03333
3 3 3.2 E13 GG 3.5 4 3.3
3.5 3 3
E13 GG 4 4.12 3.1
4 3.5 3.6 E14 GG 2.5 4.33 4.93333
2.5 4.5 5
E14 GG 3 3.98 3.8
3 3.72 3.88889 E14 GG 3.5 3.5 3.775
3.5 3.3 3.26
E14 GG 4 0 0
4 3.33 3.26667 E14 GG 4.5 0 0
4.5 0 0
E15 GG 2.5 2.86 3.43636
2.5 4.32 4.1 E15 GG 3 3.5 2.2
3 3.67 3.43333
E15 GG 3.5 2 2.1
3.5 3.77 3.07692 E15 GG 4 3.75 3.8
4 4 4.4
E15 GG 4.5 0 0
4.5 0 0 E16 GG 2.5 3 3.2
2.5 3.75 4
E16 GG 3 3 3.3
3 3.61 3.42222 E16 GG 3.5 0 0
3.5 0 0
E16 GG 4 0 0
4 0 0 E16 GG 4.5 0 0
4.5 0 0
S71 GG 2 4.29 4.85714
2 4.12 4.25 S71 GG 2.5 4.17 3.93333
2.5 3.59 3.6
S71 GG 3 3.83 3.36
3 3.35 3.52 S71 GG 3.5 3.33 3.06667
3.5 3 2.9
S71 GG 4 0 0
4 3.5 2.4 S72 GG 2 4.1 3.74
2 4.34 3.8
S72 GG 2.5 3.81 4.15
2.5 4.2 3.925 S72 GG 3 3.75 3.4
3 3.85 3.31
264
S72 GG 3.5 3 3.2
3.5 4 3.6875 S72 GG 4 0 0
4 3.93 3.62857
S73 GG 1.5 4.43 4.08571
1.5 4.06 4.22222 S73 GG 2 4 3.2
2 3.75 3.43333
S73 GG 2.5 3.62 2.9
2.5 3.57 3.31429 S73 GG 3 3.4 2.52
3 3.67 2.5
S73 GG 3.5 0 0
3.5 0 0 S74 GG 2.5 4.17 3.93333
2.5 4.23 4.24
S74 GG 3 3.83 3.36
3 3.58 3.2 S74 GG 3.5 3.33 3.06667
3.5 3.5 2.7
S74 GG 4 3 2
4 0 0
S74 GG 4.5 0 0
4.5 0 0 S75 GG 2.5 3.33 2.94286
2.5 3.89 3.85714
S75 GG 3 3.6 3.03226
3 3.77 2.9697 S75 GG 3.5 3.94 3.63704
3.5 3.61 2.57419
S75 GG 4 3.55 3.12
4 3.33 2.3619 S75 GG 4.5 0 0
4.5 0 0
S76 GG 2 4.29 4.85714
2 4.12 4.25 S76 GG 2.5 4.17 3.93333
2.5 3.59 3.6
S76 GG 3 3.83 3.36
3 3.35 3.52 S76 GG 3.5 3.33 3.06667
3.5 3 2.9
S76 GG 4 0 0
4 3.5 2.4
E71 GG 2 3.59 3.35294
2 3 2.6 E71 GG 2.5 4.3 3.33913
2.5 3.4 3.24
E71 GG 3 3.71 2.55
3 3.25 3.1 E71 GG 3.5 3.5 3.8
3.5 4 2.9
E71 GG 4 0 0
4 0 0 E72 GG 1.5 4.83 5.15
1.5 0 0
E72 GG 2 4.75 4.41176
2 3.5 3.44286 E72 GG 2.5 4.5 3.94286
2.5 3.33 3.2
E72 GG 3 3.5 3.8
3 4 2.9 E72 GG 3.5 0 0
3.5 0 0
E73 GG 2.5 3.86 3.76957
2.5 4.13 4.18065
E73 GG 3 3.4 3.58367
3 3.48 3.52308 E73 GG 3.5 3 3
3.5 3.6 3.36
E73 GG 4 3 2.7
4 3.25 2.7 E73 GG 4.5 3 2.7
4.5 3.25 3.1
E74 GG 2.5 3.81 3.825
2.5 3.6 3.74 E74 GG 3 4 3.72727
3 3.65 3.56
E74 GG 3.5 4.25 3.33333
3.5 4 3.48 E74 GG 4 3.83 3.47
4 4.37 4.1
265
E74 GG 4.5 0 0
4.5 0 0 E75 GG 2 4.59 3.8
2 4.1 4.44
E75 GG 2.5 4.25 3.54286
2.5 4 3.42222 E75 GG 3 3.58 2.83333
3 3.9 3.18667
E75 GG 3.5 3 2.53333
3.5 3.75 2.825 E75 GG 4 0 0
4 3.5 3
E76 GG 1.5 4.17 4.4
1.5 4.5 4.7 E76 GG 2 3.83 3.47
2 4.37 4.1
E76 GG 2.5 3.5 3.07273
2.5 4.08 3.74737 E76 GG 3 3.7 2.85714
3 3.65 3.07692
E76 GG 3.5 3.5 3.8
3.5 3 2.4
S281 GG 2.5 4.18 3.87059
2.5 3.99 3.8439 S281 GG 3 3.69 3.2
3 3.87 3.57949
S281 GG 3.5 3 3.06667
3.5 3.57 3.41429 S281 GG 4 2.86 2.54286
4 4 4.2
S281 GG 4.5 0 0
4.5 0 0 S282 GG 2.5 4.64 4.85714
2.5 4.67 4.91111
S282 GG 3 4.35 4.33846
3 4.2 3.88148 S282 GG 3.5 4.25 3.2
3.5 3.75 3.78
S282 GG 4 0 0
4 3.19 3.075 S282 GG 4.5 0 0
4.5 0 0
S283 GG 2 0 0
2 0 0
S283 GG 2.5 4.19 3.83333
2.5 4.17 4.07692 S283 GG 3 4.23 3.85385
3 3.81 3.15
S283 GG 3.5 3.95 3.34545
3.5 3.8 3.28 S283 GG 4 3.5 3.6
4 3.42 3.16667
S284 GG 2.5 4.22 4.7875
2.5 4.48 4.89524 S284 GG 3 4.4 4.27097
3 4.39 4.90909
S284 GG 3.5 3.73 3.4
3.5 3.92 4.11667 S284 GG 4 4.33 3.2
4 3.37 3.65
S284 GG 4.5 0 0
4.5 4 2.6 S285 GG 2 4 4.21053
2 4.15 4.04
S285 GG 2.5 4 3.72941
2.5 4.22 4.02222
S285 GG 3 3.47 3.27059
3 3.48 3.10769 S285 GG 3.5 3.23 3.21333
3.5 3.66 3.21818
S285 GG 4 3.3 3.44
4 3.37 2.925 S286 GG 2.5 4.2 4.16
2.5 4.12 4.25
S286 GG 3 3.3 2.98
3 3.63 3.01053 S286 GG 3.5 2.86 2.54286
3.5 3.21 3.03333
S286 GG 4 3 2
4 3.4 3.48 S286 GG 4.5 0 0
4.5 0 0
266
E281 GG 2 0 0
2 0 0 E281 GG 2.5 3.5 3.6
2.5 4.33 4.5
E281 GG 3 3.5 3.4
3 4.15 3.89231 E281 GG 3.5 3.2 2.84
3.5 2.5 1.6
E281 GG 4 0 0
4 0 0 E282 GG 2 0 0
2 4.5 4.1
E282 GG 2.5 4 4.3
2.5 4.1 3.92 E282 GG 3 4.27 3.81538
3 3.83 3.5
E282 GG 3.5 0 0
3.5 3.75 3.3 E282 GG 4 0 0
4 4 3.6
E283 GG 2.5 3.92 4.13333
2.5 0 0 E283 GG 3 4.5 3
3 4 4.15789
E283 GG 3.5 3.6 3.46
3.5 3.69 3.675 E283 GG 4 3.25 3.2
4 3.5 3.7
E283 GG 4.5 0 0
4.5 0 0 E284 GG 2 4.5 3.85714
2 4 3.7
E284 GG 2.5 4.27 3.81538
2.5 3.42 3.225 E284 GG 3 3.92 3.4
3 3.37 3.075
E284 GG 3.5 0 0
3.5 3.1 3.32 E284 GG 4 0 0
4 0 0
E285 GG 2 3.47 3.93333
2 4 4.24348 E285 GG 2.5 3.44 3.7125
2.5 3.73 3.54667
E285 GG 3 3.46 2.53333
3 3.64 2.8 E285 GG 3.5 4.5 4.6
3.5 0 0
E285 GG 4 0 0
4 0 0 E286 GG 2 5 6
2 4 4.7
E286 GG 2.5 5 5.9
2.5 3.83 4.23333 E286 GG 3 3.65 3.22
3 3.5 3.67273
E286 GG 3.5 0 0
3.5 3 3 E286 GG 4 0 0
4 0 0
LATENCY
Face-M1
GROUP
LAD LT.
LAD RT.
GROUP
RAD LT.
RAD RT.
GROUP GG LT.
GG RT.
N 11.5 8.5
N 8.3 8.5
N 10.5 8.7 N 8.5 8.3
N 8.5 9.7
N 8.7 9.1
N 9.5 8.9
N 8.7 13.9
N 9.1 8.9 N 9.5 8.5
N 8.7 11.3
N 12.1 12.1
N 11.1 13.5
N 9.5 10.9
N 9.1 9.9
267
N 13.3 8.9
N 8.5 13.3
N 16.5 14.1
S1 12.7 8.7
S1 8.7 10.1
S1 15.7 13.1 S1 9.5 8.7
S1 9.1 8.7
S1 12.7 10.9
S1 9.3 8.7
S1 9.3 15.7
S1 12.3 12.7 S1 10.9 8
S1 9.1 10.4
S1 12.3 12.7
S1 12.3 8.3
S1 11.5 13.1
S1 11.5 13.5 S1 10.9 8.7
S1 9.1 10.4
S1 12.3 12.7
E1 16.1 10.1
E1 10.1 10.1
E1 12.7 14.1 E1 9.7 8.7
E1 8.3 13.1
E1 12.9 13.7
E1 11.9 9.7
E1 9.1 10.3
E1 9.5 14.1 E1 9.1 8.3
E1 8.3 8.7
E1 10.1 12.1
E1 11.5 8.9
E1 9.5 10.9
E1 11.7 9.3 E1 11.9 8.7
E1 8.3 11.1
E1 11.7 14.1
S7 10.1 8.3
S7 8.5 9.9
S7 9.3 9.1 S7 9.3 8.3
S7 8.3 9.1
S7 10.7 10.3
S7 10.9 9.1
S7 9.5 12.1
S7 8.9 10.1 S7 10.1 8.5
S7 8.3 10.3
S7 12.5 10.3
S7 9.7 8.5
S7 12.7 10.3
S7 10.5 10.1 S7 10.1 8.3
S7 8.3 10.3
S7 9.1 10.3
E7 12.1 8.7
E7 9.5 10.3
E7 13.1 16.7 E7 9.3 9.1
E7 8.3 9.7
E7 9.9 12.3
E7 13.1 12.7
E7 9.9 16.7
E7 9.9 10.3 E7 8.7 8.3
E7 8.3 8.9
E7 12.3 12.5
E7 9.7 8.1
E7 8.3 9.5
E7 11.1 10.9 E7 10.9 8.7
E7 8.3 9.5
E7 11.9 10.9
S28 9.5 8.7
S28 8.5 9.1
S28 10.1 10.5 S28 9.5 8.9
S28 8.7 12.1
S28 10.7 9.1
S28 12.5 8.5
S28 8.3 9.1
S28 9.1 9.7 S28 12.3 9.1
S28 8.7 9.3
S28 11.1 10.7
S28 8.3 8.3
S28 8.3 9.1
S28 9.7 10.3 S28 10.7 8.5
S28 8.3 11.3
S28 10.5 10.3
E28 13.3 9.5
E28 9.5 16.9
E28 14.5 16.9 E28 9.1 8.3
E28 8.7 10.3
E28 12.3 12.9
E28 11.7 8.5
E28 9.5 9.3
E28 12.3 8.9 E28 10.7 8.9
E28 8.7 10.1
E28 9.7 8.5
E28 11.5 9.3
E28 8.3 9.9
E28 12.3 13.1
268
E28 9.7 9.1
E28 9.7 12.7
E28 13.7 11.5
Face-S1
GROUPS
LAD LT.
LAD RT.
GROUPS
RAD LT.
RAD RT.
GROUPS GG LT.
GG RT.
N 11 8.3
N 9.5 11.3
N 9.7 9.1 N 9 8.7
N 8.3 9.7
N 10.7 10.7
N 9.5 9.7
N 8.3 10.7
N 10.7 9.1 N 11.7 8.5
N 9.1 11.3
N 11.7 10.1
N 11.1 9.7
N 9.5 10.9
N 12.3 10.9 N 12.7 8.9
N 9.7 13.3
N 12.7 12.1
S1 9.7 8
S1 9.3 9.1
S1 11.1 11.1 S1 10.3 8.7
S1 9.3 10.7
S1 12.9 11.9
S1 9.3 8.7
S1 8.1 14.7
S1 11.9 11.7 S1 9.9 8
S1 9.3 10.7
S1 12.7 12.1
S1 11.7 8.3
S1 9.7 13.1
S1 10.7 12.9 S1 10.9 9.3
S1 9.1 10.4
S1 12.7 12.1
E1 12 8.3
E1 8.7 10.7
E1 12.1 12.1 E1 11.7 8.3
E1 8.3 10.1
E1 12.1 11.3
E1 9.9 8.3
E1 8.7 10.7
E1 10.1 12.1
E1 9.1 9.7
E1 8.7 10.1
E1 9.7 10.7 E1 10 8.3
E1 9.1 9.7
E1 10.7 10.3
E1 11.9 8.7
E1 9.7 9.1
E1 10.7 13.7
S7 10.7 8.3
S7 8.7 10.1
S7 10.3 10.1 S7 12.3 8.3
S7 8.3 12.7
S7 9.9 9.7
S7 9.3 8.3
S7 9.1 9.1
S7 9.1 11.3 S7 10.3 9.1
S7 9.7 10.1
S7 12.1 10.7
S7 10.3 8.5
S7 9.7 10.7
S7 10.7 10.1 S7 8.7 8.3
S7 8.3 10.3
S7 9.7 10.1
E7 10.3 8.3
E7 8.7 11.3
E7 11.9 14.7
E7 10.3 8.1
E7 9.1 9.7
E7 10.7 11.7 E7 11.3 9.7
E7 8.7 10.3
E7 10.1 11.3
E7 10.1 9.7
E7 8.7 10.7
E7 11.7 11.3 E7 8.7 8.3
E7 8.3 9.5
E7 11.7 9.7
E7 9.3 8.7
E7 8.1 9.7
E7 12.1 11.3
S28 12.3 9.3
S28 9.1 10.3
S28 10.3 9.5
269
S28 9 8.7
S28 8.3 11.7
S28 9.7 11.1 S28 10.7 8.3
S28 8.3 10.7
S28 10.7 9.7
S28 12.1 8.7
S28 9.1 10.3
S28 9.1 10.1 S28 10.7 9.1
S28 8.3 9.1
S28 11.3 11.9
S28 10.3 8.3
S28 8.3 9.3
S28 12.3 10.7
E28 12.7 9.5
E28 8.7 12.7
E28 13.1 14.3 E28 11.3 8.3
E28 8.7 11.3
E28 11.7 12.9
E28 9.7 8.5
E28 9.1 11.7
E28 11.7 9.7 E28 10.7 8.9
E28 8.3 10.1
E28 10.3 10.1
E28 10.5 9.3
E28 8.3 10.7
E28 11.7 12.7
E28 9.7 9.1
E28 9.1 12.7
E28 12.1 9.1
270
EXPT. 3.
vonFrey face
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
E-rt 8 8 8 8 6 6 8 8 8 8 10 8 8 8 E-rt 8 8 8 8 6 6 6 8 8 8 8 8 8 8 E-rt 8 8 8 8 6 6 6 6 8 8 8 8 8 8 E-rt 8 8 8 8 6 6 6 6 8 8 8 8 8 8
E-rt 10 8 8 8 6 6 6 6 8 10 10 10 10 10 E-rt 8 8 8 6 4 4 6 6 8 8 8 6 8 6 E-rt 8 8 8 8 6 6 6 6 8 8 8 8 10 8
S- rt 10 10 10 10 10 10 10 10 10 10 8 8 10 10 S- rt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S- rt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S- rt 10 10 10 10 10 10 10 10 10 10 8 8 10 10 S- rt 8 8 8 6 6 6 6 6 6 6 6 8 8 8 S- rt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S- rt 8 8 8 8 8 8 10 10 10 10 8 8 8 8
E-lt 8 8 8 8 8 8 8 8 8 10 10 8 8 8 E-lt 8 8 8 8 6 8 8 8 8 8 10 8 8 8 E-lt 8 8 8 6 6 6 6 8 8 8 10 8 8 8 E-lt 8 8 8 8 6 6 6 8 8 8 8 8 8 8 E-lt 10 10 10 10 6 6 6 8 8 10 10 8 10 10 E-lt 8 8 8 6 4 4 6 6 8 8 8 8 8 8 E-lt 8 8 8 8 6 6 6 6 8 8 8 8 8 8
S-lt 10 10 10 8 8 8 8 8 10 10 8 8 10 10 S-lt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S-lt 8 8 8 8 8 8 8 8 8 8 8 8 8 8
S-lt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S-lt 8 8 8 8 8 8 8 8 8 8 8 6 8 8 S-lt 8 8 8 8 8 8 8 8 8 8 8 8 8 8 S-lt 10 10 10 8 10 8 8 8 10 10 8 10 10 10
vonFrey –upper lip
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
271
E-rt 10 10 8 8 6 8 8 8 10 8 10 10 10 10 E-rt 10 10 10 10 6 6 8 8 8 10 10 8 10 10 E-rt 8 8 10 8 6 6 8 8 8 10 8 8 8 8 E-rt 10 10 10 8 6 6 8 8 10 10 10 10 10 10 E-rt 10 10 10 8 8 8 8 8 8 10 10 10 10 10 E-rt 8 8 8 8 6 6 6 6 8 8 8 10 8 8 E-rt 10 8 10 10 8 8 8 8 8 10 10 10 10 10
S- rt 10 10 10 10 10 10 10 10 10 10 10 10 10 10 S- rt 10 8 10 8 10 10 10 10 10 10 10 10 10 10
S- rt 10 10 10 10 10 10 10 8 8 10 8 8 10 10 S- rt 8 10 8 10 8 8 8 10 8 10 10 10 10 10 S- rt 10 10 10 10 10 10 10 8 10 10 8 8 8 8 S- rt 10 8 8 8 8 10 8 10 10 10 10 10 10 8 S- rt 10 10 10 10 10 10 10 8 10 10 10 10 10 10
E-lt 8 10 10 10 10 10 10 10 10 10 10 10 10 10 E-lt 10 10 10 10 6 6 8 8 8 10 10 10 10 10 E-lt 10 10 10 8 8 8 8 8 10 10 10 10 10 10 E-lt 8 8 8 8 6 6 8 8 8 10 8 8 8 8 E-lt 10 10 10 8 8 8 8 8 8 10 10 10 10 10 E-lt 10 10 10 10 6 6 6 6 8 10 8 8 8 8
E-lt 10 10 10 10 8 8 8 8 8 10 10 10 10 10
S-lt 10 10 10 10 10 10 10 10 10 10 10 10 10 10 S-lt 10 10 10 10 8 10 10 10 10 10 10 10 10 10 S-lt 10 8 8 10 8 10 10 10 10 10 10 10 10 10 S-lt 10 10 10 10 10 10 10 10 8 8 8 8 8 8 S-lt 10 10 10 10 10 10 10 10 10 10 10 10 10 10 S-lt 8 8 8 8 8 8 8 8 10 10 10 10 10 10 S-lt 10 10 10 10 10 10 10 8 10 10 10 10 10 10
vonFreymaxillary incisor gingival
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
E-rt 6 6 6 4 4 4 4 4 6 6 6 6 6 6 E-rt 4 4 4 2 2 2 2 4 4 4 6 4 4 4 E-rt 6 6 6 4 2 4 4 4 6 6 6 6 6 6 E-rt 6 6 6 2 2 2 2 2 4 6 6 6 6 6
272
E-rt 6 6 6 4 4 4 4 4 6 6 6 6 6 6 E-rt 6 6 6 4 2 2 2 2 6 6 6 6 6 6 E-rt 4 4 4 4 2 2 2 2 4 4 4 4 4 4
S- rt 6 6 6 6 6 6 6 6 6 6 6 6 6 6 S- rt 6 6 4 4 4 4 4 4 6 6 6 6 6 6 S- rt 6 6 6 6 6 6 6 6 6 6 6 6 6 6 S- rt 4 4 4 4 4 4 4 4 4 4 4 6 4 4 S- rt 6 4 6 6 6 6 6 6 6 6 6 6 6 6 S- rt 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S- rt 6 6 6 6 6 6 6 6 6 6 6 6 6 6
E-lt 6 6 6 4 4 4 4 4 6 6 6 6 6 6 E-lt 4 4 4 2 2 2 2 4 4 4 4 4 4 4 E-lt 6 6 6 4 4 4 4 4 6 6 6 6 6 6 E-lt 6 6 6 2 2 2 2 2 6 6 6 6 6 6 E-lt 6 6 6 4 4 4 4 4 6 6 6 6 6 6 E-lt 6 6 4 4 2 2 2 2 6 6 6 6 6 6 E-lt 4 4 4 4 2 2 2 2 4 4 4 4 4 4
S-lt 6 6 6 6 6 6 6 6 6 6 6 6 6 6 S-lt 6 4 4 4 4 4 4 4 6 6 6 6 6 6 S-lt 6 6 6 6 6 6 6 6 6 6 6 6 6 6
S-lt 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S-lt 6 6 6 6 6 6 6 6 4 4 6 4 6 6 S-lt 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S-lt 6 6 6 6 6 6 6 6 6 6 6 6 6 6
Thermal duration
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
E-lt 0 0 0 0 1.1 1.1 1.1 0.9 0.5 0 0 0 0 0 E-lt 0 0.5 0 0 0.5 0.5 0.5 0.5 0.5 0 0 0 0 0
E-lt 0 0 0 0 1.1 1 0.9 0.9 0 0 0 0 0 0 E-lt 0 0 0.5 0.5 1 1 1 0.9 0.5 0 0 0 0.5 0 E-lt 0 0 0 0 0.5 1 0.5 0 0 0 0 0 0 0 E-lt 0.5 0 0.5 0.5 1.2 1.2 1 1 1 0.5 0.5 0 0 0 E-lt 0.5 0.5 0.5 0.5 1.1 1 1 1 0 0 0 0 0.5 0
S-lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0
273
S-lt 0 0.5 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 0 0 0 0.5 0.5 0.5 0.5 0.5 0 0 0 0 0 S-lt 0.5 0 0.5 0.5 0.5 0.5 0 0 0 0 0 0 0 0 S-lt 0 0.5 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 0 0 0 0.5 0 0.5 0 0.5 0 0 0 0 0
E-rt 0 0.5 0 0 1.2 1.1 1.1 1 0.7 0.5 0 0 0 0 E-rt 0 0 0 0 1.2 1.2 1.2 1.2 1.2 0.5 0.5 0.5 0 0 E-rt 0 0 0 0 1.1 1.1 1 1 0.8 0 0 0 0 0 E-rt 0 0 0.5 0.5 1.2 1.2 1.2 0.9 0.9 0 0 0 0.5 0.5
E-rt 0.5 0.5 0.5 0.5 1.1 1.1 1.1 1 1 0 0 0 0 0 E-rt 0 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0 0 E-rt 0.5 0.5 0.5 0.5 1.2 1.2 1.2 1.1 0.9 0 0 0 0.5 0.5
S-rt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S-rt 0 0 0.5 0.5 0 0 0 0 0 0 0 0 0 0 S-rt 0 0 0 0 0 0 0 0 0 0 0 0 0.5 0 S-rt 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0 0 0 0.5 S-rt 0 0 0 0 0 0 0 0 0 0.5 0.5 0 0 0 S-rt 0 0 0 0 0.5 0.5 0.5 0 0 0 0 0 0 0 S-rt 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0.5 0 0 0 0 0.5
Thermal score
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
E-lt 0 0 0 0 3 3 3 2 1 0 0 0 0 0 E-lt 0 1 0 0 1 1 1 1 1 0 0 0 0 0 E-lt 0 0 0 0 3 2 2 2 0 0 0 0 0 0 E-lt 0 0 1 1 2 2 2 2 1 0 0 0 1 0 E-lt 0 0 0 0 1 2 1 0 0 0 0 0 0 0 E-lt 1 0 1 1 3 3 2 2 2 1 1 0 0 0 E-lt 1 1 1 1 3 2 2 2 0 0 0 0 1 0
S-lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 1 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S-lt 0 0 0 0 1 1 1 1 1 0 0 0 0 0 S-lt 1 0 1 1 1 1 0 0 0 0 0 0 0 0 S-lt 0 1 0 0 0 0 0 0 0 0 0 0 0 0
274
S-lt 0 0 0 0 1 1 1 0 1 0 0 0 0 0
E-rt 0 1 0 0 3 3 3 2 1 1 0 0 0 0 E-rt 0 0 0 0 3 3 3 3 3 1 1 1 0 0 E-rt 0 0 0 0 3 3 2 1 2 0 0 0 0 0 E-rt 0 0 1 1 3 3 3 2 2 0 0 0 1 1 E-rt 1 1 1 1 3 2 2 2 2 0 0 0 0 0 E-rt 0 0 0 0 1 1 1 1 1 1 1 0 0 0 E-rt 1 1 1 1 3 3 3 3 2 0 0 0 1 1
S-rt 0 0 0 0 0 0 0 0 0 0 0 0 0 0
S-rt 0 0 1 1 0 0 0 0 0 0 0 0 0 0 S-rt 0 0 0 0 0 0 0 0 0 0 0 0 1 0 S-rt 0 0 1 1 1 1 1 1 1 0 0 0 0 1 S-rt 0 0 0 0 0 0 0 0 0 1 1 0 0 0 S-rt 0 0 0 0 1 1 1 0 0 0 0 0 0 0 S-rt 1 1 1 1 1 1 1 0 1 0 0 0 0 1
Thermal percentile rate
Groups
day -1
1 hrs.
3 hrs.
6 hrs.
24 hrs.
day 2
day 3
day 4
day 5
day 6
day 7
day 14
day 21
day 28
E-lt 20 0 0 40 80 80 60 60 60 0 20 40 0 20 E-lt 0 60 40 0 60 60 60 60 60 20 40 0 0 20 E-lt 20 20 0 0 80 80 60 80 40 40 20 20 20 0 E-lt 20 20 60 60 80 80 60 60 60 20 0 20 20 0 E-lt 0 0 20 0 60 80 60 40 40 20 40 0 0 20 E-lt 60 20 60 60 100 100 80 80 80 60 60 20 20 0 E-lt 60 60 60 60 100 80 80 60 40 40 20 0 60 0
S-lt 0 0 20 20 40 20 20 20 20 40 20 0 0 20 S-lt 40 60 0 0 20 20 40 20 20 0 0 20 20 0
S-lt 0 20 20 20 20 40 20 20 40 20 40 20 20 0 S-lt 20 20 0 40 60 60 60 60 60 20 0 20 0 20 S-lt 60 0 60 60 60 60 20 20 0 0 20 0 20 20 S-lt 0 60 0 0 0 20 20 0 20 20 20 20 0 0 S-lt 0 0 20 0 60 60 60 0 60 20 0 0 0 0
E-rt 0 60 40 40 100 100 100 80 60 60 40 40 0 20 E-rt 40 20 20 20 80 80 80 80 80 60 60 60 20 20
275
E-rt 40 0 20 20 100 100 80 60 80 20 20 0 20 0 E-rt 0 0 60 60 100 100 80 80 80 20 40 20 60 60 E-rt 60 60 60 60 100 80 80 80 80 20 20 20 0 0 E-rt 20 0 0 0 60 60 60 60 60 60 60 20 20 20 E-rt 60 60 60 60 100 100 80 100 80 40 20 0 60 60
S-rt 40 20 0 20 20 20 40 40 20 40 20 0 20 0 S-rt 0 20 60 60 20 0 20 0 0 20 0 20 0 0 S-rt 0 0 0 20 40 40 20 20 0 0 20 40 60 0 S-rt 20 20 60 60 60 60 60 60 60 0 0 20 40 60 S-rt 20 0 20 20 0 20 0 0 0 60 60 20 0 0
S-rt 0 20 0 0 60 60 60 20 20 20 0 0 20 0 S-rt 60 60 60 60 60 60 60 40 60 20 20 0 0 60