the effects of shaking on the eye and central nervous ... · shaking did not immediately cause...

The effects of shaking on the eye and central nervous system of

mice and Barbados Green Monkeys

by

Jin Han (Patrick) Kim

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

Copyright by Patrick J.H. Kim 2009

ii

Abstract

The effects of shaking on the eye and central nervous system of

mice and Barbados Green Monkeys

Patrick J.H. Kim

Master of Science

Department of Laboratory Medicine and Pathobiology

University of Toronto

2009

Shaken baby syndrome is a clinicopathologic syndrome characterized by a triad of findings:

subdural hemorrhage, retinal hemorrhage and axonal injury. Although shaking is widely

believed to cause the triad, it is not yet entirely clear if shaking without head impact can produce

the triad. Initial attempts to test the effect of shaking in mouse pups were unsuccessful as neither

controlled continuous vibration nor pulse acceleration caused any of the components of the

triad. With no other convenient modeling system available, a pilot study with three adult

subhuman primates was conducted. Although a conclusive statement cannot be made, manual

shaking did not immediately cause hemorrhagic injuries to the primates’ brains and eyes. Future

studies should test for delayed development of axonal injury. In addition, a comparative

anatomical study should also be conducted to test the validity of the adult primate as a model

system for human infant injuries.

iii

Foundations

It is those who know little, and not those who know much, who so positively assert that this

or that problem will never be solved by science.

- Charles Darwin (1809 ~ 1882), Introduction to The descent of man and selection in relation to

sex (1871)

iv

Acknowledgements

I thank the members of my advisory committee for their support and guidance: Drs. C.

Bergeron (chair), M. Johnston, and M.S. Pollanen (supervisor). I also thank Dr. T.H. Rose, Ms.

E. Bidgoli and Ms. A. Mainiero for reading and editing the earlier drafts of this thesis. Special

thanks go to Drs. K. Cunningham, I. Kitulwatte and D.N. McAuliffe for valuable discussions

regarding general issues in forensic pathology.

I acknowledge the Department of Laboratory Medicine and Pathobiology for the visiting

trainee graduate award. I was fortunate to have the help of Mr. D. Kang and Ms. M. Currie,

histotechnologists, who answered countless questions I had about processing animal tissues. I

would like to thank Ms. B. Anders and Ms. R. Perri for their support in procurements. I also

thank all other members at the Forensic Pathology Unit, Office of the Chief Coroner for Ontario

for their daily support.

Special appreciation goes to Ms. J. Manias for her technical assistance and Dr. S. Nag for

graciously providing laboratory space and equipments for immunostaining.

I would like to thank Ms. T. McCook and L. Penny at Division of Comparative Medicine for

their assistance in animal handling. Also, Dr. K. Banks at DCM provided invaluable help in

establishing animal protocols. I would also like to thank everyone at BPRC for their hospitality

and assistance. Mr. D. Clutterbuck provided expert assistance in autopsy and photography. All

gross photographs for the Barbados Green Monkey study were taken by Mr. Clutterbuck.

Finally, my very special gratitude goes to my supervisor, Dr. M.S. Pollanen for his constant

support of this thesis through challenges and unexpected delays. This project is dedicated to my

family.

v

Part of the materials in this thesis was presented at the Canadian Association of

Neuropathologists annual meeting in 2007 in a talk “Non-Impact Head Injury in Infants –

Mouse Model of Shaken Baby Syndrome”, which won the Morrison H. Finlayson award.

vi

Table of contents

Abstract ii

Foundation iii

Acknowledgements iv

Table of contents vi

List of tables xi

List of figures xii

Abbreviations xiv

Chapter 1. Introduction and literature review 1

1.1. Introduction 1

1.2 Infant head and neck 3

1.2.1 Anatomical considerations 3

1.2.2. Pattern of head and neck injuries in child abuse 3

1.2.2.1. Injuries by blunt impact 3

1.2.2.2. Injuries by sudden accelerations of the head 4

1.2.2.3. Typical head and neck injuries seen in child abuse 4

1.3. Rise of Shaken Baby Syndrome 6

1.3.1. Historical considerations 6

1.3.2. Caffey’s Lecture in 1972 7

1.3.3. Retrospective case series vs. Anecdotal case reports 9

1.4. Study of Shaken Baby Syndrome 12

1.4.1. Animal models of Shaken Baby Syndrome 12

1.4.2. Mechanical models of Shaken Baby Syndrome 13

1.4.3. Evidence-based reviews of the retrospective case series 14

1.5. Current points of controversy 18

1.5.1. Pathologic nature of ‘pure shaking’ 18

1.5.1.1. Immediate traumatic injuries due to shearing and traction 18

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1.5.1.2. Hypoxic and ischemic encephalopathy due to brainstem injury 19

1.5.2. Specificity of the triad 19

1.5.2.1. Short fall debate 22

1.5.2.2. Confounding medical conditions 23

1.5.3. Nature of subdural hemorrhages 24

1.6. SBS in criminal justice system 26

1.6.1. Harris appeal and Shaken Baby Syndrome cases review in Britain 26

1.6.2. Goudge inquiry in Ontario 27

Chapter 2. Experimental design 28

2.1. Hypothesis 28

2.2. Research Objective 28

2.2.1. Overall 28

2.2.2. Specific aims 29

2.3. Rationale 30

2.3.1. Advantages of animal model 30

2.3.2. Indication from previous studies 30

2.3.3. American Academy of Pediatrics technical report (2001) 31

2.4. Material and methods 33

2.4.1. High frequency vibration of postnatal mice 33

2.4.1.1. Mice 33

2.4.1.2. Shaking apparatus 33

2.4.1.3. Displacement measurement 36

2.4.1.4. Anesthesia and euthanasia 36

2.4.1.5. Tissue processing and histology 36

2.4.2. Manual shaking of Barbados Green Monkeys 40

2.4.2.1. Barbados Green Monkeys 40

2.4.2.2. Accelerometer 41

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2.4.2.3. Anesthesia and euthanasia 41

2.4.2.4. Postmortem examination 47

2.4.2.5. Tissue processing and histology 47

2.4.2.6. Immunohistochemistry 48

2.5. Experimental protocol 49

2.5.1. Mice 49

2.5.1.1. Constant vibration 49

2.5.1.2. Pulse acceleration 50

2.5.1.3. Hinge-point 50

2.5.1.4. Displacement measurement 51

2.5.2. Barbados Green Monkeys 52

2.5.2.1. Sham (Negative control) 52

2.5.2.2. Anterior-posterior shaking 52

2.5.2.3. Lateral shaking 53

2.6. Ethics of animal use 56

2.6.1. Mice 56

2.6.2. Barbados Green Monkeys 56

Chapter 3. Results: Mice 57

3.1. Summary of findings 57

3.2. Constant vibration study 57

3.3. Pulse acceleration study 58

3.4. Hinge-point study 58

3.5. Vibration frequency and displacement 63

Chapter 4. Results: Barbados Green Monkey 66

4.1. Summary of findings 66

4.2. Physiological responses after shaking 67

ix

4.3. Anatomical and histologic findings 73

4.3.1 Brain and cervical spinal cord 73

4.3.2. Eyes and retina 102

4.3.3. Neck 102

4.3.4. Fingertip bruising 102

4.4. Accelerometry data 111

4.4.1. Sham animal 111

4.4.2. AP animal 111

4.4.3. Lat animal 111

Chapter 5. Discussion 119

5.1. Mice 119

5.1.1. General discussion 119

5.1.2. Negative findings: What do they mean? 120

5.1.3. Role of mouse model in future investigations of Shaken Baby Syndrome 122

5.2. Barbados Green Monkeys 124

5.2.1. General discussion 124

5.2.2. Negative findings: What do they mean? 125

5.2.3. Role of Barbados Green Monkey model in future investigations of Shaken Baby Syndrome 128

5.3. Back to Shaken Baby Syndrome debate: significance of experiments performed 130

5.3.1. True non-impact head injury protocol 130

5.3.2. Mechanical properties of Barbados Green Monkey manual shaking 130

5.3.3. Empirical data for Shaken Baby Syndrome discussion 131

Chapter 6. Future directions 133

6.1. Primate model of Shaken Baby Syndrome 133

6.1.1. Axonal injury 133

6.1.2. Describing head and neck movement during shaking 134

x

6.1.3. Ethical consideration 135

6.1.4. Anthropomorphic model 135

6.1.5. Mechanical properties of tissues and finite element modeling 136

6.2. Hypoxia as cause of subdural hemorrhages 137

6.2.1. Perinatal and neonatal intradural hemorrhages 137

6.2.2. Implication for Shaken Baby Syndrome ‘unified hypothesis’ 137

6.3. Medical investigations of Shaken Baby Syndrome 139

6.3.1. Retrospective and prospective studies using APP immunostaining 139

6.3.2. Cerebrospinal fluid analysis 139

6.4. Possible mechanism of Shaken Baby Syndrome triad development 140

Chapter 7. References 141

Appendix 150

A. Experimental protocols 150

A-1. Routine H&E staining protocol 150

A-2. Routine LFB/H&E staining protocol 152

B. Ethics 154

B-1. Revised Barbados Green Monkey manual shaking research proposal 154

B-2. Response to reviewers’ comments 165

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List of tables

Table 1-1. Summary of cases used by Caffey in 1972 article

Table 2-1. Selection of Barbados Green Monkey for the study

Table 2-2. Barbados Green Monkey Anesthesia/Euthanasia records

Table 4-1. Barbados Green Monkey morphometric dimensions

Table 6-1. Proposed experimental groups for the study of axonal damage from manual shaking

of Barbados Green Monkey

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List of figures

Figure 1-1. Photomicrographs of traumatic axonal injury (low/high power, APP)

Figure 1-2. Flow chart of two possible mechanisms of shaking injuries

Figure 2-1. Experimental setup of high frequency vibration of postnatal mice

Figure 2-2. Whole mount of postnatal mice head and neck

Figure 2-3. Overall photograph of an Barbados Green Monkey

Figure 2-4. Mounting of accelerometer

Figure 2-5. Experimental setup of manual shaking of Barbados Green Monkeys

Figure 3-1. Photomicrographs of A. Dural space, B. Retina, C. Cerebral cortex

Figure 3-2. Focal subarachnoid hemorrhage

Figure 3-3. Displacement over time plot of the shaking apparatus

Figure 4-1. Anterior-posterior shaking

Figure 4-2. Lateral shaking

Figure 4-3. Brain and cervical spinal cord of Sham animal. Gross View.

Figure 4-4. Brain and cervical spinal cord of Anterior-posterior animal. Gross View.

Figure 4-5. Brain and cervical spinal cord of Lateral animal. Gross View.

Figure 4-6. Coronal sections of Sham animal brain after formalin fixation

Figure 4-7. Horizontal sections of Sham animal brainstem after formalin fixation

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Figure 4-8. Coronal sections of Anterior-posterior animal brain after formalin fixation

Figure 4-9. Horizontal sections of Anterior-posterior animal brainstem after formalin fixation

Figure 4-10. Coronal sections of Lateral animal brain after formalin fixation

Figure 4-11. Horizontal sections of Lateral animal brainstem after formalin fixation

Figure 4-12. Photomicrographs of cervical spinal cord of Barbados Green Monkey

Figure 4-13. Photomicrographs of pons of Barbados Green Monkey

Figure 4-14. Photomicrographs of medulla of Barbados Green Monkey

Figure 4-15. Photomicrographs of cerebral cortex of Barbados Green Monkey

Figure 4-16. Photomicrographs of posterior corpus callosum of Barbados Green Monkey

Figure 4-17. Retina after formalin fixation.

Figure 4-18. Photomicrographs of retina.

Figure 4-19. Posterior neck dissection.

Figure 4-20. Fingertip bruising found of body surfaces.

Figure 4-21. Accelerometry tracing from Sham treatment

Figure 4-22. Accelerometry tracing from Anterior-posterior animal shaking

Figure 4-23. Accelerometry tracing from Lateral animal shaking

xiv

Abbreviations

APP -amyloid precursor protein

AAP American Academy of Pediatrics

AP Anterior-posterior

BGM Barbados Green Monkeys

BPRC Barbados Primate Research Center

CSF Cerebrospinal fluid

CVP Central venous pressure

DAB 3,3’-Diaminobenzidine

G Gravitational acceleration unit (1G = 9.80665 m/s2)

H&E Hematoxylin-eosin stain

ICP Intracranial pressure

IDH Intradural hemorrhages

IM Intramuscular

IV Intravenous

LACC Local Animal Care Committee

Lat Lateral

LFB Luxol-fast-blue stain

PBS Phosphate buffered saline

RBC Red blood cell

RH Retinal hemorrhages

SBS Shaken baby syndrome

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SDH Subdural hemorrhages

TAI Traumatic axonal injury

tDAI Traumatic diffuse axonal injury

1. Introduction and literature review

1.1 Introduction

When a person dies, it is in the public interest to understand how the person came to die. This

is especially true in the case of deceased babies where public interest may be highly emotional,

and legal action may follow if any unlawful actions contributed to death. During death

investigations, forensic pathologists conduct autopsies and provide key evidence that may

influence the direction of the investigations. By performing a postmortem examination, a

pathologist seeks to determine the cause, and mechanism of death as well as facilitate a

determination of the manner of death. A postmortem examination consists of a series of steps

starting from the scene investigation and collection of the history, external and internal

examination of the body, ancillary testing on samples collected from the examination, and

interpretation of the findings to create a knowledgeable and balanced opinion.

In the past ten years, a considerable degree of controversy in the field of pediatric forensic

pathology in Toronto has come to public exposure by a series of wrongful conviction claims. As

Commissioner Goudge stated in his report for the Inquiry into Pediatric Forensic Pathology in

Ontario, the problematic issues that arose did not solely represent “a simple failure of any

individual, but in addition it represents systemic issues” 1. The bases of the controversy are

intricate and fundamental differences in the methods of gaining and interpreting knowledge in

the fields of medicine, science and the criminal justice system.

During the past three years at the University of Toronto and the Provincial Forensic

Pathology Unit, Ontario, I have focused on the topic of shaken baby syndrome (SBS) which

illustrates Commissioner Goudge’s statement very clearly. SBS is a combination of a clinical

and pathological findings in infants that are believed to be violently shaken by their caregivers 2.

1

In my effort to understand the SBS controversy, I came to appreciate that each group that

receives and interprets such findings made by forensic pathologists has a specific knowledge

base and unique requirements that influence how that information will be utilized. From the

scientists’ perspective, the goal is to discover the truths about the natural world by objectively

asking questions, and testing thoughfully developed hypotheses through experimentation. Due

to the very nature of this approach, existing frameworks to understand the truth (theories) are

questioned when new observations do not conform to the accepted perspective and the cycle of

inquiry begins anew. In medicine, the goal is to save lives, and the knowledge gained through

repetitive experience is just as important as scientific knowledge. When a new scientific

paradigm does not fit the previously accepted dogma, medicine reacts in a conservative manner

until a new body of experience confirms or refutes the scientific knowledge. In contrast, the

justice system uses expert opinions as a foundation of understanding that will be evaluated along

with other factual evidence from the death investigation. However, the justice system as a whole

or the expert witnesses themselves may fail to recognize that opinion evidence is subject to

change with advances in science and medicine. Failure to recognize the ever changing base of

scientific and medical knowledge could lead to devastating consequences as illustrated by

wrongful convictions associated with SBS.

This dissertation represents my effort to address the SBS controversy in an objective and

scientific manner. In chapter one, I have reviewed the literature to present the contentious issues

at hand and how they have affected the criminal justice system. In the subsequent chapters, I

have detailed my approach to the investigation of the issues surrounding the effect of shaking on

human infants with murine and Barbados Green Monkeys (BGM) models. A discussion of the

experimental results is found in chapter five which forms the basis of the future directions in

chapter six.

2

1.2 Infant head and neck

1.2.1 Anatomical considerations

The temporally disproportionate development of the anatomy of the head and neck in human

infants leads to its distinct morphology and susceptibility to external mechanical forces, such as

through shaking. The initially large head-to-trunk ratio of the neonate decreases until it reaches

that of the adults. In addition, the neck musculature continues to develop during infancy to

support the large cranium in bipedal locomotion. Other anatomic differences between the infants

and the adults include greater tensile strength of the scalp due to its higher collagen content,

patency of cranial sutures that gradually ossify over time, continued development of the

meningeal layers as the dura is not yet firmly attached to the inner table. Furthermore, the

relatively high water content of the brain decreases during the early years of life as a result of

continuous myelination of the axons. These developmental patterns lead to distinct

physiological properties during infancy that are most prominent immediately after birth and

gradually diminish with time as the child physically matures into an adult. The mechanically

important characteristics of an immature head and neck anatomy are: 1. A large head supported

by undeveloped neck structures, 2. A greater range of movement for the brain within the

cranium, and 3. A scalp which can stretch greatly without being restricted by the cranium.

1.2.2. Pattern of head and neck injuries in child abuse

1.2.2.1. Injuries by blunt impact

Blunt impact can be defined as a transfer of energy by a (typically) non-penetrating contact

event. The impact energy is transmitted to the different layers underneath the impact site. The

extent of tissue damage is determined by the force transmitted by the impact event and by the

injury threshold of the tissue. The extent of injury caused by the same impact varies for different

3

tissues. In addition, anatomical and morphological features must be considered to correlate with

the type of force that caused the observed injuries. The aforementioned anatomical properties of

infants have been thought to alter the extent of the blunt impact injuries from what would be

seen in adults after similar impact events. These differences pose a great challenge in

ascertaining the impact event from the pattern of injuries. Some of these factors include: 1. A

large head supported by a weak neck, which results in a higher susceptibility to the acceleration

after the impact, 2. A pliable cranium allows for more space for the brain to move independently

of its underlying structures following impact, potentially resulting in further damage, and 3.

Finally, as a consequence of the scalp’s increased elasticity and tensile strength, the extent of the

damage to the scalp could be inconsistent with the injuries seen on the structures underneath.

1.2.2.2. Injuries by sudden acceleration of the head

Most investigations of injuries caused by sudden acceleration of the head have been

conducted regarding motor vehicle crashes. It is now understood that, in adults, both linear and

rotational acceleration can cause damage to the head and neck without direct impact.

Acceleration injuries include: 1. Stretch injuries to the brainstem, 2. Traumatic diffuse axonal

injury, and 3. Fractures of vertebrae in severe accelerations. These injuries can occur in

combination with impact (impact-acceleration) and when they do, it is hard to determine the

contribution of each mechanism to the injuries.

1.2.2.3. Typical head and neck injuries seen in child abuse

Child abuse may be defined as “a large, complex group of human behaviours characterized

by traumatic interactions between parents or other caretakers and the infants and children of all

ages under their care, as well as between strangers and children during casual contact” 3. It is

not limited to physical trauma; other forms of maltreatment such as emotional as well as sexual

4

abuse, and various forms of neglect could also be part of child abuse. The American Academy

of Pediatrics (AAP) recognizes failure to thrive as another sign of child abuse 4. In chronic child

abuse, it is not uncommon to find at postmortem examination multiple bruises of different ages

on the scalp and face, skull fractures, retinal hemorrhage (RH), chronic and acute subdural

hemorrhages, generalized brain swelling and cerebral contusions of varying ages. Other injuries

to the body include long bone and rib fractures, metaphyseal fractures, visceral injuries and

burns. Although many of these injuries are primary consequences of specific physical

mechanisms, it is important to note that the broad timeframe of child abuse could lead to chronic

and secondary manifestations of the primary injuries.

5

1.3. Rise of the Shaken Baby Syndrome (SBS)

1.3.1. Historical considerations

Although the famous French forensic pathologist Ambroise Tardieu first described the

patterns of child abuse in 1860 5, it was not until early in the twentieth century that physicians

started to recognize child abuse as a separate entity. While most injury patterns were similar to

those of adults and thus generally reflected well understood mechanisms of infliction, some

unique injuries were seen in these infants that did not follow the pattern of direct impact

inflicted injuries. The term ‘whiplash injuries’ were used to describe the set of clinical findings

in abused babies that was thought to be caused by a whiplash motion of the head due to sudden

acceleration or deceleration. These clinical findings included gross hemorrhages and contusions

over the surface of the brain and upper cervical cord, cerebral concussion, and subdural

effusions 6.

From 1964 to 1969 Ommaya et al. published a set of studies in which the heads of primates

were subjected to sudden acceleration, causing a constellation of injuries similar to that of

‘battered babies’ 7-12. In 1971, Guthkelch published a case report in which he found subdural

hemorrhages in babies who had been shaken and noted a similarity to Ommaya’s experimental

findings 13. This, he felt, strongly correlated with a shaking mechanism. However, since the

findings that were supposedly caused by a whiplash mechanism were reported in the context of

battered babies, other cranial findings such as skull fractures and scalp bruising were present

and were often used as an indicator of an abusive event. It should be mentioned that other causes

of such intracranial injury in infants such as birth trauma were already known at this time.

6

1.3.2. Caffey’s Lecture in 1972

In 1972, Dr. J. Caffey gave an Abraham Jacobi lifetime achievement award lecture entitled

“On the theory and practice of shaking infants: Its potential residual effects of permanent brain

damage and mental retardation” 14. In this lecture, Caffey first described the metaphyseal bone

lesions in infants that were then believed to be pathologic features of shaking. Caffey then

reviewed the clinical and pathological findings of abusive trauma from multiple previous reports

along with three cases of his own. Of 29 cases of ‘whiplash-shaking’ (which Caffey mistakenly

reported as a total of 27 cases), bone lesions were found in six cases, surgically treatable

subdural hemorrhage (SDH) in three cases, and retinal hemorrhage (RH) in three cases. Results

from only one postmortem examination were mentioned in these cases of traumatic brain injury

(Table 1-1). Reporting on these seemingly uncommon incidents, Caffey primarily drew from his

experience and the Ommaya study 10 to conclude that in ‘whiplash-shaking’ causes 1. Bilateral

subdural hematoma caused by ‘indirect acceleration traction stresses’ are frequently

undiagnosed 2. Bilateral retinal lesions are indicative of subclinical chronic subdural hematoma

and 3. Early cerebral edema could lead to diffuse gliosis and cause infantile obstructive

hydrocephalus. Caffey also argued that the bilaterality of SDH and RH and the lack of external

signs of impact were suggestive of a non-impact origin such as ‘whiplash-shaking’. Caffey

described at length examples of child abuse and stated that infant shaking ‘appears to be

practiced widely’ and the number of incidents ‘cannot be even estimated satisfactorily’. He

concluded that ‘whiplash-shaking is always potentially pathogenic to some degree’, and when

repeated, ‘even relatively mild shaking…are probably more pathogenic than … more violent

and conspicuous shakings during wilful assault’.

7

Table 1-1. Summary of 29 cases mentioned in Caffey’s 1972 article

Case history Findings

Example 1 (15 cases)

An infant-nurse admitted killing three infants and injuring 12 more by shaking. Shaking and pounding was done to help infants burp

“Traumatic brain injury” in one fatal case

Example 2 (4 cases)

Two cases of young children being shaken, a case of infant being shaken and banged against the crib, and a case of an infant being shaken and beaten to death

No mention of pathologic findings

Example 3 (3 cases)

a) A case of an infant who died after being shaken to stop paroxymal coughing b) A case of an infant who was found convulsing and vomiting; parents admitted to shaking c) A case of an infant with broken femur; parents admitted to shaking

a) SDH

b) SDH, RH, bruising on forearms that fit adult hands

c) No other pathologic findings mentioned

Example 4 (1 case)

A case of young infant who was gripped by legs and shaken upside down

Multiple massive involucra and metaphyseal avulsions in femurs and tibias

Example 5 (1 case)

“Believed to be whiplash-shaken” Compression fractures of vertebral bodies

Example 6 (3 cases)

a) Two cases of infants who were yanked upwards to prevent from falling b) A case of an infant who was shaken and swung onto the bed by 8 year old sibling

a) Massive involucra of the radius and ulna in one, and avulsion metaphyseal fracture and traumatic involucra of tibia in another b) Traumatic involucra of both femurs

Example 7 (2 cases)

a) A case of an infant who was grabbed by the legs and swung around b) A case of an infant who was gripped by the thorax and shaken violently

a) SDH and RH b) RH

Total (29 cases)

Heterogeneous history with many with concurrent impact events

1 Traumatic brain injury 3 SDH 3 RH Remainder with bone lesions

8

1.3.3. Retrospective case series vs. Anecdotal case reports

In his 1972 lecture, Caffey stated that the findings of subdural hemorrhage, retinal

hemorrhage, and diffuse gliosis are due to the mechanical shearing or traction force generated

from shaking. However, none of his 29 case examples could qualify as pure shaking incidents

where the possibility of an impact event could be ruled out. Most cases had a history of

concurrent impact whether or not evidence of an impact could be found on examination.

Subsequent case reports also claimed that the reported findings are due to inertial force applied

during shaking. However, it is now understood that these cases might have had evidence of

impact that was missed or disregarded as a contributory factor at the original examination.

Although inadequate by today’s standards, the series of articles by Ommaya et al. 7-12 and

Caffey 14 was enough to raise awareness of SBS among physicians. The newly created clinical

entity of SBS was now considered to be a ‘previously ignored’ clinical presentation of child

abuse. Many retrospective studies using a group of known physical abuse cases were published

confirming the presence of SDH and RH along with axonal injuries which were detected by

silver stain on microscopic slides of the brain 15-20. From these studies, traumatic Diffuse Axonal

Injury (tDAI) was proposed as a histological correlate of the shaking injuries, and the swelling

of the brain originally described was then considered as a secondary response to such injuries.

The triad was now redefined to be 1. SDH, 2. RH and 3. tDAI. This had very significant legal

implications since infants with tDAI could not be expected to be alert for any period of time

after the initial injury (lucid interval). tDAI by definition involves disruption of the reticular

activating system that would render the infants immediately unconscious. As result, the finding

of tDAI at postmortem examination implies that the last caregiver who witnessed the infant

alive is the one to blame for the demise. The finding of tDAI was regarded as independent

9

pathologic evidence of shaking and many convictions were made without confessions, based on

the finding of tDAI at postmortem examination.

There are two major shortcomings of these retrospective studies which need to be

understood. First is the inherent fundamental limitation of retrospective studies. Retrospective

studies rely on the indirect correlation between the majority of findings present in a group of

cases and the criteria used to select such a group. It is a very powerful approach to defining a

clinical entity if the selection criteria have been designed to confer specificity. However, the

methods by which the findings were observed cannot be ascertained with this approach, and

even with well-devised selection criteria, it can only illustrate a strong correlation at best. As

mentioned previously, retrospective studies in the 1980s and 1990s did not have proper selection

criteria to correlate the shaking mechanism with the observed injuries. In the first group of

reports, the study criteria included all suspected abuse cases and simply attributed all cases

without other significant findings to shaking 21, 22. The other group of studies analyzed fatal

infant head trauma and attributed those without evidence of impact to the result of shaking 23, 24.

With such broad criteria, it is not inconceivable that the findings are heterogeneous and do very

little to isolate the possible mechanisms for the injuries present. Furthermore, both of these

approaches are inadequate in ascertaining the mechanism of injury, since the ‘evidence’ of

shaking does not derive from the findings themselves but rather from the assumption that these

injuries are caused by shaking. These studies should serve as the starting point in the search for

mechanisms responsible for injuries that cannot be explained by known mechanisms such as

impact to the head.

Another approach in medicine to define or refute a clinical entity is by the accumulation of

anecdotal reports. By examining cases that do not fit the existing paradigm, anecdotal case

reports may help in refining clinical entities. If enough anecdotal reports are accumulated, the

10

existing entity may cease to be accepted altogether and a newer entity could be defined. When

infant head injuries in the context of child abuse were beginning to be recognized as a separate

entity, numerous anecdotal reports of birth trauma presenting in suspicious circumstances were

also published. Once SBS was defined and the triad was being used as pathognomonic of SBS,

many anecdotal reports were soon published and formed the basis for the controversies which

are still being debated now. These anecdotal reports may be grouped into following categories;

1. Cases of infant head trauma caused by an impact-related mechanism presenting with the triad

but with no sign of impact 25, 2. Cases presenting with the triad of SBS where alternate causes

are known (such as disease conditions or resuscitation efforts) 26-38, or 3. Cases demonstrating

the potential misdiagnosis of the triad in other disease processes 39-44. Anecdotal case reports

constantly evaluate the validity of a medical entity where the mechanism is not known.

Unfortunately, similar to the process of elimination, it is an indirect approach and cannot be

used to directly confirm whether shaking could cause the triad.

11

1.4. Study of SBS

1.4.1. Animal models of SBS

The first animal models of inertial head injury were published in 1964 7. Ommaya

demonstrated an experimental concussion caused by occipital impact acceleration in monkeys.

The study presented a series of linear acceleration thresholds for experimental cerebral

concussion, and proposed that these curves could be used as a baseline for non-impacting

impulsive loads. Throughout the late 1960s and early 1970s, Ommaya et al. reported a series of

experiments where whiplash motion was modelled as a single episode of acceleration delivered

by a mechanical actuator 7, 9, 45-48. Gennarelli et al. 18 and others 16, 49-53 further characterized this

model. It showed that all putative markers of SBS (except retinal hemorrhages which were

unexamined) were produced by a single rotational acceleration to the head on the coronal plane

(side-to-side on lateral axis) to the head. It is interesting that the rotational acceleration on the

sagittal plane (which is commonly believed to be a type of acceleration an infants’ head would

experience in an abusive shaking event) only produced a concussion while the lateral rotational

acceleration produced more severe effects including SDH and coma. Histological methods using

silver impregnation correlated diffuse axonal injury with the acceleration injuries. It should be

noted that in 1981, skull fracture, SDH and hypoxic encephalopathy were described as part of

the main findings of acceleration injuries, whereas in an article published in 1982 using the

same model system, none of the animals had such findings. This set of studies has been cited as

the strongest scientific evidence of the pathogenic nature of shaking to date. However, such an

important discrepancy in the results from this model system has not been properly tested by

other researchers due to the difficulties in conducting primate experiments. Since 1998 a few

studies have used the swine model to reproduce Gennarelli’s primate experiments with variable

12

results 54-56. The need for a conclusive animal model is well recognized by all participants in the

SBS controversy 57-61.

1.4.2. Mechanical models of SBS

Most anthropomorphic simulations have been done using scale models of adults, and the data

has been extrapolated to give an injury threshold for infants. In 1987, Duhaime et al. published a

seminal study which showed that the acceleration injury threshold established by Gennarelli’s

primate experiments could not be reached by shaking an anthropomorphic model irrespective of

the range of neck characteristics used 62. On the other hand, impact of the head of the

anthropomorphic model generated accelerations that were in a similar range to the Gennarelli

primate model injury thresholds. In the same paper, postmortem examination findings of 13

fatal infant head injuries that were diagnosed as SBS were reviewed. All 13 cases had findings

that were suggestive of impacts, such as scalp bruising or a skull fracture. The findings of this

study were reaffirmed by another anthropomorphic study in 2003 63, where the authors

concluded that:

These findings suggest that inflicted impacts against hard surface may be more frequently associated with

clinically significant inertial brain injuries than vigorous shaking or falls from less than 1.5m. In addition, there are

no data showing that maximum change in angular velocity and peak angular acceleration of the head experienced

during shaking and inflicted impact against unencased foam is sufficient to cause SDHs or primary TAIs in an

infant.

The main criticism against such studies is the issue of scaling. The argument centers on the

premise that the mechanical properties of the infants are not a simple linear scale reduction to

that of adults. As discussed earlier, infants have very distinct anatomical and physiological

properties but there is very scant data available that demonstrates the contribution of such

distinct characteristics to altering the mechanical scaling. Because of this, most

13

anthropomorphic studies to date have employed an approach that included theoretical extremes

of a given mechanical property, resulting in data that could not be conclusively correlated to

human infant shaking events.

1.4.3. Evidence-based reviews of the retrospective case series

Another shortcoming of the retrospective studies from the 1980s and 1990s is not due to an

imperfect scientific basis but rather a technical one. In these studies, silver-based stains of brain

tissue were used in neuropathology to detect axonal lesions along with H&E to show other

changes within the brain. More recently, immunostaining of the brain for β-amyloid precursor

protein (βAPP) expression has been accepted as the new norm for brain injuries (Figure 1-1) 64-

68. βAPP is a neuronal protein with various native functions including synaptic formation and

repair 69. It is mostly known as the precursor form of the β-amyloid protein (transmembrane

domain of βAPP) that forms characteristic plaques in Alzheimer’s disease. βAPP is normally

transported from neuronal cell bodies to the synapses by axoplasmic flow 70. Disruption of the

axons produces an accumulation of βAPP, histologically characterized by an ‘axonal retraction

ball’ formation. It is also reported that βAPP expression increases after axonal injury 66. βAPP

immunostaining is much more specific than silver stains. In 2001, Geddes et al. published a

two-part retrospective study of 53 non-accidental head injuries in infants and children which

showed that there is no significant difference in pathology between the shaken only group and

the impact group (These papers are sometimes called Geddes 1 and 2) 71, 72. Also, βAPP

immunostaining of the cases confirmed only two cases of tDAI present in the shaking group. In

2003, vascular changes following hypoxic/ischemic encephalopathy were proposed as a possible

alternate causal mechanism to the ‘shaking triad’. In these studies, it was discovered that the

expression of βAPP in the brain is not limited to traumatically damaged axons but can also be

expressed by the axons in response to direct and/or indirect effects of hypoxia (This paper is

14

sometimes called Geddes 3) 73. Following Geddes’ paper, the evidentiary nature of the previous

retrospective reports was questioned, and more evidence-based reviews of the previously

reported SBS cases were published 59, 74, 75. In these reviews, at times more opinionated than

objective, the conclusions varied from “cause of death in the SBS victims was a global cerebral

ischemia induced by a multifactorial process” in one review 74 to “whiplash shaking without

impact is the cause of death of this subset of infant homicides” in another review 59. The

opinions varied greatly on most of the critical points of the SBS diagnosis and they are

discussed in the following section.

15

Figure 1-1. Photomicrographs of traumatic axonal injury in human brain. A. Numerous axonal retractions balls (brown) are seen. (100x magnification) B. High power view of the axonal retraction balls. Slight positive staining of the neuronal bodies is also seen. (400x). βAPP immunostaining using DAB method.

16

1.5. Current points of controversy

1.5.1. Pathogenesis of the triad - Pathologic nature of ‘pure shaking’

Currently there are two hypotheses which may account for the development of the triad after

shaking without an impact component (i.e. pure shaking). First is the traditional hypothesis

which focuses on the inertial load to the various anatomical interfaces from repeated

acceleration and deceleration. The second hypothesis attributes the triad to secondary

permeability changes due to hypoxia/ischemia that is caused by initial brainstem damage

(Figure 1-2) or another inciting cause of hypoxia. No direct experimental evidence that supports

either of these two hypotheses has been reported, while many retrospective studies have been

aimed at confirming one over the other.

1.5.1.1. Immediate traumatic injuries due to shearing and traction

In this hypothesis, the supposed fragility of the infant structures is key when they are exposed

to a great inertial loading through repeated acceleration/decelerations generated by shaking.

According to this hypothesis, the bridging veins which cross the dura-arachnoid interface would

break under the inertial load to cause SDH, while the traction generated by the vitreous fluid

would cause retinal hemorrhages. Also, undermyelinated infant axons are more susceptible to

damage under an inertial load. It is postulated that the injuries to the axon would be diffuse due

to the generalized inertial load. This hypothesis explains the frequent bilaterality of

hemorrhagic lesions in suspected SBS cases as well as the presence of the diffuse axonal

injuries.

The critical weakness of this hypothesis is that the injury threshold of any of the structures

involved in such a mechanism is not known, or studied in the context of each other. Only a few

mechanical parameters of infant tissue are available. None of the studies have measured such

18

parameters or considered the contribution from connected structures. This hypothesis cannot

account for the frequent absence of neck injuries in suspected SBS cases where some studies

have suggested that the injury threshold for neck or vertebral injuries is much lower than for

intracranial injuries.

1.5.1.2. Hypoxic ischemic encephalopathy due to brainstem injury

In this hypothesis, violent shaking of the infant head causes damage to the brainstem and

upper cervical spinal cord via stretching or hinge trauma (pinching) within a confined space. As

a result, apnea due to damage to the brainstem then triggers a hypoxic response. This in turn

leads to changes in vessel permeability that could allow leakage of blood within the dura, and

then into the subdural space or retina. This hypothesis was first suggested in 1999 76. Recent

retrospective studies using βAPP have shown that the axonal lesions that were previously

thought to be traumatic were hypoxic in nature 32, 73, 77.

This alternative hypothesis (also called the ‘unified hypothesis’) fits well with the typical

presenting history of supposed SBS infants who are usually found in a state of apnea or “vital

signs absent” by the caregiver. However, an exhaustive resuscitation effort usually follows such

a discovery, and hypoxic/ischemic encephalopathy is a frequent finding in post-resuscitation

infants. In such cases, the assessment of the relative contribution to injury by the traumatic

mechanism versus the hypoxic remains highly controversial 78, 79.

1.5.2. Specificity of the triad

Putting the pathogenic nature of pure shaking aside, the next biggest question is the

specificity of the triad. This question was brought to light by the accumulation of anecdotal

reports where mechanisms other than shaking alone resulted in the triad of findings. In addition,

a retrospective review of previously diagnosed SBS cases revealed that many such cases have

19

Figure 1-2. Comparison of traditional and unified hypothesis. Green arrows: Traditional hypothesis. Red arrows: Unified hypothesis. (Modified from Geddes 3, 2003)

20

some evidence of impact to the head that was originally undetected or described as a co-finding

of ‘shaken-impact syndrome’. Part of this controversy can be explained because SBS was first

defined in the context of general physical child abuse where an impact to the head was not an

exclusionary criterion. For any professional involved in the assessment of the triad, it is

important to remain aware of other known mechanisms that could result in the triad.

1.5.2.1. Short fall debate

In 2001, Plunkett published a study where 18 fatal head and neck injuries in infants and

children involving playground equipment were collected from the Consumer Product Safety

Commission database 34. Of these18 cases, 12 were witnessed by a non-caregiver to fall from

equipment at various heights between 0.6 and 3 metres. The results showed that SDH and RH

are not specific indicators of the shaking mechanism since 13 of 18 cases had SDH, whereas 4

of 6 cases where the retina was examined had RH. Also, 12 of 18 infants or children had a lucid

interval which indicated that the tDAI might occur at much higher forces than SDH.

The study faced an immediate backlash where many of the study’s limitations were pointed

out. Spivack argued that injuries from a contact force of a fall are fundamentally different than

injuries from inertial forces of shaking or impact following shaking 80. Levin argued that RH in

accidental falls is extremely rare as the author “had to search literally tens of thousands of

records” to identify the four cases 81. These two comments clearly demonstrated the fundamental

division in a field where some professionals would contest the facts, methodology or limitations

stated in an article and dismiss the findings of any study that did not conform to their paradigm.

In the author’s reply to these comments, Plunkett pointed out that the physics of a fall or ‘slam’

is identical and the number of cases examined for RH was 6 out of 18 cases where a

22

funduscopic retinal examination was performed. In his word of caution, Plunkett ended a letter

to the editor with the following comments 82:

I hope that my study encourages us to re-examine our concepts regarding traumatic brain injury (TBI) and the

relative importance of inertial or impulsive loading (whiplash) and contact. Dr. Caffey’s “theory,” accepted for

almost 30 years, taught in medical schools, approved as an ICDA-9 “codable disease” and testified to as “truth” in

court, is based on a misinterpretation of early pioneering experiments performed for the automotive and space

industry. Ommaya published a landmark study in 1968 showing that TBI could be produced in rhesus monkeys by

acceleration of the head alone (with the midneck as a fulcrum) and no contact. However, the level of acceleration

he used to cause these injuries was 10,000–100,000 r/s2, with the lower limit being the concussion threshold. (Ten

thousand r/s2 at a radius of 6 inches is 5,000 f/s2 or 156 G’s). Caffey called Ommaya after his (Caffey’s) 1972

article was published and discussed it with him. Ommaya told him that he (Caffey) was misinterpreting his

(Ommaya’s) studies, but Caffey either didn’t understand or forgot to tell us. This misinterpretation is repeated in

Caffey’s 1974 article. And here we are today. A WWII paratrooper aphorism concerning chute-deployment failure

says it best: “It is not the fall that kills you. It’s when you hit the ground.”

This undoubtedly led to more studies which either re-examined the injury database 83, 84 or

used anthropomorphic models to confirm the forces involved in short falls 63, 85, 86.

Unfortunately, a fundamental misunderstanding of Ommaya’s experimental results still forms

the basis of many of the articles published on SBS which have claimed to be authenticated by

the vast clinical experience of physicians.

1.5.2.2. Confounding medical conditions

There are many anecdotal clinical reports where at least one of the triad has been described in

other natural and more benign medical conditions. SDH has been described in a variety of

circumstances including birth trauma 39, 87, impact trauma that does not have any other external

findings, and as a complication of a shunting procedure for hydrocephalus 27. Although reports

are rare, cerebrovascular thrombosis has been described as a delayed injury presentation of head

23

and neck trauma 34, 88-91. Also, SDH has been described as a complication of non-traumatic

conditions such as glutaric aciduria type-1 33, 92, hemodialysis 93, rhinocerebral mucormycosis 30,

Menkes disease 35 and more. Also, the possibility of misinterpretation of subdural effusion or

mass as SDH has been well reported.

Some physicians now believe that RH may occur in any circumstance where the intracranial

pressure (ICP) is increased 94, 95. This also includes circumstances where venous outflow is

obstructed leading to secondary increase in ICP. Accidental head injuries resulting in increased

ICP could show RH and in rare circumstances, RH has been documented in cases where

resuscitation efforts involved chest compression 96.

1.5.3. Nature of subdural hemorrhages

Although SDH has been described for many types of cranial trauma, the very nature of the

subdural space is still not uniformly agreed upon. Initially believed to be a cavity between the

dura and arachnoid, newer ultrastructural studies showed that it is a loosely filled interface

between the dura and arachnoid that is prone to cavitation 97-99. Fluid channels within the dura

itself have also been demonstrated 100. Whether it is tearing of bridging veins or leakage of blood

after hypoxia, there are important questions regarding SDH that need to be addressed to

accurately correlate it to the chain of events that might cause this bleeding.

SDH can be diagnosed directly (at surgery or at postmortem examination) or indirectly (by

imaging). Once developed, an acute SDH can either be reabsorbed or persist to become a

chronic SDH. In classical medical teaching, acute SDH is described as a collection of freshly

clotted blood along the contour of the brain surface, without extension into the depths of sulci

101. Once the original bleeding is limited, SDH may organize in a sequence where the clot is

24

lysed followed by highly vascularised granulation tissue formation and fibroblast growth into

the hematoma. The SDH could then retract leaving a thin layer of reactive connective tissue 102.

The age of the SDH is highly important in legal proceedings where multiple, temporally

distint causes of SDH are involved. Since the development and resolution of SDH are both slow

processes, the aging of multiple SDHs of different ages can pose a great challenge. Hemosiderin

and fibrin detection methods can aid in determining the age of SDH 102, but the resolution of the

temporal sequence is still in the range of few days to weeks. Some physicians suggest that the

aging of SDH can also be complicated by a phenomenon called ‘re-bleeding’ 42, 103, 104. This

suggests that the vascularised granulation tissue is prone to bleed even with minor trauma that

would not normally result in SDH formation. It could then be mistaken for an acute SDH where

greater forces are thought to be required.

25

1.6. SBS in criminal justice system

SBS has been hotly debated in the criminal justice system in many countries since the

controversy surrounding its specificity has been raised. Since many SBS convictions were based

on the pathologic evidence (presence of the triad), the possibility of potential miscarriages of

justice triggered large scale reviews in Britain and Canada. The British review was conducted

between 2004 and 2006 while the review of SBS in Ontario, Canada is still in process as of June

2009. Below are the summaries of both reviews to date.

1.6.1. Harris appeal and SBS cases review in Britain

Following two contentious infant homicide cases, a large scale review of infant homicides

was conducted. Following this review, a group of 88 cases was identified as SBS and were

further reviewed by AG Lord Goldsmith 105. At the same time, the Court of Appeal heard four

cases involving SBS, the most famous of which was the case of Lorraine Harris. Harris was

accused of killing her four month old son by shaking and was later convicted and sentenced to

three years imprisonment. The baby had been found having difficulty breathing, and Harris

admitted shaking the baby in an attempt to help. A home visit was made by a general

practitioner who did not find RH or any signs of abuse. However, the baby was found vital signs

absent about an hour later and resuscitated by the emergency crew. Upon arrival at the hospital,

the baby was noted to have an extensive bilateral RH and later died in hospital.

The appeals court examined the extensive areas of controversy in the SBS debate. The court

heard from Dr. Geddes about the ‘unified hypothesis’, and examined the fine points of the

controversy in detail. All of the current topics were discussed including the degree of force

required to cause injuries such as SDH, short falls, and the significance of RH. In the end, the

court quashed the conviction of Harris on the ground that fresh evidence heard in the appeal

26

might reasonably have affected the jury’s decision to convict, therefore rendering the conviction

unsafe.

With regard to the triad and the degree of force required to cause the triad, the appeals court

stated that “in cases where the triad alone is present, triad alone cannot automatically or

necessarily lead to a conclusion that the infant has been shaken”. However, the court also stated

that the triad remains a strong pointer to SBS. The court also acknowledged that although the

force required to cause the triad in the vast majority of the cases is more than rough handling,

there are rare or very rare cases where such injuries could be caused by little force.

1.6.2. Goudge inquiry in Ontario

The Inquiry into Pediatric Forensic Pathology was established to conduct a systemic review

and assessment of pediatric forensic pathology in Ontario from 1981 to 2001. The inquiry was

in part triggered by a series of cases in which the possibility of wrongful conviction was raised

due to the errors made by a pathologist who conducted several postmortem examinations. Many

challenging cases of pathologic diagnosis in forensic settings were reviewed. Among his

recommendations, the commissioner noted that there is a “significant evolution in pediatric

forensic pathology relating to shaken baby syndrome and pediatric head injuries warrants a

review... because of the concern that there may have been convictions that should now be seen

as miscarriages of justice”. After the release of the commissioner’s report 1, a review was

announced of previously diagnosed SBS cases in the province of Ontario. This review is

currently in progress.

27

2. Experimental design

2.1. Hypothesis

a. High frequency, low amplitude angular acceleration of the postnatal mouse head can

produce the triad (SDH, RH and axonal injury) and be a model for SBS

b. Manual shaking of adult BGM can produce the triad and be a model for SBS

2.2. Research objective

2.2.1. Overall

As discussed above, the four main approaches to the fundamental question of whether

shaking can cause injuries are: retrospective review, anecdotal case report, biophysical modeling

and experimental animal models. Both retrospective review of cases and anecdotal case series

provide valuable input to the debate but cannot ultimately answer the question due to their

inherent limitations. Retrospective studies rely on the history of a putative shaking event to

make a causal connection between shaking and injuries. Indisputably, anecdotal case evidence

does support a linkage between the triad and historical evidence of shaking. However, recent

experimental approaches using anthropomorphic models have not provided corroborative data in

support of SBS. In fact, one major drawback to anthropomorphic models is the inability to

produce the triad, thus limiting its value in answering the fundamental question. A well-

designed animal model could describe the mechanical parameters while providing the

pathophysiological mechanisms of injuries. Past attempts at modeling SBS have produced

inconclusive data due to several factors. The models have failed to reproduce the precise

shaking motion of the head and have utilized impact-related methods such as impact-

28

acceleration 9, 106, 107 and fluid percussion 108-114. The aforementioned baboon-macaque model

also fails to meet this criterion as only a single large acceleration was applied to the animals.

In the first part of the study, experiments were designed to test the feasibility of using mouse

pups as a model of SBS. If feasible, the model will represented a convenient method to

scientifically address the controversies regarding the specificity and characteristics of shaking

injuries in human infants. In the second part of the study, a small number of adult BGM will be

manually shaken to experimentally test the effect of inertial forces on the head and neck of an

animal that is anatomically and physiologically similar to humans.

2.2.2. Specific aims

Specific aims of the experiments were

I. To determine if high frequency, low amplitude vibration of mouse pups under general

anesthesia can cause subdural hemorrhage, retinal hemorrhage and diffuse axonal

injury/hypoxic encephalopathy as are seen in human infant head injury without evidence of

impact.

II. To determine if manual shaking of Barbados green monkeys (Cercopithecus aethiops) under

general anesthesia can cause subdural hemorrhage, retinal hemorrhage and diffuse axonal

injury/hypoxic encephalopathy as are seen in human infant head injury without evidence of

impact.

and

III. To describe physical parameters such as frequency and acceleration of both anterior-

posterior and lateral manual shaking of BGMs.

29

2.3. Rationale

2.3.1. Advantages of animal model

Animals have a distinct advantage in modeling human diseases, as they are a true living

system where complex physiological responses to experimental variables can be measured.

Responses may be evaluated temporally (progression), which allows for further studies on how

the responses could be interrupted (treatment) or prevented. Once the experimental protocol

establishes in an animal a condition that resembles the human disease, this can be used as a

model to study various aspects of the human disease by adjusting different variables in a

controlled manner. However, a fundamental assumption underlies animal models of human

disease: that the differences between the human and the animal can be ignored. Also, another

common assumption in animal models of human injury is that the mechanism of injury itself is

irrelevant as long as the presentation of the injury in the animal is similar to the human injury. In

establishing animal models for SBS, the potential effect of these common assumptions must be

carefully evaluated, as the mechanism of injury itself is in question. Species differences in

craniocervical anatomy result in vastly different ranges of head and neck movements in

response to external forces. As well, most currently available animal models of head injury

could not be used to study SBS because the mechanisms of injury infliction in these models

include an impact component.

2.3.2. Indication from previous studies

There is very limited literature available on animal models of SBS. In rodent models used by

Bonnier et al., linear shaking of postnatal pups reportedly gave rise to some RH and focal white

matter lesions including some atrophy, although the changes were not clear from the

photomicrographs published 115, 116. If these results could be replicated, it would represent a

30

convenient model to test the effect of inertial force injuries in a large sampling group. However,

this model did use mechanical means (linear rotating shaker) rather than manual shaking to

generate the inertial force, which is not a true representation of SBS where the force is applied

manually.

On the other hand, the baboon-macaque model of Gennarelli 16, 49, 52, 53 showed that either an

indirect impact angular acceleration or a single pneumatic rotational acceleration of a primate’s

head could cause SDH, concussion and axonal injuries. The experiments showed that the lateral

rotational acceleration of the head (side-to-side acceleration/deceleration of the head) with the

neck being the fulcrum caused more damage than the anterior-posterior rotational acceleration

(front-to-back acceleration/deceleration of the head). This primate model holds an advantage

over the rodent model, as the animals used are more anatomically similar to humans. The larger

size of the animals also permits some direct comparison of the shaking parameters without

scaling of the data.

Anthropomorphic studies also generated some very valuable data that were considered in

designing my experiments. First, anthropomorphic studies showed that the maximum

acceleration that could be generated manually is around 12 G which is well below the injury

threshold reported in Gennarelli’s model 62, 63. Secondly, they demonstrated that the key

difference between shaking and impact was the duration of the peak force applied to the head.

The difference in acceleration between shaking and impact was explained almost exclusively by

the difference in the peak force duration.

2.3.3. American Academy of Pediatrics technical report (2001)

In this technical report regarding SBS 2, the AAP states that a shaking event that generates

SBS ‘must be so violent that individuals observing it would recognize it as dangerous and likely

31

to kill the child’. This also sets the guideline for what is considered to be shaking in a modeling

system. Any model of SBS should address all aspects of this statement. For the purpose of

establishing an animal model for SBS, the AAP statement could be rewritten as ‘the model for

SBS should involve manual shaking that applies repeated oscillation with great enough force

that onlookers would see it as dangerous and likely to kill the animal’.

32

2.4. Material and methods

2.4.1. High frequency vibration of postnatal mice

2.4.1.1. Mice

CD-1 mice are outbred laboratory mice that are frequently used in experiments where no

specific genetic characteristics are required. General weight and size for male and female adults

are 25 to 30 g and 20 to 25 g respectively. Mature female CD-1 mice give birth to a litter of 8 to

12 pups after 21 days of gestation. Postnatal day 8 pups are considered to be developmentally

similar to full term human newborn infants 117. Pups open their eyes and start foraging for food

around postnatal day 15 and can be weaned by day 21. In this experiment, eight untimed

pregnant mice were purchased from Charles River laboratory and housed at the Division of

Comparative Medicine at University of Toronto. Animals had free access to food and water, and

nest material was provided. The birthdates of the pups were recorded by monitoring the cage

each morning.

2.4.1.2. Shaking apparatus

A standard analog vortex mixer capable of speeds up to 3000 rpm was used as shaking

apparatus in this experiment (Fisher Scientific, Catalog #02-215-365). Cotton fitted 50 ml

conical bottom tubes were used as a restraint that permitted a sitting position of the pups while

free head movement was allowed (Figure 2-1). In the article by Bonnier et al. 115, a horizontally

rotating shaker was used to ‘shake’ mice at 900 rpm in a way that did not limit head movement

or cause chest compression. However, the precise information regarding the apparatus and

parameters used in the Bonnier experiments could not be obtained and thus, my experiments

should not be considered as replication of the Bonnier study.

33

Figure 2-1. Experimental setup of high frequency vibration of postnatal mice. A. Vortex mixer with cotton-fitted tube on top. B. Cotton-fitted tube with P15 mouse in situ.

34

2.4.1.3. Displacement measurement

The vibration frequency, displacement of the head of the analog vortex mixer and top of the

tube were measured using a laser-optical electronic system at one-thousandth second intervals

(Micro-Epsilon, Model #. optoNCDT 1401). Data output was then saved to a comma-separated

values file format and calculations were made using a Microsoft Excel program.

2.4.1.4. Anesthesia and euthanasia

Animals were anesthetized prior to shaking by the inhalation of vapourized Isoflurane (5%

v/v) until no reflex retraction was observed upon pinching of a distal hind limb. In cases where

animals were observed to regain consciousness during the procedure, shaking was immediately

terminated and more isoflurane was promptly given. All animals were euthanized by overdose

of Isoflurane. Once euthanized, all animals were immediately submerged in 10% buffered

formalin for fixation. For larger animals of advanced age (postnatal day 21 (P21) and up), an

incision was made in the abdomen to facilitate fixation.

2.4.1.5. Tissue processing and histology

After fixation, serial whole mount sections of the head and neck were prepared by overnight

decalcification in 10% formic acid followed by coronal serial sections in five millimetre slices.

Sections were then processed for paraffin embedding in a Shandon Excelsior tissue processor™

(Thermo Electron Corporation). The overnight processing cycle started with two 30 minute

changes of 10% buffered formalin, then six one hour changes of alcohol in sucessively higher

concentrations (starting at 60%, last two changes in absolute ethanol) to dehydrate. Three one

hour changes of Clearene® were followed by three 80 minute immersions in 61°C paraffin.

Tissues were left immersed in 61°C paraffin until embedding.

36

Blocks were cooled to room temperature and sectioned at 5 μm, floated on a warm water

bath, mounted on glass slides coated with egg white albumin to prevent loss during staining, and

baked at 60 degrees overnight. Both eyes were included in the coronal head sections and

examined histologically (Figure 2-2). Slides were then stained with routine hematoxylin-eosin

(H&E) (Appendix A-1).

37

Figure 2-2. Whole mount sections (5 μm thickness) of mouse head and neck regions. Left-to-right: Eyes to cervical spine. H&E stain.

38

2.4.2. Manual shaking of Barbados green monkeys

2.4.2.1. Barbados green monkeys

Although Barbados green monkeys (BGM, C.aethiops) are not native to the Caribbean island

of Barbados, they were brought to the island as pets on slavery ships during the 17th century.

BGM have since naturalized and are considered an agricultural pest with an established wild

population of over 14,000. C.aethiops, old-world monkeys that are phylogenetically closely

related to vervets, have been extensively used for medical research including polio vaccine

production. The Barbados Primate Research Centre (BPRC) was originally established to

control the wild population. It is an accredited primate research facility with veterinary and

technical support. In this study, four male adult BGM were initially screened and medical

records of each animal were retrieved. Three animals were then chosen based on their health

status and weights: B4830 for sham treatment, B0098 for anterior-posterior (AP) shaking, and

B2038 for lateral (Lat) shaking (Table 2-1).

Table 2-1. Selection of BGM for the study

Cage Number Animal ID Weight* (kg) Height** (mm)

2A1 B2038 5.90 469.9

2A3 B4830 5.50 508.0

2A6 B0098 5.68 482.6

* Weight of the animals at initial health screening. Differ slightly from the weight recorded at the time of experiment.

** Height is measured from crown-to-rump.

40

The weights of the animals resemble the 50th percentile of two month old human infants. A

day before the experiments, vital signs of the animals were recorded and the head, wrist and

ankle of the animals were shaved under ketamine sedation to facilitate mounting of

accelerometers and the placement of an intravenous (IV) catheter (Figure 2-3) by the facility

veterinarian.

2.4.2.2. Accelerometer

In the previous anthropomorphic experiments, acceleration of the manual shaking was

reported to be in the range between 8 to 9 Gs with the maximum reaching 12 G 63. Two triaxial

accelerometers were used in this study to measure acceleration during shaking (Microstrain Inc.

Model# G-Link-10G). These accelerometers record up to 10 G and designed to withstand up to

the maximum of 500 G. Thus, the integrity of the measurements is preserved even after peaks

over the maximum recording range. The maximum range of these accelerometers was slightly

below the maximum peak acceleration reported in the anthropomorphic studies. However, these

were the only directly mountable wireless accelerometers available (small form factor, wireless)

on the market. The accelerometers were directly mounted onto the top of the animal’s head and

the wrist using medical tape and gauze (Figure 2-4). Data output was originally recorded by

proprietary software from the manufacturer, then exported as a comma-separated values file

format. The calculations and accelerometry traces were made using a Microsoft Excel program.

2.4.2.3. Anesthesia and euthanasia

Animals were given an intramuscular injection of ketamine by the facility veterinarian while

their movements were restricted using a pull out mechanism of the cage. After a complete loss

of motor control was observed, the animal was transferred to the surgical suite where vital signs

were recorded. An IV catheter was placed in the great saphenous vein and a valium/ketamine

41

cocktail was administered intramuscularly as an anesthetic agent. The state of anesthesia was

confirmed by examining jaw tone. Anesthetic agent was administered intramuscularly as needed

throughout the procedure (Table 2-2). The animals were euthanized by injection of pentobarbital

through the IV catheter at the end of the experiment by the facility veterinarian.

Table 2-2. Anesthesia and euthanasia records of BGM

Animal ID Drug Dose Route Time

Sham

Ketamine 0.6 ml IM 9:15 AM

Ketamine/Valium 2.0 ml IM 9:28 AM



Pentobarbital Sodium 2.5 ml IV 10:56 AM

AP

Ketamine 0.6 ml IM 1:56 PM

Ketamine/Valium 2.5 ml IM 2:02 PM




Pentobarbital Sodium 2.5 ml IV 4:29 PM

Lat

Ketamine 0.6 ml IM 8:45 AM





Pentobarbital Sodium 2.5 ml IV 11:18 AM

42

Figure 2-3. Preanesthetized BGM with IV catheter placement. Animal was sedated with IM injection of ketamine in cage. Once sedated, head, wrist and ankle were shaved to facilitate the mounting of the accelerometers and IV catheter placement.

43

Figure 2-4. Mounting of accelerometer on BGM. Wireless accelerometer unit was secured onto BGM head by gauze and medical tapes. Tapes were carefully placed to avoid limiting jaw movement.

45

2.4.2.4. Postmortem examination

A postmortem examination was performed on all animals. External photographs were taken

at the beginning of each postmortem examination to document any visible injuries, and gross

dissections and findings were photographed throughout. A layered dissection of the posterior

neck was performed to detect neck injuries. The brain was removed in continuity with the

cervical spinal cord using a posterior approach. The eyes were removed in continuity with the

retrobulbar optic nerves after orbital roof removal. The eyes, brain, spinal cord and clivus were

fixed in 10% buffered formalin before being transported to the University of Toronto in a semi-

wet state according to international regulations on shipping of biological specimens (not

immersed in formalin but wrapped in damp cloths in a liquid tight container).

2.4.2.5. Tissue processing and histology

The brain and the spinal cord were serially sectioned in the coronal plane. All regions of the

brain and cord were sampled, with larger sections bisected or trisected to fit within blocking

cassettes. The globes of the eyes were bisected to reveal the retina, floated on 10% buffered

formalin and photographed. Eyes were embedded in toto in a direction where the longitudinal

cross section profile of the eyes (semi-disk with optic nerve entry in middle sections) could be

seen. The clivus was decalcified in 1% formic acid solution for one week before serial

sectioning along the parasagittal plane. The sections were then processed for paraffin

embedding in the manner described above. Blocks were sectioned at 5 μm, floated on a warm

water bath, mounted on glass slides coated with egg white albumin to prevent loss during

staining, and baked at 60 degrees overnight. Mollifex® solution was used for on spot

decalcification of the clivus blocks. All slides were histologically examined after routine H&E

staining and all neural tissues were also examined with Luxol fast blue (LFB)/H&E staining.

47

2.4.2.6. Immunohistochemistry

Formalin fixed, paraffin embedded brain and spinal cord tissues were sectioned 5μm and

mounted on Fisher FrostedPlus© slides. Sections were deparaffinized in two five minute

changes of xylene, rehydrated in a series of absolute and a successively lower concentration of

ethanol (two three minute changes in absolute alcohol, 3 minutes in 90% alcohol, 2 minutes

each in 70% and 50% alcohol), then brought to distilled water. Slides were submerged

completely in a beaker containing citrate buffer adjusted to pH 6.0 and boiled for 10 minutes for

the antigen retrieval. The beaker was then removed from the heat and allowed to cool on the

benchtop for 20 minutes. Sections were further cooled by washing in a change of phosphate

buffered saline (PBS) on a shaker for three minutes. Endogenous peroxidase activity was

blocked by treatment with 0.3% methanolic peroxide (0.5 ml of 30% H2O2 in 50ml methanol)

immersion for 20 minutes and washed with distilled water for three minutes on a shaker. Slides

were then rinsed in two three minute changes of PBS (pH 8.0). Excess buffer was removed by

careful blotting and a circle was drawn around the section with a hydrophobic pen to localize the

antibody solutions. Non-specific antibody binding sites were blocked by incubating sections

with 250 μl of normal goat serum (Jackson #005-000-121, 1/20, 15 minutes) in a wet chamber.

The block solution was then rinsed away by careful blotting with a Kimwipe and the section

incubated for 2 hrs at a room temperature with 250 μl per slide of diluted antibody (Zymed #13-

0200, 1/180). After primary antibody incubation, slides were washed three times in PBS (3 mins

each on shaker). Antibody binding was detected using a biotin-streptavidin detection system

(Biotinylated Goat-antiMouse, Jackson #115-135-003, 1/850, 30 minutes; Strepavidin

conjugated with horseradish peroxidase, Jackson #016-030-084, 1/650, 30 minutes) with 3,3’-

diaminobenzidine as the chromogenic substrate.

48

2.5. Experimental protocol

2.5.1. Mice

2.5.1.1. Constant vibration

In this study, two groups of mouse pups at different postnatal ages (P8 and P15, n=20 and 19

respectively) were subjected to varying degrees of shaking under anesthesia to examine the

effect of the shaking frequency. Standard sterile technique was used to handle the mice and a

circulating warm water blanket was used to help maintain body temperature. Mice were

anesthetized, then their weights were recorded using a top-loading balance. An identification

number was marked on their backs using a permanent marker. Mice were then placed in a

cotton-fitted 50ml conical bottom tube in a sitting position and the chest movements were

restricted by the careful placement of cotton balls. Mice were then shaken using an analog

vortex mixer for 30 seconds at various speeds between 1000 rpm to 3000 rpm facing forward.

Animals were then removed from the tube and allowed to regain consciousness before being

returned to the cage. Two control animals of the same age were subjected to a sham treatment

where they were anesthetized and returned to the cage after regaining consciousness. Mice were

immersed in soiled bedding before being returned to the cage to reduce the chance of maternal

rejection. Any animals that died during the procedure were fixed in 10% buffered formalin

immediately. All surviving animals and the control animals were then euthanized by an

isoflurane inhalation overdose 48 hours after the procedure. Once euthanized, all animals were

immediately submerged in 10% buffered formalin for fixation. The same set of experiments was

repeated at a 90° clockwise rotated position (n=15 at each time point) to examine the effect of

the rotational plane.

49

2.5.1.2. Pulse acceleration

In this experiment, a single angular rotational acceleration experiment as described in the

literature (baboon-macaque model of SBS) was combined with vibrational shaking. This

modification was designed to capture the shaking motion more accurately by allowing multiple

angular accelerations to be applied to the brain. Anesthetised pups were divided into three

groups by age (P8, P15 and P21, n=5 for each time point) and then subjected to pulsating

vibrations (continuous pulsation between 20 to 50 Hz, one minute in total) using the shaking

apparatus described above. Two control animals of the same age were subjected to a sham

treatment where they were anesthetized and returned to the cage after regaining consciousness.

The surviving and the control pups were euthanized 48 hours after the procedure. For P21 mice,

an incision was made in the abdomen after euthanasia to facilitate satisfactory fixation. The

same set of experiments was repeated with the position rotated 90° clockwise (n=5, 5 and 4

respectively) to examine the effect of the rotational plane.

2.5.1.3. Hinge-point

In this experiment, both constant vibration and pulse acceleration experiments were repeated

with modification of restraint to change the location of the hinge-point of the head movement.

P22 pups (n=11) were restrained in a manner that allowed no head movement, simulating a

shaking event where a baby is grabbed by the head. This was achieved by allowing the mice to

sit deeper into the tube with the neck extended and carefully placing cotton balls to restrict

movements within the tube. Two control animals of the same age were subjected to a sham

treatment where they were anesthetized and returned to the cage after regaining consciousness.

50

2.5.1.4. Displacement measurement

Direct measurement of mouse head acceleration could not be made as continuous contact of

the laser beam with the mouse head could not be established during the vibration. Instead, the

displacement measurements were made by aligning a laser beam with the top of the conical

bottom tube. The rotational acceleration was then calculated by approximating the movement of

the vortex head as a circular motion (acircular = 4π2rf2, where acircular is uniform circular

acceleration, r is radius of the orbit and f is frequency of the circular motion).

51

2.5.2. Manual shaking of Barbados green monkeys

2.5.2.1. Sham (Negative control)

The animal was preanesthetized using a ketamine injection and the state of general anesthesia

was induced with an intramuscular injection of valium/ketamine cocktail. Once anesthesia was

confirmed, the initial vital signs and morphometric parameters were recorded. Small, wireless

accelerometers were mounted directly onto the forehead and wrist using gauze and medical

tapes. The animal was firmly held by the chest just below the scapulae in the back and below the

ribcage in the front with the surrounding skin pulled tight (Figure 2-5). The animal was gently

moved in a rocking motion for a minute to simulate parental cradling of an infant. The rocking

motion was first made in AP axis and repeated in lateral axis (sagittal and coronal plane,

respectively). General anesthesia was maintained for an hour after rocking with monitoring of

vital signs. The animal was the euthanized by an intravenous pentobarbital injection.

Funduscopic examinations were also made during the survival interval. The animal underwent

postmortem examination as described above.

2.5.2.2. Anterior-posterior shaking

This animal was used to establish a baseline for the AP shaking (front-to-back,

acceleration/deceleration of the head on sagittal plane) which is commonly believed to be the

type of shaking involved in SBS. After induction of anesthesia and mounting of the

accelerometers, the animal was firmly held by the chest in the manner described above and

manually shaken in an anterior-posterior fashion for a minute continuously at the maximum

speed, producing maximum head displacement at each flexion and extension. General

anesthesia was maintained for an hour while vital signs were continuously monitored.

Funduscopic examinations were also made during the survival interval. The animal was then

52

euthanized by intravenous injection of pentobarbital. The animal underwent postmortem

examination.

2.5.2.3. Lateral shaking

The same procedure as the AP shaking described above was repeated on the third animal

where the only difference was that the animal was shaken in the lateral axis (side-to-side,

acceleration/deceleration of the head on coronal plane). Lateral shaking produced more damage

in the Gennarelli baboon-macaque model when compared to AP shaking.

53

Figure 2-5. Experimental setup of manual shaking of BGM. The animal is held at chest level with scaled background.

54

2.6. Ethics of animal use

2.6.1. Mice

The experimental protocol was approved by University of Toronto Local Animal Care

Committee (LACC, Protocol #2000-6688). All standards set out in the Care and Use of

Experimental Animals guideline by the Canadian Council on Animal Care were followed. A

facility veterinarian was available for consultation throughout the experiment.

2.6.2. Barbados green monkeys

Application for animal use was made to the University of Toronto LACC, which was

externally reviewed on the applicant’s request (Appendix B-1). Revisions were made along with

the submission of the response to the reviewers (Appendix B-2). The protocol was approved by

LACC in a revised form (Protocol #2000-7466). The protocol was then reviewed by the

institutional animal care and use committee at BPRC and a joint experimental protocol (Protocol

#281008A) was established. The facility veterinarian was in attendance throughout the

experiments to assist with all animal handling.

It is understood that the use of primates in medical research is very contentious. All

alternative means of testing should be considered. It is further accepted that the ethics of animal

rights are a legitimate and important barrier to the use of primates in many experimental studies.

However, the application was made in the belief that the potential knowledge gained from the

proposed experiment outweighs the ethical arguments against it.

56

3. Results: Mice

3.1. Summary of findings

By using several different experimental settings for shaking the heads of mice, we have been

unable to produce meningeal, brain or spinal cord injuries of any type. Mouse pups were

subjected to a high frequency vibration (20 to 50 Hz) under anesthesia while restrained in a

manner that minimized chest compression but allowed free head movement. In both continuous

high frequency vibrational shaking and pulse acceleration of the head, no central nervous system

injury was detected in the whole mount section of the head (Figure 3-1). In contrast to previous

reports, change in rotational plane did not result in more severe trauma. When attempts were

made to deliver vibration directly to the head by restricting both head and neck movement, no

injuries were seen.

3.2. Constant vibration study

Two groups of mouse pups at different postnatal ages (P8 and P15, n=20 and 19 respectively)

were subjected to shaking for 30 seconds under anesthesia at a constant frequency. When

subjected to 20 to 50 Hz of continuous high frequency vibration, neither group of mouse pups

showed any signs of injuries. No SDH, RH, hypoxic injuries or evidence of traumatic axonal

injury were seen with routine microscopic examination. The same set of experiments was

repeated at a 90° clockwise rotated position (N=15 for each time point) and also produced no

injuries. There were three intra-procedural deaths. One pup had a focal microscopic

subarachnoid hemorrhage (Figure 3-2) without SDH, RH or brain swelling. No abnormalities

were found in the other two animals. No signs of functional neurological damage was seen

immediately after the shaking as all animals that survived the shaking procedure had locomotion

57

comparable to the control animals. No maternal rejection was seen and no sign of

developmental delay or malnutrition was observed.

3.3. Pulse acceleration study

Three groups of anesthetized pups (P8, P15 and P21, n=5 for each time point) were subjected

to continuous pulsating vibrations between 20 to 50 Hz for 1 minute using the shaking apparatus

and repeated five times. The same set of experiments was repeated at a 90° clockwise rotated

position (n=5, 5 and 4 respectively) to examine the effect of the rotational plane. No SDH, RH,

hypoxic injuries or evidence of traumatic axonal injury were seen on routine microscopic

examination. There were 10 intra-procedural deaths in which no injuries were found upon

histological examination. No signs of functional neurological damage were seen immediately

after the shaking as all animals that survived the shaking procedure had locomotion comparable

to the control animals. No maternal rejection was seen and no sign of developmental delay or

malnutrition was observed.

3.4. Hinge-point study

The P22 pups (n=11) were shaken while restrained in a manner that restricted the head and

neck movements. Histological examination revealed no evidence of injuries or hypoxia.

However, this procedure resulted in a higher mortality rate than the previous procedures as 5 out

of 11 pups were killed during the vibration. This is likely due to the higher level of chest

compression needed to restrain the pups during the procedure. No signs of functional

neurological damage were seen immediately after the shaking as all animals that survived the

shaking procedure had locomotion comparable to the control animals. No maternal rejection

was seen and no sign of developmental delay or malnutrition was observed.

58

Figure 3-1. Photomicrographs of postnatal mouse. A. Meninges with no SDH (50x). B. Retina showing no hemorrhages (200x). C. cerebral white matter with no axonal damages (400x). H&E stain.

59

Figure 3-2. Photomicrograph of a local SAH found in intra-operative death (200x). Hemorrhage was limited to subarachnoid space and thought to be non-lethal. H&E stain.

61

3.5. Vibration frequency and displacement

Vibrational frequency closely matched the frequency range provided by the manufacturer

(25-50Hz). There was a general agreement between the experimental acceleration calculated

from the displacement data (~20 G at maximum) and the circular approximated theoretical

acceleration (6-25 G). Figure 3-3 shows an initial one second plot of displacement measured

with the vortex machine dial set on 6. The vibration frequency measured was 40Hz, with

consistent amplitude of 5 millimeters.

63

Figure 3-3. Displacement over time plot of the shaking apparatus

64

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

Displacement (millimeters)

Tim

e (s

econ

ds)

Dis

pla

cem

ent

of

vort

exer

hea

d o

ver

tim

e

65

4. Results: Monkey

4.1. Summary of findings

Overall, violent, manual shaking in a manner that conforms to the American Academy of

Pediatrics description of the shaking event in SBS did not cause any immediately hemorrhagic

lesions in eyes, dura, cerebrum, brainstem or spinal cord of the animals. Also, there were no

injuries to the posterior neck musculature or the clivus. Fingertip bruises were noted on the torso

of both animals that were subjected to shaking. The animals used in this study were comparable

in weight to 2-month-old human infants (Table 4-1).

Table 4-1. BGM morphometric dimensions at time of autopsy

Animal ID

Head circumference*

(mm)

Neck circumference

(mm)

Chest circumference**

(mm)

Shoulder to elbow

(mm)

Elbow to

wrist (mm)

Hip to

knee (mm)

Knee to

ankle (mm)

Sham 280 225 340 170 140 170 170

AP 270 180 350 170 160 170 170

Lat 285 210 340 140 120 170 170

*Head circumference measured over supraorbital ridge-ear-occiput after shaving of the head.

**Chest circumference measured directly over nipples with the fur firmly pressed against the skin.

Anterior-posterior shaking lasted 43 seconds with 125 shakes while lateral shaking lasted 53

seconds with 178 shakes. The maximum frequency reached for AP shaking was 3.2 Hz with an

average frequency of 2.9 Hz. The maximum frequency reached for Lat shaking was 3.7 Hz with

an average frequency of 3.4 Hz. Both types of shaking achieved full extension/flexion and

saturated the accelerometer recordings along the axis of the shaking motion (Figure 4-1). The

video recording clearly showed that the movement of head during lateral shaking was not

limited to the coronal plane. The head followed ‘figure 8’ pattern in its motion where the neck

66

was repeatedly hyperextended or hyperflexed on top of being extended in the coronal plane

(Figure 4-2). The accelerometer data confirmed this secondary movement axis of the lateral

shakes as the acceleration peaks on the sagittal (AP) axis also reached the recording range limit

of the accelerometer (~10 G). Incidental findings at postmortem examination include an old

amputated digit and a 7 cm retroauricular, suboccipital scalp scar on the sham animal. Also, a

shaving abrasion of 3 cm in diameter was found on the suboccipital scalp of both the sham and

Lat animal. The Lat animal had an old scar on the left knee.

4.2. Physiological responses after shaking

Heart rate and temperature were monitored throughout the experiment including one hour

survival interval after shaking under general anesthesia. A slight drop in body temperature was

observed in all animals including the sham animal. However, it should be noted that the room

temperature was maintained at 18°C during the procedure and it is unclear if the slight

hypothermia was due to the procedure (anesthesia and/or shaking) or the environment. There

was a slight elevation in the heart rate immediately after shaking for both the AP and Lat

animals. Breathing was also shallower immediately after shaking, but both the heart rate and

breathing rate was within the normal range and returned to the baseline within few minutes.

At 38 minutes after shaking, a slight twitching of the left ear was observed in the AP animal

along with fasciculation of the left masseter muscle. A loud snoring was also observed with a

small amount of clear, foamy discharge from nose and mouth. The twitching and snoring

continued for approximately one minute, then disappeared. A slight shivering was observed at

43 minutes after shaking. No RH was seen upon funduscopic examination at any time point

during the survival interval although the examination became difficult due to the presence of

tears.

67

The Lat animal had a similar course during the survival interval. Shivering was observed at

26 minutes after shaking with non-specific movements at 33 minutes after shaking. These

movements lasted about one minute, then disappeared. Possible deviation of the eyes was seen

at 41 minutes after shaking and snoring was observed at 42 minutes after shaking.

68

Figure 4-1. Still frames from AP animal manual shaking video. Frames show various head and neck positions at A. full extension to B. full flexion. Atypical extension was also observed (C) where head was seen slightly rotated to the left.

69

Figure 4-2. Still frames from Lat animal manual shaking video. Head and neck positions during lateral shaking showed much more complex movement where the exception of E, all head positions had a component of sagittal extension/flexion, rotation and lateral flexion.

71

4.3. Anatomic and histological findings

4.3.1 Brain and cervical spinal cord

At postmortem examination, no scalp contusions or subgaleal or meningeal hemorrhages

were seen in any of the shaken animals. No SDH, subarachnoid hemorrhage or extradural

hemorrhage were seen on the surface of the exposed spinal cord (Figure 4-3 to 4-5). After

fixation in 10% buffered formalin, horizontal sections of brainstems and coronal sections of

brains were made and were free of visible macroscopic hemorrhages (Figure 4-6 to 4-11).

Histologically, the brain and the spinal cord were examined with both H&E and LFB/H&E

stains (Figure 4-12 to 4-16). No microscopic SDH or subarachnoid hemorrhages were seen in

any of the animals. As expected in highly mobile animals, axons of the corticospinal tracts in all

animals including the sham animal appear to be enlarged compared to that of the humans. βAPP

immunostaining revealed no visible accumulation of βAPP in axons in all of the sections

examined. Some weak neuronal staining of βAPP was seen in all sections. Sections examined

with βAPP immunostaining from each animal were: upper cervical spinal cord, pons, medulla,

superior cerebral hemisphere at the level of anterior commisure, and posterior corpus callosum.

A known βAPP positive human brain tissue sample was used as a positive control for the

immunostaining procedure (Figure 1-1), while incubation with PBS buffer solution without the

primary antibody was used as a negative control for each animal. Minimal non-specific

background staining was seen in all sections (Figure 4-12, inlay).

73

Figure 4-3. Brain and cervical spinal cord of Sham animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C. Brain and cervical spinal cord after fixation. Inferior view.

74

Figure 4-4. Brain and cervical spinal cord of AP animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C Brain and cervical spinal cord after fixation. Inferior view.

76

Figure 4-5. Brain and cervical spinal cord of Lat animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C Brain and cervical spinal cord after fixation Inferior view.

78

Figure 4-6. Coronal sections of sham brain after formalin fixation.

80

Figure 4-7. Horizontal sections of sham brainstem after formalin fixation.

82

Figure 4-8. Coronal sections of AP brain after formalin fixation.

84

Figure 4-9. Horizontal sections of AP brainstem after formalin fixation.

86

Figure 4-10. Coronal sections of Lat brain after formalin fixation.

88

Figure 4-11. Horizontal sections of Lat brainstem after formalin fixation.

90

Figure 4-12. Photomicrographs of cervical spinal cord of BGM. A. to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. Inlay is negative control of immunostaining procedure where the section was incubated with PBS buffer without βAPP antibody. D. to F. AP animal. G. to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.

92

Figure 4-13. Photomicrographs of pons. A. to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D. to F. AP animal. G. to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.

94

Figure 4-14. Photomicrographs of medulla. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.

96

Figure 4-15. Photomicrographs of frontal cerebral cortex. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 200x.). No hemorrhagic or axonal injuries were seen.

98

Figure 4-16.Photomicrographs of posterior corpus callosum. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 400x). No hemorrhagic or axonal injuries were seen.

100

4.3.2. Eyes and retina

No RH or optic nerve sheath hemorrhages were seen after the removal of the orbital roof and

enucleation. Globe of the eyes were bisected to reveal retina, and the examination while floating

in 10% buffered formalin revealed no RH (Figure 4-17).

Histologically, no microscopic RH or perioptic nerve sheath hemorrhages were seen in any

of the sections examined using the H&E stain (Figure 4-18).

4.3.3. Neck

Layered posterior neck dissection revealed no gross structural damage (Figure 4-19). Serial,

parasagittal sections of clivus from all three animals showed no hemorrhages or structural

damage upon microscopic examination.

4.3.4. Fingertip bruising

Subcutaneous bruises extending to the myofacial plane were observed on both shaken animals

(Figure 4-20). These bruises were located over bony prominences at the points where the

animals were held during shaking such as the scapula, thoracic spinous processes and the ventral

torso, inferior to the nipples. The shapes of the bruises ranged from circular to irregular and the

sizes measured from 0.5 cm in diameter to 4 cm by 1 cm.

Hemorrhages representing the subcutaneous bruises extended into the fibroadipose tissue but

not into muscles (Figure 4-20 C).

102

Figure 4-17. Retina after formalin fixation. Globe of the eye was bisected to reveal retina on posterior hemisphere. Photographs were taken while posterior globe was floated on 10% buffered formalin. A. and B. Sham animal (left (A) and right eyes (B)). C. and D. AP animal. E and F. Lat animal. No grossly visible RH was seen.

103

Figure 4-18. Photomicrographs of retina. No microscopic RH was seen. A and B. Sham animal. C and D. AP animal. E and F. Lat animal. (All 200x).

105

Figure 4-19. Posterior neck dissection. Layered dissection of posterior neck muscles revealed no structural damage or bleeding. A and B. Sham animal. C and D. AP animal. E and F. Lat animal.

107

Figure 4-20. Fingertip bruising found on body surfaces. A. Skin bruising over ribcage just below nipples. B. Bilateral bruising over scapulae found after skin reflection. C. Photomicrograph of myofacial hemorrhage associated with skin bruising. H&E stain, 100x.

109

4.4. Accelerometry Data

4.4.1. Sham animal

Both front-to-back and lateral rocking of the sham animal generated a maximum tangential

acceleration of 1 to 2 G with mean frequency of ~1 Hz. Figure 4-21 shows a plot of the

tangential accelerations measured while being gently rocked in AP direction. In this plot, blue

line denotes a tangential acceleration in front-to-back direction and red line denotes a tangential

acceleration in side-to-side direction. The sweep number corresponds to number of times the

measurements are taken by the accelerometer.

4.4.2. AP animal

In AP shaking, most of the acceleration peaks measured in front-to-back direction reached

the upper recording limit of the accelerometer. Figure 4-22 shows a plot of the tangential

accelerations measured while being violently shaken in AP direction. In this plot, blue line

denotes a tangential acceleration in front-to-back direction and red line denotes a tangential

acceleration in side-to-side direction. Accelerations on side-to-side direction reached the

maximum of ~5 G. Overall, the animal was shaken front to back 125 times in 43 seconds of

manual shaking (mean frequency: ~2.9 Hz). Maximum frequency reached was ~3.2 Hz.

4.4.3. Lat animal

In Lat shaking, most of the acceleration peaks measured in the side-to-side direction reached

the upper recording limit of the accelerometer. Figure 4-23 shows a plot of the tangential

accelerations measured while being violently shaken in lateral direction. In this plot, the blue

line denotes a tangential acceleration in front-to-back direction and the red line denotes a

tangential acceleration in side-to-side direction. Interestingly, some of the peak accelerations in

111

the front-to-back direction also reached the limit while the rest of the peaks in this direction had

a mean value of ~8 G. Overall, the animal was shaken side-to-side 178 times in 53 seconds of

manual shaking (mean frequency: ~3.4 Hz). Maximum frequency reached was ~3.7 Hz.

112

Figure 4-21. Accelerometry tracing from Sham treatment.

113

-2

-1.5

-1

-0.5

0

0.5 45

00

4700

49

00

5100

53

00

5500

57

00

5900

61

00

6300

65

00

Acceleration (G)

Swee

p nu

mbe

r (6

24 sw

eeps

/sec

ond)

Acc

eler

atio

n tr

ace

of S

ham

ani

mal

AP

Axi

s L

ater

al A

xis

114

Figure 4-22. Accelerometry tracing from Anterior-posterior animal shaking.

115

-15

-10 -5 0 5 10

15 45

00

4700

49

00

5100

53

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Figure 4-23. Accelerometry tracing from Lateral animal shaking.

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5. Discussion

5.1. Mice

5.1.1. General discussion

Rodents have been used for modeling human head injuries. Relative ease of handling and

lower operational cost have contributed to their wide use in various medical research. Previous

rodent models of head injuries focused primarily on impact trauma or impact-induced angular

acceleration trauma. In these models, a wide range of brain injuries from mild concussion to

diffuse axonal injury has been documented. However, the utility of these models in describing

repeated inertial injuries of shaking is low, since neither the mechanism (non-impact induced

rotational acceleration) nor the manner (repeated oscillatory motion) is reliably replicated in

these models.

There are only a few rodent studies that evaluated a non-impact mechanism of brain injury.

Non-impact induced angular acceleration combined with hypoxia has been reported to cause

brain lesions in adult rats 118. Bonnier’s articles in 2002 and 2004 represent one of the very few

rodent head injury studies that combined the mechanism and manner (through the use of s linear

rotating shaker at 900 rpm for 15 seconds) to be addressed in modeling SBS. In this model, RH,

focal axonal injuries and focal white matter hemorrhages were reported, but the assessment of

the figures provided showed the general overstaining for most immunostains. It is also puzzling

that no astrocytic reaction was seen despite the injuries mentioned.

There was a total of 18 intra-procedural deaths in the three studies.16.5% mortality rate in

our study is lower than the 27% mortality rate reported in Bonnier’s study. Without any positive

intracranial findings, there are two main causes that could account for the intra-procedural

deaths. The first is the possibility of accidental anesthetic overdose. Since the anesthetic agent

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(isoflurane) was vaporized and administered via inhalation, the amount of the agent that entered

the animal largely depended on the animal’s pulmonary function. The lungs of younger pups are

not yet fully developed and individual variation may have been great. The second possible cause

of intra-procedure death is mechanical asphyxia due to the restraint used to immobilize the

animal within the tube. This may have prevented chest expansion during inhalation. The higher

mortality rate in the pulse acceleration (10 of 29) and hinge-point study (5 of 11) supports this

speculation as more adjustment of the fitting was required for the pulse acceleration study (5

repetitions) and a larger amount of cotton was used in the hinge-point study to limit both chest

and head movements.

5.1.2. Negative findings: What do they mean?

In this experiment, high frequency vibration of postnatal mice did not result in any injuries

commonly thought to be caused by shaking. There are three potential explanations why this

experimental setup did not result in injuries.

First is the difference in craniocervical anatomy between human infants and mouse pups.

During vibration, the head of the mouse generally followed the movement of the tube which

closely approximates a circular motion. The mice’s heads were observed to ‘jerk’ out of the

circular path from time to time, particularly when the vortex machine was changing speeds

(start/end and pulse acceleration). Since these pups were anesthetized, any voluntary movement

could be ruled out. These jerking motions could potentially be explained by the craniocervical

anatomy of the mice. The foramen magnum in quadrupedal animals is positioned in the occiput,

oriented vertical to the ground 119. The position of the foramen magnum gradually moves

towards the inferior surface of the occipital bone in higher order animals. The vertical position

of the foramen magnum and atlanto-occipital joint of rodents limits their range of neck

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extension. If greater range is required the trunk has to move together with the head. Also, in

younger pups, head and neck development are incomplete and the neck circumference is larger

than the head circumference. This also does not permit true shaking of the head where the neck

needs to act as a fulcrum. The jerking movements observed during high frequency vibration

could be interpreted as movement of trunk when the sudden change of acceleration exceeded the

extension limit of the neck.

Second, the force applied to the mouse pups may not have been high enough to cause head

and neck injuries. It is believed that brain weight is a critical factor in determining the force

required to create injuries by acceleration/deceleration in an inverse relationship where greater

non-impact acceleration would be required to cause injuries to a smaller brain. The brain weight

of the postnatal mice used in this study was not recorded since whole mount sections of the head

and neck regions were prepared without removal of the brain. However, with the overall weight

of the pups ranged from 6 g to 20 g, the brain weight would not exceed a few grams at the most.

In other animal studies of non-impact head injury, at least 350 krad/sec2 of angular acceleration

was required to cause a mild traumatic brain injury such as concussion in a rat model 106 (brain

weight = ~5 g), whereas in a piglet model 55 (brain weight = ~35 g), 110 krad/sec2 was sufficient

to cause SDH and axonal injuries. When scaled for the brain weight of 500 g, concussion, SDH

and tDAI threshold from Gennarelli’s baboon-macaque model 62 were 100 krad/sec2, 350

krad/sec2 and 400 krad/sec2 respectively. Applying the proposed inverse relationship, it is

expected that the amount of angular acceleration required to cause some type of injury in

postnatal mice model would exceed these figures. Although the angular acceleration was not

directly measured in this study, it is reasonable to project from the calculated tangential

acceleration that the high frequency vibration used did not apply enough force to cause any head

and neck injuries.

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Last, the lack of findings in mice pups might be a true indication that shaking does not cause

head and neck injuries. With the exception of Bonnier studies, all other animal models of non-

impact human head injury that resulted in positive findings employed forces that are orders of

magnitude greater than the forces generated by manual shaking in anthropomorphic studies.

With no experimental evidence showing that damage could result from such lesser force, the

negative findings in this study add to a body of evidence that the accelerations that are

comparable in magnitude to the accelerations from manual shaking does not cause head injuries.

However, due to limitations of the model discussed above, the results of this study should not be

considered definitive evidence that manual shaking does not cause head and neck injuries in

human infants.

5.1.3. Role of mouse model in future investigations of shaken baby syndrome

During the development of the experiment, it was a challenge to come up with a shaking

apparatus that would not cause chest compression or other trauma to the body while delivering

sufficient non-impact force to the head of the mouse pups. We could not think of any alternate

solution that could achieve this goal. Other means of accelerating the head of the animals

available in literature, such as impact-acceleration 106, 107 or fluid percussion 108-114 cannot be

applied to mouse pups as they would cause catastrophic damages to the pups overall at the level

of force projected to cause internal head and neck injuries. This limitation effectively eliminates

the possibility of using postnatal mice in replicating mechanism of manual shaking.

Despite the inability of replicating the shaking mechanism itself, the convenience of using

small animals such as mice should be utilized in studying the pathobiology of injury. For

example, the specific molecular mechanism for vessel permeability changes in the setting of

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hypoxia or ischemia is yet to be fully understood and could be studied by designing experiments

where hypoxia or ischemia is induced by means other than shaking.

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5.2. Barbados green monkeys

5.2.1. General discussion

Manual shaking of BGM in this study was designed to detect immediate hemorrhagic injuries

and possible physiologic responses suggestive of axonal damage. Several non-specific

physiologic responses that could be suggestive of axonal damage was observed during one hour

survival including hypothermia, twitching and deviation of eyes. Unfortunately, βAPP

immunostaining did not show any signs of axonal injury (no axonal positivity). This does not

rule out the possibility of such injury as the survival interval might not have been sufficient for

broad development of detectable βAPP accumulation in injured axons. Although it is widely

recognized that the time interval between cerebral injury and expression of βAPP is at least two

hours, there is growing evidence in the literature that it may be detected at earlier time intervals

after the injury 120. Longer time intervals allow broad development of βAPP-stainable axonal

retraction balls. However, they also allow secondary changes to the brain via generalized

swelling after initial traumatic injuries, leading to non-perfusion anoxia and florid hypoxic

ischemic encephalopathy. Therefore, allowing sufficient time for βAPP development could give

rise to confounding results that overshadow true injuries produced by shaking. This is indeed the

case for the most of the head-injured babies where ascertaining the mechanism of the initial

injury can be quite challenging without clear findings of an impact event.

In Gennarelli’s baboon-macaque model, lateral rotational acceleration generated significantly

more severe damage than did anterior-posterior (sagittal) rotational acceleration of the same

magnitude. However, Gennarelli did not offer any possible explanation why lateral acceleration

on the coronal plane resulted in more severe injuries. In manual shaking of BGM, although there

were no detectable injuries, lateral shaking generated more complex head movement than did

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AP shaking. As shown in Figure 4-23, tangential acceleration on both the sagittal and coronal

axes reached over 10 G, and where combined, tangential acceleration could be significantly

more. This secondary movement axis in lateral shaking may be due to the adaptation of

craniocervical junction in bipedal animals. The range of lateral flexion movement in higher

order animals, especially bipedal animals, is all but lost and this may create a secondary axis of

motion 121. The exact magnitude of combined tangential acceleration cannot be calculated since

the maximum peak values could not be recorded . This should be addressed in future

experiments using an accelerometer with a higher recording range.

Findings of fingertip bruising on the skin surface where the monkeys were held for shaking

shows that the force required to hold the animal during shaking is significant. Although findings

of non-specific punctate bruising is relatively common in chronically abused babies, recognizing

their significance as a possible sign of manual shaking could provide additional corroborative

evidence for the pathologist in providing a balanced opinion. Thus, a layered dissection of both

the anterior and posterior torso and arms (in case the baby was held by the arms) should be

performed in all unexpected deaths of infants as subcutaneous bruises on such locations could

be a possible sign of the shaking event.

5.2.2. Negative findings: What do they mean?

Dissimilarity due to developmental parameters

A frequent argument against using adult animals in modeling SBS is the belief that the

immature brain and meninges have different mechanical properties than those of adults. During

the external revision of the BGM experimental protocol, this question was raised by one of the

reviewers.

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“Scientifically, what this model does not address is the difference between the immature brain and its

coverings in the two-month-old human infant and that of the adult non-human primate brain. The infant

brain and its vessels would be expected to be more mobile and more fragile than an adult brain.”

As described in the introduction, human infants have distinct developmental parameters that

could potentially affect the response to external forces. A tensile scalp, pliable cranium,

underdeveloped meninges and blood vessels, and lack of the myelinations all could have

mechanical ramifications. These differences are most pronounced in the perinatal and neonatal

periods and are considered to be a contributing factor to the predominance of suspected SBS

cases in these age groups.

Currently available mechanical studies of infant vessel properties do not conclusively answer

this difficulty. These studies are limited in their inability to reproduce the complex mechanical

relationships that the infant brain and its vessels are exposed to including brain dimensions, the

anatomical location of vessels and axis of angular displacement. One of the aims of the BGM

study was to describe mechanical displacement parameters of manual shaking. For this purpose,

having dimensionally (size and weight) similar animals offered more relevant information that

could be compared to anthropomorphic studies in the literature.

Weight of the brain and the acceleration

In an anthropomorphic study using a scaled model of 1-month-old human infants (brain

weight = assumed to be 500 g), manual shaking generated a maximum of 2.64 krad/sec2, a

simulated fall from 0.9 metre onto concrete surface generated 89.4 krad/sec2, while inflicted

impact against a benchtop surface generated 173 krad/sec2. An older anthropomorphic study

using a similar scaled model showed that manual shaking could generate peak tangential

acceleration of 9.29 G with mean angular acceleration of 1.14 krad/sec2 while impact against a

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metal bar generated 428 G and 52.5 krad/sec2 respectively. When combined, these figures

suggest that manual shaking simply could not generate enough angular acceleration to cause any

injuries 62, 63.

The weights of the brains of the BGM used in this study were 76.0 g (AP animal) and 61.0 g

(Lat animal). From the injury thresholds above, it is not hard to project that manual shaking

cannot generate the acceleration thought to produce injuries. Unfortunately, the peak tangential

acceleration during manual shaking of BGM could not be determined due to the saturation of

the accelerometer’s upper recording limit. Without a measured maximum peak tangential

acceleration value, interpretation of the acceleration data obtained in this study is limited.

However, the accelerometry trace showed that the some peak individual oscillations were not

saturated and the tangential accelerations of those peaks were clustered around 10 G. Thus, it is

likely that peak tangential acceleration during manual shaking of BGM did not reach the

magnitude of the reported injury thresholds.

Craniocervical anatomy of Barbados green monkeys

Phylogenetically, monkeys are closely related to humans. It is generally accepted that their

anatomy resembles that of humans more closely than does the anatomy of non-primates. BGM

are essentially bipedal in their modality, thus the position of the foramen magnum is near the

base of the skull similar to humans. Also, the anatomy of the craniocervical junction permits full

extension and flexion of the neck, similar in the range that is observed in human. This permits a

true whiplash type of shaking motion to be replicated in BGM where, without support of neck

musculature after general anesthesia, the movement of the head is isolated from the trunk. This

craniocervical anatomic similarity is one of the most important benefits of using primates for the

initial experiments of SBS where several important mechanical parameters and physical

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responses could be studied. However, a thorough anthropological comparison study of BGM to

human must be carried out to document the subtle differences that would be required in

interpreting the data gathered from BGM studies. The general aims of such study are outlined in

the next chapter.

Fundamental question: Can shaking cause head and neck injuries?

The very fundamental question in the SBS debate still remains unanswered. Both traditional

and unified hypotheses hinge on the assumption that the shaking can cause some type of injury

(primary injury) that initiates cascade of responses (secondary injury). In this experiment, a

violent manual shaking of animals that are similar in dimensions to human infants did not cause

any acutely detectable hemorrhagic injuries. The pertinent negative results from this

experiments are: 1. Shaking did not cause shearing of bridging veins that is thought to cause

SDH in SBS, 2. Shaking did not cause immediate RH from vitreous-retinal traction, 3. No apnea

was observed immediately after shaking, and 4. No evidence of axonal injury was detected after

one hour survival interval under general anesthesia after shaking. On the other hand, the

limitations of this study were: 1. Mature animals were used instead of infantile/juvenile animals,

2. Brain weights were significantly less than those of human infants, 3. Accelerometer could not

capture the maximum peak acceleration, and 4. Insufficient time was allowed for the delayed

marker of injuries. Some of these limitations could be directly or indirectly addressed by the

approaches discussed in the following chapter.

5.2.3. Role of BGM model in future investigations of SBS

As discussed above, primates hold a distinct advantage over other animal models in studying

SBS. There are many physiological and mechanical aspects of the SBS debate that still need to

be described. First of all, the development of βAPP after a longer survival interval must be

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addressed using the same protocol established in this study. Along with the axonal injury study,

many of other studies that require experimental data input from primate studies are discussed in

the following chapter.

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5.3. Back to the shaken baby syndrome debate: Significance of experiments performed

5.3.1. True non-impact head injury protocol

This BGM study represents one of the first to use a non-impact mechanism of applying

rotational acceleration in animals. No previous animal studies have employed true shaking,

where the head undergoes fast repetitive oscillatory motion with the neck being the fulcrum.

Baboon-macaque studies by Gennarelli 16, 18, 50, 52, 53 and piglet studies by Margulies 54-56 both

failed in this regard, where only a single (or multiple, separate single accelerations over 15

minute time interval) large scale acceleration was applied to create the head injuries. Also, in

this BGM study, the force was delivered manually which also is a novel approach in animal

studies of SBS. By incorporating these two factors, this study meets the criteria of the violent

shaking event described by AAP 2 and what pediatricians would believe to cause the triad. This

is important because previous animal models that were able to generate part of the triad were

rejected since they failed to meet the description of a ‘shaking event’ in the context of child

abuse.

5.3.2. Mechanical properties of Barbados green monkey manual shaking

The frequency and tangential accelerations recorded in this study generally validate the

anthropomorphic data from Duhaime and Prange studies 62, 63. At full strength, an adult male

experimenter was able to generate frequency less than 4 Hz with the projected maximum

tangential accelerations in the realm of anthropomorphic data. However, without properly

measured maximum acceleration, further investigations using accelerometers with higher

calibrated recording range are required for meaningful comparison between BGM model and

anthropomorphic devices. The BGM study performed here still represents an improvement from

the anthropomorphic approach where no ‘worst case scenario’ assumptions on head and neck

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mechanical properties were needed. In addition, our BGM model not only allowed physical

recording of the mechanical parameters, but it also allowed correlation of such data with the

actual injuries created (had there been some type of detectable injuries). This is a unique aspect

of manual shaking protocol where precise control of forces applied is nearly impossible, yet

with the proper instrumentation, similar type of mechanical data recorded from the baboon-

macaque or piglet studies could be recorded.

5.3.3. Empirical data for shaken baby syndrome discussion

In the introduction, an example of rhetorical exchange in the current SBS debate was given.

The debate has reached a point where studies of SBS may not be analyzed rationally for their

methodology but simply criticized or applauded based on conclusions reached by the study and

on which side of the debate the reader stands. In one such exchange, opposite groups in the

debate have accused each other of ‘living in a glass house’ or doing ‘junk science’ 122, 123. This is

a serious subject, the consequences of which are as serious as infanticide and potential

miscarriage of justice, and opinions of ‘junk scientists’ or medical professionals ‘living in

glasshouses’ are not sufficient. There are multiple urgent steps that need to be taken to refocus

this highly charged debate into a rational, scientific one. A critical evaluation of retrospective

and anecdotal medical evidence for their study methodology such as selection criteria,

postmortem examination protocols and analytical methods is required. At the same time,

carefully designed scientific studies must be conducted to test the various hypotheses that could

arise from such re-evaluations.

This BGM study represents the first step in that direction. Although inconclusive due to the

limitations stated above, the model incorporated many aspects that are discussed in the field. It

is one of the first producing experimental data that can be critically evaluated, not based on the

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‘vast experience’ of the professionals who have worked in this field for many years. I recognize

that their contribution to the field has been invaluable and their input is still very much required

in both designing and evaluating the experimental studies. The BGM protocol used in this study

could be adapted and modified to gain more experimental data that will only help the

understanding of SBS. The body of experimental data can then be used to validate other studies

of SBS or to modify study designs to be more relevant to the issues being evaluated.

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6. Future directions

6.1. Primate model of SBS

6.1.1. Axonal injury

The experiments performed did not produce any detectable injuries. Thus, the question of

whether or not manual shaking can cause head injuries is still unanswered. The BGM study in

this thesis has established an experimental protocol that could be adapted with two different

survival intervals, allowing sufficient time for βAPP development should there be axonal

injuries (Table 6-1).

Table 6-1. Proposed experimental groups for the study of axonal damage from manual shaking of BGM

Animal ID

Axis of displacement

Survival interval between shaking and euthanasia (Hrs) Other

Sham 1 n/a One Anesthesia followed by euthanasia only Sham 2 n/a Six

AP 1 Anterior-Posterior One

AP 2 Anterior-Posterior Six

Lat 1 Lateral One

Lat 2 Lateral Six

The presence of widespread axonal injuries after a longer survival interval without immediate

hemorrhagic injuries in BGM could imply two possibilities. First, if the axonal injuries are truly

traumatic in origin, it could suggest that the shearing of the axon is the mechanism of injury and

since the threshold of vessel injuries are higher in adult BGM than in human infants, only

axonal injuries are detected. Secondly, if the axonal injuries are secondary consequences of

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hypoxic ischemic event, the mechanism of which these axonal injuries and other components of

the triad develop after shaking must be further studied. Both traditional and unified hypotheses

could explain the development of hypoxia (generalized brain swelling after diffuse axonal injury

or hypoxia/ischemia following apnea from brainstem injury) but the mechanism of which SDH

or RH could develop is not yet known.

6.1.2. Comparative anatomy and head and neck movement during shaking

One of the critical factors in determining behaviour of the head and neck movement during

application of the force is the shape of the craniocervical junction. A study by Penning

compared the craniocervical junctions of humans and other animals and their impact on various

neck movements 121. Careful measurements of the locations and joint surface angles in humans

and other animals were taken from different components of the craniocervical junction including

the occipital condyles, foramen magnum, atlas and axis vertebrae. The study then measured the

range of motions allowed in these animals and found important differences between

quadrupedal mammals and human. The study also included lateral flexion data from a primate

(Orangutan) that showed the most similarity to the human compared to other animals. Compared

to quadrupedal animals, humans have largely lost this lateral flexion. Also in human, this

motion is coupled with the atlanto-axial rotation.

A similar type of analysis is needed to confirm the anatomical similarities between human

infants and BGM. The study should include thorough measurements of the location and angles

of the components of the craniocervical joint. The range of motions including sagittal extension,

flexion and translation (occipito-axial translation and head-trunk translation), rotation about the

atlanto-occipital and atlanto-axial joints, and lateral flexion should be evaluated with or without

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the supporting neck musculature. This study could be done in conjunction with the axonal injury

study proposed above to minimize the number of animals required.

6.1.3. Ethical consideration

The proposed study represents a minimum number of animals required to confirm the

original findings and to determine if manual shaking could generate axonal damage. It also

gives an opportunity to use a higher-range accelerometer to measure maximum peaks of

tangential accelerations. Once baseline mechanical displacement parameters are established,

attempts of scaling could be made for future studies of smaller but developmentally and

physiologically similar animals.

6.1.4. Anthropomorphic model

The general lack of knowledge about properties of the neck of human infants has led to

problems in designing biofidelic anthropomorphic devices. In the study previously mentioned,

Prange et al. had to design a neck with negligible resistance to err on the side of a worst case

scenario 63. A hinge was used as the centre of rotation and provided no resistance to either

extension or flexion in sagittal plane. However, the hinge did not provide any range of motion in

other directions and the location of the centre of rotation had to be estimated. This resulted in no

information being provided regarding movements in coronal plane and likely overestimated the

rotational acceleration.

The design of biofidelic devices could benefit greatly if the craniocervical anatomy of

primates is confirmed to be similar to humans. In that case, the resistance of the neck in all

directions could be measured in primates either under anesthesia or during postmortem

examination to reduce the effect of developed neck musculature of adult primates. Also, such

study could determine the precise location of the centre of rotation which would provide more

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realistic estimation of the rotational acceleration. Combined with the information from the

comparative anatomy study above, a more complex hinge mechanism could be designed that

could account for the movements in both the sagittal and coronal planes.

6.1.5. Mechanical properties of tissues and finite element modeling

A reductionist approach in building in silico model of SBS in the past has been limited by the

lack of experimental data about mechanical properties of the tissues and the interface involved.

Most studies of mechanical properties of tissues have been limited to testing of selective tissue

types (vessels, scalp, skull, etc.) with no regards to the properties of the modular systems these

components build together 124. Also, the finite element modeling of SBS has been hampered by

the lack of experimental data the model should aim to emulate 125-129.

The development of an animal model of SBS could provide key information required in such

a reductionist approach. While an animal model could provide physiologic and pathologic

information, the finite element models could be used to complement the inherent shortcomings

of the animal models. Using in silico models, the subtle species differences identified in

comparative anatomic studies and the age specific differences in tissue properties could be

tested for their effect on shaking.

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6.2. Hypoxia as cause of subdural hemorrhages

6.2.1. Perinatal and neonatal intradural hemorrhages

In recent months, a series of papers were published that suggested a link between age related

meningeal development and the propensity of SDH in hypoxia. In a retrospective, observational

study, Cohen and Scheimberg analyzed postmortem examination findings of 25 fetuses of 24

weeks of gestation or older that died in utero and 30 infants of one month of age or younger,

selected for the presence of intradural hemorrhage (IDH) 130. Both falco-tentorial and parietal

IDH were examined grossly and histologically. A strong association was found between the

presence and degree of hypoxia, the amount of IDH and the presence of SDH especially in

neonates. The paper also suggested that the IDH and SDH are mechanistically linked by

eventual weakening of the dural border cell layer next to the arachnoid membrane upon

accumulation of IDH. Interestingly, Squire et al. demonstrated the lack of intradural space

(dural channels) in neonates which is thought to contribute to the occurrence and severity of

IDH in this age group. These spaces could function as a reserve cavity for reflux from various

venous sinuses present in dura 100, 104, 131. With these two studies combined, a correlation could

be made for the propensity of hypoxia related IDH and SDH in younger infants.

6.2.2. Implication for the shaken baby syndrome ‘unified hypothesis’

In 2003, Geddes 3 has proposed the ‘unified hypothesis’ which proposed that hypoxia could

be the underlying cause of SDH seen in previously diagnosed SBS cases 73. However, Geddes

failed to demonstrate a mechanistic link between hypoxia and SDH other than the typical

presenting history of apnea in these SBS cases and the presence of brainstem injuries in some of

the cases reviewed. The ‘unified hypothesis’ has since been challenged in court (discussed in

Chapter 1) and in the literature. However, the ‘unified hypothesis’ can now gain some support

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with the newly proposed link between hypoxia and IDH/SDH in neonates if it can be

demonstrated that manual shaking can cause enough damage to the brainstem to cause apnea

leading to hypoxia.

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6.3. Medical investigations of SBS

6.3.1. Retrospective and prospective studies using βAPP immunostaining

Geddes 2 72 and a study by Oehmichen 74 represent the retrospective efforts being made to

evaluate the incidence of tDAI in previously diagnosed SBS cases using βAPP. These studies

are limited in their scope as number of cases is limited in each study location and therefore

could be difficult to generalize. Continuing efforts should be made with this approach as it can

provide data to compare to results of the experimental studies. Both retrospective and

prospective analysis of cases should be carried out using standards such as case selection

criteria, types of investigations performed in each case and standard grading of the positive

findings. Ideally, such studies should involve multiple centres from different regions to

maximize the sample size and increase the consistency of the analysis.

6.3.2. Cerebrospinal fluid analysis

In clinical settings where an infant is in serious medical condition without a known cause,

sampling of the cerebrospinal fluid (CSF) is quite common. CSF is sterile in healthy individuals

and is isolated from the blood circulation by blood brain barrier. Blood tinge in a CSF sample is

an indication of cerebral bleeding where the presence of bacteria in CSF is an indication of

meningitis or generalized sepsis. Advanced techniques are available to study the components of

fluids and their relationships; those techniques could be applied to the study of CSF. In general,

even slight change in components of CSF could be measured against the normal state using

molecular techniques. CSF collected in clinical settings could be analyzed to identify candidate

markers for each type of injury (hypoxia or axonal injury) at different time points and could be

utilized as an sensitive analytical test of head injury.

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6.4. Possible mechanisms for Shaken Baby Syndrome triad development

1. Although our experimental findings must be interpreted in the context of the limitations

discussed, our results did not support the traditional hypotheses proposed to explain subdural

hemorrhages and retinal hemorrhages following infliction of injury.

2. The presence of axonal injury could not be confirmed histologically, as the survival

interval required for the broad development of the injury marker was insufficient. The

experimental protocols should be repeated with a longer survival interval after shaking.

3. Although the maximum acceleration of the monkey’s head could not be directly measured,

it is likely that manual shaking cannot generate enough force to cause the pattern of injuries

identified in previous animal studies. However, movement of the head during lateral shaking is

complex and needs to be studied further.

4. There is growing evidence that IDH and SDH can develop in the setting of hypoxia in

neonates. This premise might represent the mechanistic link between hypoxia and SDH in the

‘unified hypothesis’.

5. It is still inconclusive as to whether manual shaking can cause the triad in human infants.

Although manual shaking does not appear to generate sufficient force to cause immediate

diffuse injuries, it is still unclear if focal brainstem injury could be produced to account for the

development of the triad pattern of injury.

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7. References

1. Goudge, S. Inquiry into Pediatric Forensic Pathology in Ontario. Ontario: Ontario Ministry of the Attorney General, 2008.

2. American Academy of Pediatrics Committee on Child Abuse and Neglect. Shaken baby syndrome: Rotational cranial injuries - technical report. Pediatrics 2001;108:206-10.

3. Helfer, ME, Kempe, RS, Krugman, RD. The battered child. Chicago: University of Chicago Press, 1997.

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Appendix A-1. Routine Hematoxylin and Eosin staining protocol

Procedure

1. Deparaffinize in Clearene® – 5 changes, 5 minutes in total

2. Remove Clearene® with absolute alcohol – 5 changes, 10 dips each with thorough

draining between changes

3. Wash in running tap water until alcohol is removed – 10 dips

4. Put in Harris’ Hematoxylin – 5 minutes with occasional agitation, Harris’ Hematoxylin

should be filtered before each use

5. Wash off excess Hematoxylin in running tap water – 10 dips

6. Differentiate in acid alcohol – 5 dips

7. Wash well in running tap water – 10 dips

8. Blue in lithium carbonate – 2 minutes

9. Wash well in running tap water – 5 minutes, check nuclear staining under microscope.

If overdifferentiated, repeat from step 4. If underdifferentiated, repeat from step 6.

10. Stain in eosin – 2 minutes with frequent agitation

11. Rinse off excess eosin in running tap water – 10 dips

12. Dehydrate in absolute alcohol – 5 changes, 10 dips each

13. Clear in Clearene® – 5 changes, 10 dips each

14. Mount with Permount®

150

Solutions

1. Harris’ Hematoxylin

Hematoxylin 14 gram

95% Ethanol 120 ml

Aluminum ammonium sulphate 240 gram

Distilled water 2,400 ml

Mercuric oxide 6 gram

Glacial acetic acid 48 ml

Mix the alum and water and bring to a boil. Remove from heat and add the hematoxylin

dissolved in alcohol. Bring to a rapid boil again. Remove from heat and let it settle for

one minute. Add mercuric oxide very slowly at first. Bring it to boil again, the cool

quickly in cold water. Add glacial acetic acid.

2. Acid alcohol – 1% hydrochloric acid in 70% ethanol

3. Saturated Lithium Carbonate

Lithium carbonate 12 gram

Distilled water 1000 ml

4. Eosin – 1% Eosin Y in distilled water and add a few crystals of thymol (2-isopropyl-5-

methylphenol)

Eosin 30 gram

Distilled water 3000 ml

151

Appendix A-2. Routine Luxol Fast Blue/H&E staining protocol

Procedure

1. Deparaffinize in Clearene® – 5 changes, 5 minutes in total

2. Remove Clearene® with absolute alcohol – 5 changes, 10 dips each with thorough

draining between changes

3. Wash in running tap water until alcohol is removed – 10 dips

4. Stain in Luxol Fast Blue solution for 4 hrs at 60°C. Use Paraflim to cover the solution.

5. Rinse in 70% ethanol and wash in distilled water until alcohol is removed – 10 dips

6. Differentiate in lithium carbonate – 30 seconds

7. Wash well in distilled water – 10 dips

8. Differentiate in 70% ethanol – 30 seconds

9. Wash well in distilled water – 10 dips

10. Check staining with the microscope – Myelin and RBC should be blue, the rest of the

tissues should be colourless. If overstained, repeat from step 6.

11. Follow routine H&E protocol with following modifications – Harris hematoxylin 3

minutes, lithium carbonate 1 minute, and eosin 1 minute.

12. Rinse off excess eosin in running tap water – 10 dips

13. Dehydrate in absolute alcohol – 5 changes, 10 dips each

14. Clear in Clearene® – 5 changes, 10 dips each

15. Mount with Permount®

152

Solutions

1. Luxol fast blue

Luxol fast blue 0.1 gram

95% ethanol 100 ml

10% acetic acid 0.5 ml

2. Lithium carbonate (for step 6)

0.05% lithium carbonate

153

ANIMAL USE PROTOCOL AND RESEARCH ETHICS BOARD REVISED PROPOSAL FOR PEER REVIEW

TITLE A PRIMATE MODEL FOR THE SHAKEN BABY SYNDROME

INVESTIGATOR Michael S. Pollanen MD PhD FRCPath DMJ(Path) FRCPC

INSTITUTION Department of Laboratory Medicine and Pathobiology

University of Toronto SUMMARY OF REVISIONS MADE

1. The total number of proposed animals remains UNCHANGED 2. The episodic shaking experimental group has been ELIMINATED 3. Eliminated animals are REALLOCATED for a survival interval

experimental group. 4. There is NO LUCID INTERVAL in any of the animals. All animals will be

under general anesthesia for all experimental manipulations with no conscious survival interval (i.e., all animals with be euthanized under general anesthesia).

SUMMARY OF RESEARCH PROPOSAL A controversy has been developing in pediatric and forensic medicine over the past 10-15 years – whether shaking an infant can cause fatal head injury. It has traditionally been held that shaking causes fatal and serious cerebral injury – the shaken baby syndrome (SBS). Initial opposition to the SBS concept was not generally viewed as a serious or credible challenge to the received view that SBS is a specific form of abusive head trauma. Currently, however, there is growing scepticism in the forensic pathology community, and some believe that the SBS concept may not so firmly evidence-based as once held. This growing scepticism has found empirical support in the peer-reviewed literature, mostly from descriptive retrospective neuropathologic studies on putative cases of SBS and some biomechanical modeling experiments. However, there has been no definitive experimental data to provide key data to inform the underlying issue: does shaking cause injury? To date SBS has not been reproduced in an animal model. At this time some researchers believe that we are at an impasse – until SBS can be reproduced in a relevant animal model we will be suspended in the current state of oppositional scientific debate with little empirical evidence that can provide data-driven conclusions. In essence, we are currently at a point where there is a complete block to further scientific development in our understanding of SBS. The medical and scientific community needs a decisive experiment to provide new data to inform the debate.

154

Martian Pathologist

Text Box

Appendix B-1

One clearly defined way forward is determining if shaking can cause the putative markers of SBS in an animal model that closely resembles an infant. The putative markers are the so-called SBS ‘triad’: subdural hemorrhage, retinal hemorrhage, and brain swelling. On this basis, I propose a ‘proof-in-principle’ experiment to determine if shaking can induce the triad in a small number of primates. This primate experiment would provide significant new knowledge to inform the discourse around SBS. RESEARCH PROPOSAL Background General Background Physical abuse of children is the leading cause of serious pediatric head injury. The shaken baby syndrome (SBS, or abusive head trauma), is widely viewed as a common and frequently fatal form of child abuse usually seen in children younger than two years of age, but occasionally observed in children up to five years of age1-3. The early descriptions of SBS in the 1970s suggested that the mechanism of head injury was a ‘whiplash’ motion of the head caused by rapid back and forth displacement of the infant’s head while the perpetrator was shaking the infant by grasping its trunk4, 5. In the American Academy of Pediatrics Technical Report on SBS, it is stated that the act of shaking is “so violent that individuals observing it would recognize it as dangerous and likely to kill the child”6. Despite authoritative claims of professional organizations such as the American Academy of Pediatrics and the National Association of Medical Examiners6, 7, a controversy has been developing in forensic medicine over the past 10-15 years – whether shaking on infant can cause fatal head injuries in infants. This debate has polarized the medical community into two camps: (i) Reputable physicians who strongly view the current medical evidence as

definitively supportive of shaking as a mechanism of injury that can be fatal or lead to permanent neurological impairment7-21.

(ii) Reputable physicians and non-medical scientists who view the current

medical and biomechanical evidence as either entirely non-supportive of shaking as a mechanism; or, at least, have doubt that the current evidence strongly supports the conclusion that shaking causes injury1, 22-34.

It is clearly and widely held in the medical community that shaking causes fatal and serious cerebral injury. It is undisputed that the concept of SBS would be identified by most pediatricians as a mainstream concept. In fact, the initial opposition to the SBS concept was not generally viewed as a serious or credible

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challenge to the established view that SBS was a coherent concept. However, there is growing scepticism about SBS in the forensic pathology community. Some forensic pathologists now question if the SBS concept is as firmly established on an adequate evidence-based as is widely believed. This scepticism has found some empirical support in the peer-reviewed literature, mostly from descriptive retrospective neuropathologic studies on putative cases of SBS21, 24, 25, 35; and some biomechanical modeling paradigms36-38. However, there has been no definitive experimental data to provide key data to inform the underlying issue: does shaking cause injury? At this time many physicians and scientists believe that we are at an impasse – until a relevant experimental model can be developed we will be suspended in the current state of scientific oppositional debate with little evidence that can provide data-driven conclusions. In essence, we are currently at a point where there is a complete block to any further development in our understanding of SBS. The medical and scientific community needs a decisive experiment to provide new data to inform the debate. It is generally accepted by the medical and scientific community that all currently available experimental animal models (e.g., rodent models) and biomechanical paradigms (e.g., biofidelic models) cannot provide a decisive experiment to resolve the SBS debate. All attempts to address this issue experimentally have failed to reproduce definitive results. All animal models have failed to produce the combination of lesions that are thought by many to represent markers of SBS. The biomechanical models have produced promising results, but the wider medical community has not accepted the data and continues to view that approach as only marginally relevant. Although retrospective clinicopathological and clinicoradiological studies have produced a range of important results, this approach will never be capable of providing definitive results on the key issue, since the mechanism of injury will always be scientifically disputable in any particular case (i.e., we will always ultimately rely on an assumption of validity of the history of the event, or a confession to ‘shaking’). Thus, one of the clearly defined ways forward in this debate is the development of a relevant animal model to determine if shaking can cause the putative markers of SBS as derived from autopsy studies of putative SBS cases. These putative markers comprise the so-called SBS triad: subdural hemorrhage, retinal hemorrhage, and brain swelling (i.e., cerebral edema and/or hypoxic encephalopathy with [or without] axonal lesions). On this basis, I am interested in exploring the use of a small number of primates to develop an initial experiment to determine if shaking can induce the triad. This primate experiment would provide significant new knowledge to inform the debate and may provide a ‘proof-in-principle’ answer to the SBS controversy.

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Preliminary Results We have studied putative shaking injury in a developing mouse pup model. Using several different specific experimental designs for shaking the heads of mice, we have been unable to produce meningeal, brain or spinal cord injuries of any type. Mouse pups (n = 40) were subjected to a high frequency vibration (20-50 Hz) under anesthesia while restrained in a manner that minimized chest compression but allowed free head movement. In both continuous high frequency vibrational shaking and pulse acceleration of the head, no central nervous system injury was detected in the whole mount section of the head. In contrast to previous reports, change in rotational plane did not result in more severe trauma. Attempts were made to deliver vibration directly to the head by restricting both head and neck movement, but again, no injuries were seen in this setting. The manuscript describing the details of the study is in preparation for publication. Rationale The failure to recapitulate the SBS triad in experimental animal models may relate to three important factors that all current animals models have failed to possess: (i) a relatively massive head providing significant inertia during motion; (ii) the precise craniocervical anatomy that permits a ‘shake’ to cause a rapid internal displacement of the brain and eyes, rather than simply vibrating the head on the neck; and (iii) any experimental mechanism of shaking that causes loading of head that an on-looker would unequivocally identify as shaking. The latter is not to be underestimated as an important variable since the American Academy of Pediatrics has established that this is a pivotal indicator of the lethality of shaking. On this basis, the most scientifically valid approach to studying SBS using an animal model is to use subhuman primates to determine if shaking can induce the triad. This primate experiment would provide significant new knowledge to inform the debate and may provide a ‘proof-in-principle’ answer to the SBS controversy. The animal of choice is the African green monkey. African green monkeys are among the most commonly used primates in biomedical research39, 40. These animals are small, easily handled, non-endangered, evolutionarily closely related to humans, and easily bred in captivity. Adult African green monkeys range in size from 4.1-5.5 kg and have top of the head to the base of the tail length of 426-490 cm41, 42. Based on these dimensional considerations adult African green monkeys are similar in weight to ~2 month infants. Most importantly, subhuman primates share with man important craniocervical anatomical similarities, which are not found in lower animals43.

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Research Plan Objective and Specific Aim The overall objective is to determine if SBS is a valid concept by reproducing the injuries in an animal model. Our specific aim is to determine if manual shaking of African green monkeys (C. aethiops) can cause head injuries including, subdural hemorrhage, retinal hemorrhage and diffuse axonal injury/hypoxic encephalopathy. Experimental Design In this experiment, healthy adult African green monkeys that were bred in captivity at Barbados Primate Research Centre (BPRC) will be screened for general health status and evidence of previous impact head injuries prior to the experiment. Animals with any scalp bruise or scars will be excluded from the experiment as will animals in generally poor health. General vital information of selected animals such as sex, height, weight, pulse, blood pressure and morphometric parameters including dimensions of the head will be recorded. A total of six animals will be used for this experiment: two will be used as controls and four will be subjected to manual shaking followed by euthanasia after a two hour survival interval under general anesthesia. All animals will be subjected to pre-anesthetic induction by intravenous propofol injection 30 minutes prior to the experiment. The state of general anesthesia will be maintained by isoflurane inhalation until eventual euthanasia by intravenous injection of barbiturates. The following parameters will be monitored from the initial anesthesia to euthanasia for all the animals: heart rate, temperature, oxygen saturation and breathing frequency. Any sign of deterioration will terminate the anesthesia and the animal will be promptly euthanized. Three small accelerometers will be attached to the head after induction of anesthesia and will be used to measure the acceleration experienced by the animal. Also, the shaking procedure will be recorded with a high-speed video camera to visualize the motion of the head and neck and calculate the maximal angle of the neck extension and flexion. In the experimental group, four animals will be subjected to shaking under anesthesia using the following two variables: axis of acceleration (sagittal vs. lateral) and survival interval after shaking (immediate euthanasia vs. 6 hours post-procedure survival under general anesthesia). The first animal will be shaken to establish a baseline for anterior-posterior (AP) shaking (sagittal acceleration-deceleration of the head). The animal will be firmly held by the chest and continuously shaken 20 times in anterior-posterior plane at the maximum head displacement generated manually. The animal will be euthanized by intravenous injection of barbiturates immediately after shaking. The second animal will be shaken in the anterior AP plane in the same manner described above. The animal will be kept under anesthesia for a further 6 hours after

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shaking and then euthanized. The same set of experiments will be repeated while shaking is applied in lateral plane, given the results of high-fidelity reproduction of axonal lesions in the Gennarelli-Thibault baboon-macaque model44-49. The control group will consist of a single negative control and a positive head injury control animal. The negative control animal will be subjected to pre-anesthetic induction, anesthesia and euthanasia without shaking. The positive control animal will be subjected to anesthesia in the same manner, and then the posterior aspect of the head will be subjected to controlled impact onto a flat, unyielding surface. The animal will be kept under anesthesia for 6 hours post-impact and then euthanized. After euthanasia, all animals will undergo postmortem examination using the standard autopsy protocol applied to cases of putative child abuse in the Province of Ontario. All gross findings will be documented and photographed. The brain, meninges, spinal cord, and eyes50 will be fixed in 10% buffered formalin (pH 7.4) prior to dissection. In addition to the latter specimens, the ribs and metaphyses of long bones will be studied macroscopically and microscopically. Tissue blocks will be processed for paraffin embedding. Exhaustive histologic examinations of the central nervous system, meninges, and eyes will be performed as in previous studies21, 24, 25, 51. Histologic sections from the central nervous system will be studied using conventional histologic preparations and immunohistochemistry using a variety of antibodies including: βAPP (marker for axonal injury), myeloperoxidase (neutrophils), CD-68 (macrophages), ubiquitin (extra-lysosomal proteolysis), GFAP (astroglia), and MAP-2 (dendrites). Macroscopic and microscopic data will be correlated with the data collected from the accelerometers. The average and maximal head acceleration achieved during shaking will be compared to the magnitude of acceleration and force described in biofidelic models of shaking. Expected Results The experiment will either result in the creation of some combination of subdural and retinal hemorrhages and diffuse axonal injury/hypoxic encephalopathy in monkeys, or it will not. If the results are positive, then this will be the first scientific-experimental evidence to support the existence of SBS. Thus, a positive result will provide powerful non-clinical validation of the prevailing or received view about SBS. If the results are negative, the issue will not be definitively resolved.

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Difficulties Anticipated There are no scientific barriers to the proposed research other than the possibility of a negative result that will not refute the hypothesis that SBS can be reproduced in primates. However, there are legitimate ethical considerations. There has been a justifiable movement away from the use of primates in medical research. This has been the case in head injury research since PETA animal activists brought attention to the NIH-sponsored Head Injury research program at the University of Pennsylvania in the laboratory of neurosurgeon, Dr. T. Gennarelli. It is accepted in the medical community by many researchers and by the applicant that animal rights ethics are a legitimate and important barrier to the development of many experimental models with primates. However, the considerable knowledge that can potentially be gained by this experiment is felt to balance the ethical arguments against it. Timeline The experiments will be performed at the Barbados Primate Research Centre (BPRC) over three days. Tissue removed from the animals will be analyzed at the University of Toronto over a period of several months. The study will be completed in less than one year leading to submission for publication and presentation at relevant scientific meetings.

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References 1. Whitwell HL: Head injury in the child. Edited by London, Arnold, 2006, p. 2. Gerber P, Coffman K: Nonaccidental head trauma in infants, Childs Nervous System 2007, 23:499-507 3. Trenchs V, Curcoy AI, Navarro R, Pou J: Subdural Haematomas and Physical Abuse in the First Two Years of Life, Pediatric Neurosurgery 2007, 43:352-357 4. Caffey J: On the theory and practice of shaking infants. Its potential residual effects of permanent brain damage and mental retardation, American Journal of Diseases of Children 1972, 124:161-169 5. Gennarelli TA, Thibault LE, Ommaya AK: Pathophysiologic Responses to Rotational and Translational Accelerations of the Head. Edited by 1972, p. pp. 296-308 6. American Academy of Pediatrics Committee on Child Abuse and Neglect: Shaken Baby Syndrome: Rotational Cranial Injuries - Technical Report, Pediatrics 2001, 108:206-210 7. Case ME, Graham MA, Handy TC, Jentzen JM, Monteleone JA: Position Paper on Fatal Abusive Head Injuries in Infants and Young Children, The American Journal of Forensic Medicine and Pathology 2001, 22:112-122 8. Guthkelch AN: Serious effects of shaking were described in 1971, British Medical Journal 1995, 310:1600 9. Margulies SS, Thibault KL: Infant skull and suture properties: Measurements and Implications for mechanisms of pediatric brain injury, Journal of Biomechanics 2000, 122:364-371 10. Graham DI: Paediatric head injury, Brain 2001, 124:1261-1262 11. Hymel KP, Jenny C, Block RW: Intracranial Hemorrhage and Rebleeding in Suspected Victims of Abusive Head Trauma: Addressing the Forensic Controversies, Child Maltreatment 2002, 7:329-348 12. Levin AV: For Debate: Shaken Baby syndrome, British Journal of Neurosurgery 2003, 17:15-16 13. Bonnier C, Mesples B, Gressens P: Animal models of shaken baby syndrome: revisiting the pathophysiology of this devastating injury, Pediatric Rehabilitation 2004, 7:165-171 14. Punt J, Bonshek RE, Jaspan T, McConachie NS, Punt N, Ratcliffe JM: The 'unified hypothesis' of Geddes et al. is not supported by data, Pediatric Rehabilitation 2004, 7:173-184 15. Reece RM: The evidence base for shaken baby syndrome: Response to editorial from 106 doctors, British Medical Journal 2004, 328:1316-1317 16. Healey K, Schrading W: A case of shaken baby syndrome with unilateral retinal hemorrhage with no associated intracranial hemorrhage, The American Journal of Emergency Medicine 2006, 24:616-639 17. Jenny C, Neglect CoCAa: Evaluating Infants and Young Children with Multiple Fractures, Pediatrics 2006, 118:1299-1303

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18. Margulies SS, Prange M, Myers BS, Maltese MR, Ji S, Ning X, Fisher J, Arbogast K, Christian CW: Shaken baby syndrome: A flawed biomechanical analysis, Forensic Science International 2006, 164:278-279 19. Wygnanski-Jaffe T, Levin AV, Shafiq A, Smith C, Enzenauer RW, Elder JE, Morin FD, Stephens D, Atenafu E: Postmortem Orbital Findings in Shaken Baby Syndrome, American Journal of Ophthalmology 2006, 142:233-240 20. Case ME: Abusive head injuries in infants and young children, Legal Medicine 2007, 9:83-87 21. Oechmichen M, Schleiss D, Pedal I, Saternus K-S, Gerling I, Meissner C: Shaken baby syndrome: re-examination of diffuse axonal injury as cause of death, Acta Neuropathologica 2008, 22. Jayawant S, Rawlinson A, Gibbon F, Price J, Schulte J, Sharples P, Sibert JR, Kemp AM: Subdural haemorrhages in infants: population based study, British Medical Journal 1998, 317:1558-1561 23. Geddes JF, Whitwell HL, Graham DI: Traumatic axonal injury: practical issues for diagnosis in medicolegal cases, Neuropathology and Applied Neurobiology 2000, 26:105-116 24. Geddes JF, Hackshaw AK, Vowles GH, Nickols CD, Whitwell HL: Neuropathology of inflicted head injury in children - I. Patterns of brain damage, Brain 2001, 124:1290-1298 25. Geddes JF, Vowles GH, Hackshaw AK, Nickols CD, Scott IS, Whitwell HL: Neuropathology of inflicted head injury in children - II. Microscopic brain injury in infants, Brain 2001, 124:1299-1306 26. Goldsmith W: Fatal pediatric head injuries caused by short-distance falls, American Journal of Forensic Medicine and Pathology 2001, 22:334-336 27. Uscinski R: Shaken baby syndrome: fundamental questions, British Journal of Neurosurgery 2002, 16:217-219 28. Donohoe M: Evidence-Based Medicine and Shaken Baby Syndrome. Part I: Literature Review, 1966-1998, The American Journal of Forensic Medicine and Pathology 2003, 24:239-242 29. Geddes JF, Tasker RC, Hackshaw AK, Nickols CD, Adams GGW, Whitwell HL, Scheimberg I: Dural haemorrhage in non-traumatic infant deaths: does it explain the bleeding in 'shaken baby syndrome'?, Neuropathology and Applied Neurobiology 2003, 29:14-22 30. Geddes JF, Plunkett J: The evidence base for shaken baby syndrome, British Medical Journal 2004, 328:719-720 31. Lantz PE: Response to Reece et al from 41 physicians and scientists, British Medical Journal 2004, 329:741-742 32. Lantz PE, Block RW: Junk Science and Glass Houses, Pediatrics 2004, 114:330 33. LeFanu J, Edwards-Brown R: Subdural and retinal haemorrhages are not necessarily signs of abuse, British Medical Journal 2004, 328:767 34. Miller M, Leestma JE, Barnes P, Carlstrom T, Gardner H, Plunkett J, Stephenson J, Thibault K, Uscinski R, Neidermier J, Galaznik J: A Sojourn in the Abyss: Hypothesis, Theory, and Established Truth in Infant Head Injury, Pediatrics 2004, 114:326

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35. Leestma JE: Case analysis of brain-injured admittedly shaken infants: 54 Cases, 1969-2001, The American Journal of Forensic Medicine and Pathology 2005, 26:199-212 36. Ommaya AK, Goldsmith W, Thibault LE: Biomechanics and neuropathology of adult and paediatric head injury, Journal of Neurosurgery 2002, 16:220-242 37. Goldsmith W, Plunkett J: A biomechanical analysis of the causes of traumatic brain injury in infants and children, The American Journal of Forensic Medicine and Pathology 2004, 25:89-100 38. Roth S, Raul J-S, Ludes B, Willinger R: Finite element analysis of impact and shaking inflicted to a child, International Journal of Legal Medicine 2007, 121:223-228 39. Ervin F, Palmour R: Primates for 21st century biomedicine: The St. Kitts vervet (Chlorocebus aethiops, SK). Edited by Washington DC, National Research Council, 2003, p. pp. 49-53 40. Carlsson H-E, Schapiro SJ, Farah I, Hau J: The use of primates in research: A global overview, Am J Primatol 2004, 63:225-237 41. Napier PH: Catalogue of Primates in the British Museum (Natural History) and Elsewhere in the British Isles. Part II: Family Cercopithecidae, Sub-family Cercopithecinae. Edited by London, British Museum (Natural History), 1981, p 42. Skinner JD, Smithers RHN: The mammals of the Southern African Sub region. Edited by South Africa, University of Pretoria, 1990, p 43. Penning L: Craniovertebral Kinematics in Man and Some Quadrupedal Mammals, Neuro-orthopedics 1995, 17/18:3-20 44. Adams JH, Gennarelli TA, Graham DI: Brain damage in non-missile head injury: observations in man and subhuman primates. Edited by 1982, p. pp. 165-190 45. Adams JH, Graham DI, Gennarelli TA: Neuropathology of Acceleration-Induced Head Injury in the Subhuman Primate. Edited by Grossman RG, Gildenberg PL. New York, Raven Press, 1982, p. pp. 141-150 46. Gennarelli TA, Segawa H, Wald U, Czernicki Z, Marsh K, Thompson C: Physiological Response to Angular Acceleration of the Head. Edited by Grossman RG, Gildenberg PL. New York, Raven Press, 1982, p. 47. Gennarelli TA, Thibault LE, Adams JH, Graham DI, Thompson CJ, Marcincin RP: Diffuse Axonal Injury and Traumatic Coma in the Primate, Annals of Neurology 1982, 12:564-574 48. Gennarelli TA: Head Injury in Man and Experimental Animals: Clinical Aspects, Acta Neurochirurgica 1983, Suppl. 32:1-13 49. Adams JH, Graham DI: Diffuse brain damage in non-missile head injury, Recent Advances in Histopathology 1984, 241-257 50. Gilliland MGF, Levin AV, Enzenauer RW, Smith C, Parsons MA, Rorke-Adams LB, Lauridson JR, La Roche GR, Christmann LM, Mian M, Jentzen JM, Simons KB, Morad Y, Alexander R, Jenny C, Wygnanski-Jaffe T: Guidelines for Postmortem Protocol for Ocular Investigation of Sudden Unexplained Infant Death and Suspected Physical Child Abuse, Am J Forensic Med Pathol 2007, 28:323-329

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51. Shannon P, Smith CR, Deck J, Ang LC, Ho M, Becker L: Axonal injury and the neuropathology of shaken baby syndrome, Acta Neuropathologica 1998, 95:625-631

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Response to reviewers’ comments on a proposal “A primate model for the shaken baby syndrome”

Patrick Jin Han Kim HBSc (MSc candidate) Michael S. Pollanen MD PhD FRCPath DMJ (Path) FRCPC

Department of Laboratory Medicine and Pathobiology, University of Toronto

________________________________________________________________ Overview

In recent ruling on a habeas corpus petition regarding disputed Shaken Baby Syndrome (SBS), an appeal court of the United States overturned a conviction for murder by accepting the defence expert opinion that the SBS was a flawed concept1. Also in the US, a new trial was ordered in a SBS case on the basis of new doubts about the reliability of the SBS concept. In the ruling, the court stated “that a significant and legitimate debate in the medical community has developed in the past ten years over whether infants can be fatally injured through shaking alone, whether an infant may suffer head trauma and yet experience a significant lucid interval prior to death, and whether other causes may mimic the symptoms traditionally viewed as indicating shaken baby or shaken impact syndrome.”2.

Almost every day, a news story could be found where a caregiver is charged with murder of a baby from shaking3-10. However, as one of the reviewers of the proposal pointed out, “in different parts of the world, the justice system proceeds on different bases concerning what is regarded as mainstream thinking in the area”. The origin of this confusion is from the very public disagreement between even the scientists and the experts in the field without empirical scientific evidence to support their positions11-18. Based on the evidence that is available at this moment, it is generally agreed that there are two fundamental questions regarding the shaking of the infants. First is whether or not shaking can cause subdural hemorrhage, retinal hemorrhage and diffuse brain damage, otherwise known as the ‘triad’. The question of specificity of these findings to shaking alone, which is essential in many legal proceedings, is the other fundamental question that still needs to be answered. Many important controversies such as lucid interval, the existence of traumatic diffuse axonal injury, the importance of hypoxic ischemic encephalopathy (as it could be secondary to traumatic injuries to the head or represents an alternate mechanism), and rebleeding of an old subdural hemorrhage. In this proposed pilot study, the investigators do not intend to resolve all of the issues described above. Instead, the investigators intend to establish a reproducible and relevant model of SBS to lay the foundations for the future studies that could resolve many of the issues that could not be achieved by other approaches. On this basis, we have chosen the most critical experimental variables to study in a relevant animal model: (i) axis of displacement; and (ii)

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Martian Pathologist

Text Box

Appendix B-2

time interval between shaking and euthanasia. The experimental approach captures essence of shaking motion described by American Academy of Pediatrics19. Although this means that the motion is not controlled rigorously (such as electronically-controlled motion devices), the pilot study aims to describe the mechanical parameters of the manual shaking action which there are great discrepancies in present literatures. In the following discussions, we address the specific issues raised by the reviewers.

Reviewer 1

The first reviewer shared the our view on the state of knowledge in this field and recognized the need of the proposed experiment. The reviewer raised a concern regarding a very important parameter of the proposed experiment: the time interval between shaking event and euthanasia. We have decided to modify the experimental protocol to address this issue. Reviewer 1 indicated: “From a purely technical standpoint, I wonder whether post-shaking anaesthesia for two hours is sufficient time to allow B-APP stainable axon retraction balls to form? They are regarded as taking at least two hours to do so in humans (Whitwell H., Forensic Neuropathology. Hodder Arnold. P97). It would seem wise to ensure there is sufficient time for this part of the pathology to form by perhaps extending this anaesthesia.” Although it is widely recognized that the time interval between cerebral injury and expression of βAPP is at least two hours20-22, there is growing evidence in the literature that it could be detected at earlier time intervals after the injury23. Although longer time intervals would allow broad development of β-APP stainable axonal retraction balls, it also allows secondary changes to the brain due to initial traumatic injuries via generalized swelling leading to non-perfusion anoxia and florid hypoxic ischemic encephalopathy24. Therefore, allowing sufficient time for βAPP development could give rise to confounding results that overshadows true injuries produced by shaking. Furthermore, tDAI related expression of βAPP has been disputed recently (as retrospective studies have shown that diffuse axonal injuries are not present in majority of the presumed shaken cases and those with positive βAPP immunostaining are either local axonal injuries in the brainstem known as stretch injuries or vascular (implying hypoxic) in nature25-27.

To resolve this issue, we have modified the experimental groups into two distinct time points: (i) immediate euthanasia (no survival) and (ii) 6 hour survival under general anesthesia. Immediate euthanasia will be used to access hemorrhagic injuries (RH, SDH) that do not require survival interval, and the 6 hour survival interval will be used to access the development of axonal findings as detected by immunohistochemical staining of βAPP.

Using these two distinct time points, any traumatically-induced hemorrhages detected at immediate euthanasia will lack any confounding effect from generalized hypoxic changes or alterations in vascular permeability that might

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occur in the 6 hour interval of survival. Therefore, any SDH or RH can be definitely linked to trauma alone.

On this basis, we have decided to postpone one of the research questions from the original protocol. We no longer propose to study the effect of episodic shaking. The number of animals to be used in the proposed experiments remains at six. We believe this is the minimum number of the animals required to address the experimental questions we have asked (Table 1).

Table 1. Summary of animal allotment including control animals.

Animal Number Axis of displacement

Time interval between shaking and

euthanasia (Hrs) Other

Negative control n/a n/a Anesthesia followed by

euthanasia only

Positive control n/a Six Traumatic blunt impact head and

brain injury

1 Anterior-Posterior Immediate

2 Anterior-Posterior Six

3 Lateral Immediate

4 Lateral Six

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Reviewer 2

The second reviewer raised concerns with regards to the appropriateness of the model and validity of shaking mechanism. The reviewer suggests an alternative model: a piglet model that has been used in vehicular trauma research. Although the piglet model has its advantages in modelling infant trauma, it has to be balanced with other anatomical and mechanical factors before being employed for this particular research. In general, following factors should be considered in selecting an appropriate animal model of human traumatic neuropathology28.

1. Ability to reproduce hypothetical mechanism

“In the experimental design, there is lack of detail on the frequency of the shaking “the animal will be firmly held by the chest and continue shaking 20 times in the anterior posterior plane at maximum head displacement generated manually.”” In a technical report on SBS, American Academy of Pediatrics stated that “the act of shaking leading to shaken baby syndrome is so violent that individuals observing it would recognize it as dangerous and likely to kill the child. Shaken baby syndrome injuries are the result of violent trauma.”19. At this time, the authors cannot determine the time needed to conduct 20 maximal head displacements (which will subsequently allow the rough estimation of the frequency). Part of the goals of this proposed pilot study is to establish these primary parameters that will be used for the subsequent studies. 2. Mechanical parameters/skull base geometry

“The objective and specific aim is to use non-human primates, specifically the African green monkey to reproduce the shaking injury in a model which reproduces the size and special anatomy of the human head and neck at two months. The experimental work proposed utilizes the African green monkey because “based on these dimensional considerations, adult African green monkeys are similar in weight to two months infants. Most importantly, subhuman primates share with man important cranial cervical anatomical similarities which are not found in lower animals.” This commentary indicates that the primate head would be approximately the same size as a two-month-old human infant and the relationship of the neck to the head would be similar to humans. This requires confirmation.” This comment raises two of the factors that the investigators considered in choosing subhuman primates for this proposed study. First is the dimensional similarity of subhuman primate head to that of the infants of the relevant age. Since one of the goals of this pilot study is to describe mechanical parameters of manual shaking such as frequency, maximum acceleration and trajectory, it is important to have the size and dimension closely matched. The dimensional similarity can be verified by comparing the relevant values from the growth charts of the infants29, 30 and that of the animals31, 32. More importantly, it is the geometrical consideration of the skull base, specifically the position of the

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foramen magnum and its orientation and the anatomy of the craniovertebral junction. We believe these anatomical factors shared by the African Green Monkeys are critical to success of the proposed experiment33, 34. Paleontological evidence shows that the position and orientation of foramen magnum in higher order species is linked to the evolutionary transition to the bipedalism where more ventral and horizontal which allows the position of the head to be more orthogonal to the ground35-37. This has important implication on the human head movement when compared to obligatory quadrupedal mammals (such as the piglet) as extension motion is reduced by 50 degrees, sagittal head-trunk translation is largely lost, and lateral flexion of the craniovertebral junction is also greatly reduced33. Due to these differences in mechanics of head movement, it is critical to employ anatomically similar animal in modeling shaking head injuries. Therefore, the use of African Green Monkeys in mechanical modelling of head injury is justified as they possess a clear anatomical similarity to human than the piglets. 3. Age matching/developmental stage “An alternate animal model which may have similar anatomic characteristics and be similar in the state of the development of the brain may be piglets. I understand that piglets have been used to study traumatic injury by the Insurance Institute of Highway Safety, Charlottesville, Virginia, USA. I do not know whether the information from these studies is publicly available.” There are studies published by a group in University of Pennsylvania using the swine model in this subject but generally are not accepted as true model of shaking. In these studies, the piglets are immobilized while their heads are subjected to a single, rapid (~40ms) angular acceleration controlled by actuator38-

40. It is ironic that this is the same group that initially explored baboon-macaque model with similar experimental set up and their study terminated due to ethical concerns raised by activist groups. Indeed, the subsequent swine studies essentially continued the research established the baboon-macaque model. However, the anatomical differences between human infants and piglets (described above) have resulted in the general belief that there is no valid model of shaking injuries in human infants, with exception of 11, 41-44. “Scientifically, what this model does not address is the difference between the immature brain and its coverings in the two-month-old human infant and that of the adult non-human primate brain. The infant brain and its vessels would be expected to be more mobile and more fragile than an adult brain. Our societal norm is to reduce invasive studies utilizing non-human primates, except when absolutely required. A key question is whether the brain anatomy and physiology of adult non-human primates is sufficiently similar to infant humans. The infant pig should be considered as an alternative animal model for these experiments.”

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The argument for the supposed fragility of the vessels in infants during shaking comes from the original lecture by Caffey as he proposed the mechanism of subdural hemorrhage during shaking is the tearing of the veins that transverses subdural space45. However, currently available mechanical studies of infant vessel properties do not give conclusive answer to this question as these studies are limited due to their inability to reproduce complex mechanical relationships that these vessels are exposed to such as brain dimensions, anatomical location of vessels and axis of angular displacement46. In this pilot study, one of the aims is to describe the mechanical displacement parameters of manual shaking. For this purpose, having dimensionally (size and weight of the head) similar animal, such as subhuman primates, will offer more relevant information for future studies in the field. Once baseline mechanical displacement parameters are established, attempts of scaling could be made for future studies of smaller but developmentally/physiologically similar animals. Overall, although piglet model could offer some advantages in modelling developmentally immature structures of the infant head, it does not possess anatomical (geometrical consideration of the skull base) and mechanical advantages of the primate model (ability to be shaken in a manner described by American Academy of Pediatrics and reproduce head motion of infants). As for pilot study, the study’s focus is to test plausibility of the model by limiting number of variables. Although these questions are important in the field, they could be studied better once the repeatable model is established. Conclusion The past attempts in modelling shaking head injuries in animals are limited in value due to the inappropriateness of the mechanisms that are used to inflict the injury. Although some models succeeded in reproducing the head injury38-40, 47-50, they failed to answer the fundamental question of whether or not shaking could cause these injuries. While this proposed study cannot address all questions, it serves as a starting point. The proposed experiments will help establish a direction for future research and shift the focus of the debate to empirical-experimental data. Once again, we believe the need for a primate model is justified. Much of the confusion in the field stems from the lack of agreement as to what SBS actually entails because there is no conclusive data that supports that shaking alone can produce lethal injuries. The closely related anatomical relationship between human and the subhuman primates will allow important new data to be gained from the proposed pilot study. These data can be the preliminary results for a more programmatically-focussed investigation of SBS using an experimental approach.

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the effects of shaking on the eye and central nervous ... · shaking did not immediately cause...

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