investigating the behavioural and antiapoptotic effects of

Investigating the behavioural and antiapoptotic effects of Deep

Brain Stimulation in a model of moderate-to-severe Traumatic

Brain Injury

by

Faiza Azreen Mahmud

A thesis submitted in conformity with the requirements

for the degree of Master of Applied Science

Institute of Biomedical Engineering

University of Toronto

© Copyright by Faiza Azreen Mahmud (2021)

ii

Investigating the behavioural and antiapoptotic effects of Deep

Brain Stimulation in moderate-to-severe Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a neurological event where an external mechanical force

to the brain can lead to devastating effects on physical, cognitive, and/or behavioural function in

humans. Currently, no therapeutic approach is substantially efficient in treating prolonged

secondary injury from TBI. Deep brain stimulation (DBS) is a neurosurgical procedure used to

manage debilitating symptoms in motor disorders. While DBS is demonstrated to modulate

targeted brain circuitry, its underlying mode of action is yet to be determined. This thesis

hypothesizes that high-frequency acute DBS delivered to the anterior thalamic nucleus (ANT)

will lead to recovery of memory deficits and reduce anxiety-type behaviour in a well-established

TBI rodent model, through a decrease in apoptosis. Salient findings suggest: (i) Acute

stimulation of the ANT improved spatial memory in rats with moderate-to-severe TBI, compared

to rats receiving no DBS; however, (ii) anti-apoptotic effects after ANT-DBS, as assessed via

caspase-3 measurements, were inconclusive.

iii

Acknowledgments

I would first like to express gratitude and my appreciation to Dr. Clement Hamani for his

unwavering support for the past 2 years. I am not sure what I would be doing right now without

your mentorship throughout my journey as an aspiring researcher. Thank you for granting me the

opportunity to join your lab as a technician at Sunnybrook shortly after I had completed my

bachelor’s degree. I am forever grateful for your kindness, and for helping me retain my research

aspirations in Neuroscience to this day. The past year has been difficult in terms of staying

motivated to continue my research, but I still want to pursue a path in this field because of your

guidance and ambition for all our projects. I would also like to thank Dr. Milos Popovic for giving

me this opportunity to pursue a career in Biomedical Engineering, and for allowing me to extend

my reach into (somewhat) unfamiliar territory, without hesitation. I am grateful to my committee

members, Dr. Jose Zariffa, and Dr. Suneil Kalia for their expertise and constructive criticism that

have helped me refine my thought process for this thesis. It is unfortunate that we were only able

to interact twice, and only during committee meetings. I hope that we will have more chances to

meet in the future.

I would also like to extend my deepest gratitude to Mustansir Diwan, for helping me throughout

the past 2 years and refining my technical skills in the lab with an extraordinary degree of patience.

Thank you for providing insightful advice, indulging in tangents we would go into during one of

our discussions, and the humour and jokes that followed. You have made my time in the lab a

wonderful learning experience and I have immense gratitude for all your support, even on days

when I had felt incredibly disheartened.

Thank you to Ying Meng and our amazing post-docs, Darryl Gidyk and Flavia Gouveia for all

their invaluable experimental help, especially under these current circumstances. Thank you for

teaching me with utmost patience, especially when learning stereotaxic surgery. I am sad that we

have not been able to interact with one another for half of my thesis, due to the pandemic and

current social distancing measures. I have learned a great deal from all of you about behavioural

neuroscience, and I hope to grow to be as passionate and dedicated a scientist as you all are. Thanks

to Esther Silk for being such an awesome lab-mate. It was always a pleasure hanging out and

working together when we were in each other’s presence.

Lastly, I am forever and always grateful to my beloved husband, Aadiyat. We have both come a

long way with our ambitions since high school and you are my greatest strength. I consider myself

to be very lucky to have you as my support system. You have constantly nurtured my interests and

brought me back up when my spirits were low. In both the best and worst of times, you were all

that I have had. Thank you for holding my hand throughout undergrad and our marriage. I thrive

to be a better person because we’re in this together.

iv

Table of Contents

Contents

Chapter 1 – Introduction ..................................................................................................................1

1.1 Traumatic Brain Injury (TBI) ..............................................................................................1

1.1.1 Definition and Classifying TBI ................................................................................1

1.1.2 Epidemiology ...........................................................................................................2

1.1.3 Neuropathophysiology following Acute TBI ..........................................................4

1.2 Models of Traumatic Brain Injury .......................................................................................6

1.2.1 Controlled Cortical Impact (CCI) ............................................................................8

1.2.2 Weight-drop models.................................................................................................9

1.2.3 Fluid Percussion Injury models (FPI) ......................................................................9

1.2.4 Blast and Penetrative injury models ......................................................................11

1.3 Deep Brain Stimulation......................................................................................................14

1.3.1 Overview ................................................................................................................14

1.3.2 Proposed Mechanisms of DBS ..............................................................................15

1.3.3 Deep Brain Stimulation in Traumatic Brain Injury ...............................................18

1.4 Rationale, objectives, and hypothesis ................................................................................21

Chapter 2 – General methods & materials .....................................................................................23

2.1 Animals and Surgical procedures .......................................................................................23

2.1.1 Animals .....................................................................................................................23

2.1.2. DBS electrode implantation ...................................................................................24

2.1.3 Fluid percussion injury (FPI) and acute Deep Brain Stimulation ..........................25

2.2. Behavioural assessments ....................................................................................................27

2.2.1. Novel location and novel object recognition Habituation: Open field testing .......28

2.2.2. Marble Burying ......................................................................................................28

v

2.2.3. NLR/NOR: Familiarization, novel location and novel object recognition

testing .....................................................................................................................30

2.2.4. Barnes maze ...........................................................................................................31

2.2.5 Cresyl violet staining .............................................................................................33

2.3. Neurochemical experiments...............................................................................................34

2.3.1. Caspase-3 Enzyme-linked immunosorbent assay (ELISA) ...................................35

2.4. Statistical Analyses ............................................................................................................36

Chapter 3 – Results ........................................................................................................................37

3.1 Study 1: To characterize behavioural deficits in the moderate-to-severe TBI fluid

percussion injury (FPI) model in rats.................................................................................37

3.2 Study 2: To investigate the effects of acute ANT-DBS in the FPI model through a

battery of behavioural tests associated with learning, memory and anxiety-like

behaviour............................................................................................................................44

3.3 Study 3: To test whether ANT-DBS reduces hippocampal apoptosis in FPI-exposed

rats. .....................................................................................................................................51

Chapter 4 – Discussion ..................................................................................................................53

4.1. ANT-DBS ..........................................................................................................................55

4.2. Caspase-3 ...........................................................................................................................58

4.3. Limitations and future perspectives ...................................................................................60

4.4. Conclusions ........................................................................................................................64

vi

List of Abbreviations

AD Alzheimer's Disease

ADn Anterodorsal nucleus

ALS Amyotropic Lateral Sclerosis

AM Anteromedial nucleus

ANOVA Analysis of Variance

ANT Anterior Nucleus of the Thalamus

APOE Apolipoprotein E gene

APP Amyloid Precursor Protein

AUP Animal Use Protocol

AV Anteroventral nucleus

BBB Blood-Brain Barrier

BDNF Brain-Derived Neurotrophic Factor

BUNS Barnes maze Unbiased Strategy

Ca2+ Calcium ion

CCI Controlled Cortical Impact

CHIMERA Closed-Head Impact Model of Engineered Rotational Acceleration

CT Computerized Tomography

DAI Diffuse Axonal Injury

DBS Deep Brain Stimulation

DGC Granular cells of the Dentate Gyrus

DN Cerebellar Dentate Nucleus

DNA Deoxyribonucleic Acid

DTI Diffusion Tensor Imaging

DWI Diffusion Weighted Imaging

EC Entorhinal Cortex

ELISA Enzyme-linked Immunosorbent Assay

FPI Fluid Percussion Injury

GABA Gamma-aminobutyric Acid

GCS Glasgow Coma Scale

vii

Gpi Globus Pallidus internus

HFS High Frequency Stimulation

K+ Potassium ion

LCN Lateral Cerebellar Nucleus

MB Marble Burying

MCS Minimally Conscious State

MRI Magnetic Resonance Imaging

mTBI Mild Traumatic Brain Injury

MSN Medial Septal Nucleus

Na+ Sodium ion

NMDA N-Methyl-D-Aspartate

NLR Novel Object Location Recognition

NOR Novel Object Recognition

OR Operating Room

PBBI Penetrating Ballistic-like Blast Injury

PD Parkinson's Disease

PPTg Pedunculopontine tegmental nucleus

PTSD Post-Traumatic Stress Disorder

REB Research Ethics Board

SRI Sunnybrook Research Institute

STN Subthalamic Nucleus

TBI Traumatic Brain Injury

TDP-43 Transactive Response DNA-binding protein 43

TNF Tumor Necrosis Factor

TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP nick end-

labelling

Vim Ventral intermediate nucleus of the thalamus

viii

List of Figures

Figure 1: Typical experimental set-up in TBI animal models for Fluid Percussion Injury (FPI)..

....................................................................................................................................................... 10

Figure 2: Timeline for experiments performed for both studies 1 (TBI groups; no electrode

implantation surgery was performed) and 2 (DBS-TBI groups) .................................................. 27

Figure 3: Open field arrangement for DBS-TBI animals ............................................................. 28

Figure 4: (A) Typical setup of marble burying (MB) testing cages before placing animal for

behavioural protocol. (B) MB cage after 30 minutes of behavioural testing. (C) Side-view of

buried marbles for animal ID’d as ‘Fz042’................................................................................... 29

Figure 5: (A) Novel location recognition (NLR). (B) Novel object recognition (NOR) .............. 31

Figure 6: Barnes maze .................................................................................................................. 33

Figure 7: Defensive marble burying test for TBI, sham-TBI (craniotomy, no FPI) and naïve (no

surgical procedure) groups ............................................................................................................ 38

Figure 8: Novel object recognition (NOR) in TBI, sham-TBI and naïve groups ......................... 39

Figure 9: Memory deficits observed in TBI animals compared to other groups in the novel

location recognition (NLR) test .................................................................................................... 40

Figure 10: Barnes maze data for TBI treatment and control groups displaying changes in

performance across all variables, between train and test days ...................................................... 42

Figure 11: Average Barnes maze performance in all variables (latencies, search strategy and

error count) for TBI treatment and control groups ....................................................................... 43

Figure 12: Rat brains recovered 24h post-Barnes maze testing .................................................... 44

Figure 13: Average number of buried marbles in the DBS treatment group demonstrate no

significant improvement in anxiety type behaviour, compared to sham DBS-TBI, and TBI only

groups ............................................................................................................................................ 45

ix

Figure 14: NOR performance in DBS-TBI groups demonstrate no significant difference in novel

object exploration (A) or preference (B) ...................................................................................... 46

Figure 15: NLR performance in DBS-TBI groups demonstrate no significant difference in novel

location exploration (A) or preference (B), in comparison to sham DBS-TBI, or TBI only groups.

....................................................................................................................................................... 47

Figure 16: Barnes maze data for DBS-TBI, sham DBS-TBI and TBI groups displaying changes

in performance across all variables, between train and test days ................................................. 48

Figure 17: Average Barnes maze performance across all variables (latencies, search strategy and

error count) in DBS-TBI, sham DBS-TBI and TBI only groups .................................................. 49

Figure 18: Electrode location ........................................................................................................ 51

Figure 19: Caspase-3 measurements obtained using an enzyme immunosorbent assay (ELISA)

for TBI and non-TBI hemispheres ................................................................................................ 52

1

Chapter 1 – Introduction

1.1 Traumatic Brain Injury (TBI)

1.1.1 Definition and Classifying TBI

Traumatic brain injury (TBI) is defined as a neurological event whereby damage to the

head and brain tissue results from external mechanical forces, such as those caused by rapid

acceleration or deceleration of motor vehicles, blast waves from explosive devices, or impact

from a penetrating object1,2. Due to the physical, psychosocial and cognitive impairments not

immediately visible in patients suffering TBI, it is often referred to as a ‘silent epidemic’3,4. TBI

currently remains a critically significant worldwide public health issue and is the leading cause

of mental health disorders, death and disabilities associated with trauma3. Consequently, due to

the varying degrees of head trauma, survivors of TBI suffer from long-term neuropsychiatric

sequelae of behavioural disorders, post-traumatic stress disorder, anxiety, depression, and

cognitive dysfunction5,6. Head injury, when left without proper treatment, is also reported to be a

high risk factor for developing Alzheimer’s disease (AD)7. Amyloid β-protein accumulation, a

pathological trait implicated in AD, is also found in individuals who carry the e4 allele of the

APOE gene, which is increased in expression following head injury7,8. TBI is characterized by

cellular and structural deficits as well as neurological dysfunction, including, but not limited to,

neuroinflammation, vascular injury, cortical contusions and clinical endophenotypes, as well as

diffuse axonal injury (DAI)9. In humans, cognitive impairments and pathophysiological events

following TBI are largely dependent on injury severity1.

Extensive care and rehabilitation are necessary for TBI survivors and currently, there are

no therapeutic interventions sufficiently effective in improving the outcomes and complexities of

clinical TBI10. Both primary and secondary injury mechanisms are responsible for subsequent

structural and functional damage following TBI11,12. Primary injury is associated with direct

mechanical disruptions in brain tissue following the initial injury impact. This includes blood

vessel ruptures or hemorrhaging, focal contusions, hematomas, axonal shearing and DAI13.

Secondary injury, which occurs over several hours to months following TBI, includes non-

mechanical damage that lead to complex cellular, molecular and metabolic cascading events that

2

can further exacerbate the injury and eventually lead to atrophy and neuronal cell death2,13.

Immediate cell death caused by the initial impact of trauma on the brain can be irreversible and

difficult to treat; however, current treatment interventions focusing on interrupting cascading

events caused by secondary injury may provide neuroprotection and a significant improvement

in TBI incidences1. To date, there are no known or well-established neuroprotective interventions

for treating TBI2.

Severity of injury is often used to classify TBI in clinical settings, from mild to moderate

and severe11,14. Traditionally, clinical TBI is assessed using the Glasgow Coma Scale (GCS),

where the degree of injury severity is scored out of 15 points, based on eye, verbal and motor

movements6,14,15. Mild TBI (mTBI), the least severe and most common form of TBI3,16, is

diagnosed in patients usually with a non-penetrating closed-head trauma, and receives a GCS

score of 13-159,15. Moderate and severe TBI are evaluated by scores of 9-12 and ≤8,

respectively14, and require intensive care and neurosurgical interventions11. In addition to the

GCS, clinical guidelines for evaluating the magnitude of TBI also factor in structural imaging

(e.g. computed tomography or magnetic resonance imaging scans), severity and duration of loss

of consciousness, and other neurological symptoms, such as headaches, seizures, amnesia,

attention and/or memory impairment13.

Concussion is a term sometimes interchangeably used with mTBI. While most patients

generally recover quickly from mTBI, persistent symptoms have been reported, such as post-

traumatic amnesia, slower response times, disorientation, impaired learning and other memory

deficits9,16. Diagnosis and management of clinical mTBI can be challenging due to variability in

the extent of cell death and grey matter damage, that are not easily detectable by conventional

brain imaging techniques9,17. Currently, there are no clear models available for characterizing

and validating known biomarkers and long-term cognitions associated with clinical mTBI9,11.

Further investigation is needed using different approaches, including the use of experimental TBI

models, in order to obtain comprehensive interventions to translate into the clinical setting10.

1.1.2 Epidemiology

TBI is a major cause of death and disability across different age groups. Globally, it is

estimated to cause an annual incidence of approximately 10 million deaths and/or

hospitalizations in industrialized countries3,4. Each year, an estimated 57-69 million individuals

3

have or will have experienced TBI, of which 81% are mild and 11% are moderate in severity of

injury1,3. By utilizing statistical models and available national databases, current global data

estimated nearly 60% of TBIs annually are due to road traffic collisions; 20-30% are due to fall

incidents, while the remaining 10-20% are due to war, violence, or a combination of

occupational and sports injuries3,4. The highest incidences of TBI per capita were reported in the

United States, Canada and in European regions, while South-east Asian and Western Pacific

regions demonstrated the highest overall individual and systemic burden of TBI3.

In the United States alone, it is estimated that 1.7 million people sustain a TBI each year,

leading to 52,000 deaths and 275,000 hospital admissions5,6. In addition, >40% of participants in

a state-wide population-based survey reported having at least one TBI in their lifetime, while the

US Centers for Disease Control and Prevention had reported approximately 500-800 new cases

of TBI for every 100,000 people per year between 2000-20103,11. In 2013, 2.8 million TBI-

related deaths and hospitalizations were reported in the United States, of which 79% elderly

adults and 54% children suffered from fall-related accidents, which was found to be a primary

cause of TBI, especially amongst the elderly5,6. American/Alaskan Natives and African-

Americans had previously been reported to have a 4 times higher susceptibility rate to TBI due to

violence than white males4. Differences in gender have also been reported, where males were

consistently shown to have a higher incidence of TBI (1.5 times more likely) compared to

women4,6; older adult groups >60 years demonstrated higher occurrences of TBI compared to

younger adults4,11. This increase in TBI amongst the elderly can be explained by their longer life

expectancy with increased complex comorbidities and slower hospital recovery time from injury,

compared to younger adults5. In 2015, the US Defense and Veterans Brain Injury Center had

reported more than 22,000 military service members who had sustained a TBI from previous

combat and military training; 82% of these incidences were categorized as mTBI11.

In Canada, 23,000 hospitalizations per year were due to TBI, with 8% of these

individuals succumbing to their injuries5. In Ontario, a majority of TBI from work-place injuries

were found in manufacturing and government service sectors5,18. A retrospective chart review

showed that 57.8% of work-place related TBI incidences occurred in men, with the highest

incidence of occupational TBI found in the transportation and storage industry, at 81.5 per

100,000 individuals18. Trauma from being struck by or against an object was a major mechanism

of occupational TBI, followed by fall-related injuries18.While current epidemiological data have

4

demonstrated the significant disease burden of TBI, reports from middle- and low-income

regions may be limited in accurate estimates, in comparison to reports from high-income regions,

resulting in a large disparity in global TBI incidences3,11. This is largely due to the lack of quality

and robustness in data collection from low- and middle-income regions, where health care

systems have fewer resources and greater disease burden3. Further action and changes in research

policy are necessary to gather more reliable estimates of TBI occurrences in regions with limited

resources3,6.

1.1.3 Neuropathophysiology following Acute TBI

Though mild forms of TBI are the most prevalent, the clinical impact, mortality and socio-

economic burden of moderate-to-severe TBI comprise an enormous healthcare problem.

Characterizing and validating neuropathologies associated with moderate-to-severe TBI in

animal studies would allow for further advancement of our knowledge for underlying

physiological and neurochemical responses of TBI in humans and potentially lead to the

development of novel treatment modalities11,19.

Specific features of numerous TBI animal models have supported our ongoing

investigation of the sequelae that follow acute TBI in different brain regions11,20–22. Often,

neuropathological changes of TBI would consistently result in similar effects, and can be

categorized as either focal or diffuse injury that are followed by primary and secondary phases of

neurological damage22–24. Primarily, due to severe impact caused by direct blunt force or

penetrative trauma to the brain surface, focal injuries, such as discrete cortical and subcortical

lacerations and/or contusions, intracranial bleeding, subdural hematoma and subarachnoid

hemorrhages occur22. Pathologies caused by focal injury tend to have variable results, depending

on the type of focal injury, and the neuroanatomical location in which they had appeared23. On

the other hand, diffuse injury tends to display consistent pathological events, and is not

necessarily caused by similar neurological insults; rather, there would be shearing and stretching

of brain tissue due to rotational or sudden changes in acceleration induced forces11,22. A notable

form of diffuse injury is referred to as DAI, while microvascular and hypoxic-ischemic injuries

also affect several anatomical regions of the brain24. Unlike mTBI, that is characterized by more

diffuse injuries, as had previously been denoted in both animal models of closed-head trauma

and in clinical settings16,22,25,26, most acute moderate-to-severe TBIs are heterogeneous, having

5

both focal and diffuse injuries22,24. Investigating the neurochemistry and neuropathology behind

acute moderate-to-severe TBI implicates animal models where typically, a craniotomy is

performed, and the exposed skull is subjected to direct crush impact by a well-established

impactor device (e.g. fluid percussion27, controlled cortical impact28,29, further discussed in

section 1.2).

Rapid shearing and acute disruption of axons lead to DAI, one of the most commonly

observed neuropathology features of TBIs22,30,31. Owing to its higher white to gray matter

volume ratio, large mass, size, and characteristic gyri and sulci, the structure of the human brain

is susceptible to damage caused by linear acceleration/deceleration, or rotational forces24,32. DAI

severity corresponds to the amount of deceleration force impacting on the brain22; however,

while DAI is characteristic of TBI neuropathology, challenges arise in their detection and

identification using conventional imaging techniques, such as computerized tomography (CT) or

magnetic resonance imaging (MRI) scans of affected brain regions33. Histological techniques and

novel MRI sequences and techniques, such as diffusion weighted imaging (DWI) and diffusion

tensor imaging (DTI), have clearly identified changes in axonal integrity following DAI in

patients, shortly after experiencing trauma22. Any disruption in linear microtubular and

neurofilament structure would render axons highly susceptible to axonal swellings due to the

accumulation of membrane organelles and disassembled cytoskeletal components; subsequent

interruption in axonal transport would lead to secondary neuronal disconnection between

synapses, and eventually, the initiation of Wallerian degeneration 31,34. This active process of

degeneration is characterized by cytoskeletal pathology, displaying significant calcium influx,

due to the lack of regulation of calpain-mediated proteolysis of cytoskeletal proteins24. This

excessive calcium ion influx precedes microtubule disassembly, dysfunction in the mitochondria,

and eventually prepare the mechanically injured axons for excessive stretching, cell death, as

well as gradual axotomy24,31,34. Patients that experience a progressive decline in cognitive and

motor abilities several years after the initial TBI continue to display swellings within the

damaged axons, as well as further neuronal disconnection35

Hemorrhages within the corpus callosum and rostral brainstem can be observed in

moderate-to-severe TBI, while axonal injury and neuropathology can be detected with

histological and immunostaining techniques, marking for neurofilament and amyloid precursor

proteins (APP), shortly after TBI24.

6

Initial acute trauma to the brain has shown to initiate a multifaceted series of

neurochemical changes24. DAI and its resultant damage to cell membrane integrity causes a

disruption in ion transport. For example, a high influx of calcium ions and efflux of potassium

from ion channels result in increased excitatory glutamate neurotransmitter release, which binds

to N-methyl-D-aspartate (NMDA) receptors, causing excess depolarization of neurons; this

disruption in ion balance across cell membranes leads to increased activity to restore homeostasis

and the subsequent impairment to oxidative metabolism and depletion of energy stores22–24. In

addition to the axons undergoing secondary chemical effects following DAI, moderate-to-severe

TBI consistently leads to a cascading series of neuroinflammatory events that gradually

accumulate, contributing to a higher risk for long-term neurodegenerative diseases such as

Alzheimer’s23, Parkinson’s disease (PD) as well as amyotrophic lateral sclerosis (ALS)24. The

heterogeneity of TBI has been characterized not only by secondary injury cascades involving

neuronal cell death, neuroinflammation, degradation of white matter, and microglial activation

following initial trauma, but also the presence of neuropathological markers, such as an increase

in caspase-3, APP, phosphorylated tau, or TAR DNA-binding protein 43 (TDP-43), all of which

contribute to the late onset of neurodegenerative diseases that significantly impact the quality of

life for patients who had experienced TBI23,24,31.

1.2 Models of Traumatic Brain Injury

In order to further unravel and explore the pathophysiology of TBI in humans, elucidate

causal disease mechanisms, and develop preliminary therapeutic interventions, it is necessary to

have valid animal models1,9. For any animal model being implemented, specific principles are

required to be met to avoid miscommunication, poor representation, and replication of results in

our disease or endo-phenotype of interest. Such principles include i) determining a clear

objective with respect to utilizing an animal model; ii) having based our model on established

clinical attributes that define the condition; iii) the biofidelity or the degree of accuracy between

key features of the animal model and the human condition of interest; iv) displaying a correlation

between clinical results in humans and the efficacy of pre-clinical data obtained from the animal

model to verify its validity9,36. Numerous models have been developed for replicating the various

aspects of human TBI to provide adequate paradigms for testing treatment approaches.

Validation of said models is dependent on resemblance to the human condition in terms of

behavioural deficits, pathophysiology, causation, as well as response to therapeutic strategies36,37.

7

TBI in humans is known for its considerable heterogeneity exhibited by diverse modes of

primary injury, complemented with corresponding complexities from various secondary insults

in underlying cellular pathways38. In a practical setting, due to the highly heterogenous nature of

human TBI, current reproducible experimental animal models focus on partial aspects of injury.

For every available TBI model, only a given number of processes and mechanistic properties of

TBI can be accurately replicated1,9,38. Therefore, no single TBI animal model in the current

literature is capable of reproducing all processes of TBI presented in a clinical setting38.

However, despite the absence of an all-encompassing TBI model, currently available pre-clinical

models are capable of accurately mimicking various aspects and severity of TBI, which are

instrumental in understanding their mechanisms37. Successful TBI models include weight-drop39–

41, closed cortical impact (CCI)10,28, fluid percussion injury (FPI)27,42,43, blast injury, which may

consist of diffuse1,44,45 or more focal injuries, as depicted by the penetrating ballistic-like blast

injury (PBBI) model46–48. In addition, in vitro models have also been generated to provide less

expensive and more environmentally isolated TBI experiments, having been found to predict

some in vivo results49. Both chemical and mechanical injury have been applied to immortalized

cell lines49–51 (Table 1). Despite their ease of access and repeatable characteristics, however, in

vitro TBI models still face a challenge in encompassing the many complexities of TBI,

particularly the events that follow a primary blast injury49,52, which have been well-characterized

by most in vivo models.

An extensive body of literature in pre-clinical TBI research had been acquired using the

CCI and FPI models in rodents and large animals to determine behavioural and sensorimotor

impairments, as well as underlying histopathological responses, such as increased intracranial

pressure, neuroinflammation and localized grey and white matter damage51,53–55. Hence, models

of TBI are widely discussed due to their sensitive and highly reproducible properties and will be

relevant to the experiments presented in this thesis. However, regardless of the injury model

protocol implemented in experimental TBI studies, common technical attributes, such as the

frequency of anaesthetic exposure in animal models, could potentially compromise the outcome

of most TBI models, that typically require the use of anesthesia9. There is also concern over the

lack of sensitivity and robustness of previous mild TBI models, as histological or behavioural

data demonstrate variance across mild TBI studies, and will need to be addressed with further

refinement and characterization17,37,56. Below we will briefly describe the most commonly used

8

in vivo TBI models, namely controlled cortical impact, fluid percussion injury and blast and

preventative injury. Table 1 is then provided with further details on these models as well as

additional preparations.

1.2.1 Controlled Cortical Impact (CCI)

Controlled cortical impact (CCI) injury is produced by a device consisting of a

pressurized gas (i.e. pneumatic)29 or an electromagnetically-driven10 piston that accelerates a rod

over the surgically exposed dural surface10. Key parameters of injury are controlled by

characteristics of the impactor, such as the velocity, dwell time and depth, which can be adjusted

to produce varying degrees of TBI. In a closed-head variant of the model, repetitive and mild

TBI paradigms are used with no dural exposure10,57. Alongside widespread changes in

morphology and pathology (e.g. neuroinflammation, contusion, axonal injury, cortical tissue

loss), functional deficits have been observed, including overall motor and frontal lobe function,

as well as cognitive impairments in learning and memory28,29,58–61. These deficits have been

detected in widely accepted and reliable behavioural tests, including the Barnes maze26,62, Morris

water maze63, rotarod26,64 and elevated plus maze61,65, among others. Progressive declines in

cerebral blood flow have also been reported in rats, persisting even 1 year post-CCI injury66.

Although CCI is known for producing significant cortical contusions, CCI impact can lead to

post-traumatic apnea and tissue necrosis near the injury site57. Varying one or more of the

impactor characteristics could make an injury parameter change from minor to very severe.

Hence, pilot studies are often required to establish a suitable TBI paradigm10. Due to its focal

nature, one caveat of CCI is the inability to model diffuse forms of TBI67.

Like most pre-clinical TBI models described in this chapter, CCI is limited to replicating

one or more aspects of human TBI, as opposed to the entire event10,54. Some forms of clinical

TBI occur after a rapid impact to the skull68. CCI is more useful to mimicking these types of

head injury scenarios 25,69. Another variant of the CCI model, referred to as the CHIMERA

(Closed-head impact model of engineered rotational acceleration), utilizes rotational acceleration

forces on a closed skull. This mimics closed head injury and does not require surgical

intervention to deliver the impact69. The CHIMERA-TBI model has been shown to display key

features of human TBI. These include deficits in motor activity, cognitive and behavioural tasks

9

(e.g. increased impulsivity) 25, as well as diffuse axonal injury, microgliosis, increased levels of

inflammatory markers (e.g. TNF-α) and hyperphosphorylation of the tau protein69.

1.2.2 Weight-drop models

Weight-drop models typically involve an injury caused by a guided free-falling weighted

object dropped onto either the intact skull or exposed dura of an animal38,70. Mass of the weight

and the height at which it falls can be altered to produce different levels of injury severity.

Marmarou’s widely-recognized impact acceleration model has been proposed to mimic diffuse

TBI observed in humans, and utilized a weight dropped over a plate fastened onto a rat’s

cranium39. Diffuse axonal injury is commonly observed in this model, along with widespread

bilateral damage to neurons, as well as hemorrhages and astrogliosis39,71. Other variants of this

model delivered a focal type of injury, such as those described by Feeney et al41, involving an

impact to an exposed, intact dura, or Shohami et al, whose model involved delivering a focal

blunt injury over one side of the unprotected rodent skull40. Both models were shown to induce

cortical contusions, activation of microglia and astrocytes, and damage to the blood-brain barrier

(BBB)2. While weight-drop models are easy to implement and tend to closely mimic clinical

conditions observed in human TBI, the disadvantage lies in their variability of injury severity

and high mortality rate due to respiratory depression1,39.

1.2.3 Fluid Percussion Injury models (FPI)

Fluid percussion injury (FPI) is one of the most commonly used TBI models and will be

the one implemented in the current study. First introduced as a rabbit model in 196672, FPI

involves the usage of a fluid-filled cylinder in contact with an adjustable metal pendulum, that

exerts hydraulic pressure to induce an injury that resembles blunt-force trauma9. The model

requires a craniectomy so that dura mater is exposed. Injury is induced in the form of a rapid

injection of fluid into the exposed dura. The amount of intra-cranial pressure exerted by the fluid

is controlled by adjusting the height of the pendulum (Figure 1)1,9,27,73. Following injury, FPI

yields a combination of both diffuse neuronal injury and focal cortical contusions in targeted and

surrounding regions of the brain1,16,54. Histological staining has indicated inflammation, neuronal

death and diffuse axonal injury where FPI was implemented27,73–75.

FPI has been characterized to generate either middle/midline or lateral FPI. The former is

applied closer to the sagittal suture of the skull73,76,77, whereas lateral FPI42,55 is applied to more

10

lateral cortical regions, being the most widely used paradigm1. Despite the popularity of lateral

FPI in studying mechanisms of neuronal cell death, midline FPI is recently becoming a more

preferred model for brain injury in sports-related concussions and blast-induced TBI78,79. FPI has

been practiced across multiple species, being successfully demonstrated to induce TBI pathology

and neurobehavioural deficits in rats, mice, rabbits, dogs, pigs, sheep and cats16,27,43,73,76,80–82. It

allows good replication of mild to moderate TBI but does not involve rotational or rapid

acceleration-deceleration forces, as observed in some axonal pathology of human TBI9,67. As

described above, a disadvantage of the model is the requirement of a craniectomy, which does

not mitigate a scenario commonly observed during closed-head injury83.

Figure 1: Typical experimental set-up in TBI animal models for Fluid Percussion Injury (FPI),

which rapidly exerts fluid pulse at the target site when struck by the pendulum. Adapted and

reprinted with permission from ref 1.

11

1.2.4 Blast and Penetrative injury models

Brain injuries affiliated with blast and explosive shock waves, often seen in military

personnel45, are caused by objects rapidly propelled and dispersed primarily by strong blast,

rotational and acceleration/deceleration forces, as well as chemical emissions and thermal

energy54,70,84. Blast-induced TBIs are characterized by clinically relevant neuropathological (e.g.

cerebral brain edema, phosphorylated tauopathies, chronic neuroinflammation), and cognitive

(e.g. spatial learning and memory impairments up to 1 month post-injury) responses45,65. Models

of blast-induced TBI have previously been demonstrated, where anesthetized rodents were

exposed to controlled detonations of an explosive tube, or compressed air at a fixed

distance44,84,85. Several models have been generated to cover either single or multiple

components of a blast injury. Despite their biomechanical relevance to military warfare, varied

results have been reported, which can be difficult to compare across different laboratories1,86.

Further standardization of experimental designs are still required to replicate blast models for a

better understanding of the mechanisms and biomarkers associated with blast injury45,86.

Additionally, penetrating TBI caused by firearms are also prevalent in the military and

civilians, particularly in young adults6,46. Projectile objects travelling with high energy can lead

to cavities formed in the brain87 and experimental studies have replicated this using missile48 and

modified air rifle models46,47. As penetrating ballistic injury models form large, temporary

cavities, they are notable for their acute and delayed neuroinflammatory responses, and extensive

intracerebral hemorrhage at the lesion site87. The pathological nature of a penetrating injury

model can be influenced by the path at which the projectile travels, the magnitude and energy

transferred46,47,70.

12

Table 1: Animal models of TBI (adapted and modified from refs 1,2,50,51).

Models

and

subtypes

Primary

type of

Injury Species Strengths Limitations

Refer

ences Weight-Drop

Marmarou's Diffuse

rat, mouse

Well-characterized model; Biomechanics of injury mechanism

Difficult to reproduce;

1,2,39

resemble that of human TBI

High mortality rate due to apnea

Maryland's Diffuse rat Biomechanics of injury mechanism

Not enough replication, further

71


characterization needed

Feeney's Focal rat Biomechanics of injury mechanism

Craniotomy required; High mortality rate due to

41


apnea and variable skull fracture response

Shohami's Focal rat, mouse User-friendly device; Difficult to reproduce

40,88,8

9

severity of injury is adjustable

Controlled Cortical Focal

rat, mouse, ferret,

Severity of injury is adjustable; highly reproducible; Craniotomy required

10,28,2

9,57,58

,90 Impact (CCI)

swine, monkey low mortality rate

Blast Diffuse

rat, mouse, swine

Biomechanics of injury mechanism

High variability between injuries;

44,45,8

6

resemble that of military TBI

standardization required

Fluid Percussion Injury

Middle Mixed

rat, rabbit, cat,

Severity of injury is adjustable;

Craniotomy required; variability in injury severity

43,73,7

6,91,92

dog, sheep, swine highly reproducible

between experiments; high mortality rate due to apnea

13

Lateral Mixed

rat, mouse, swine

Severity of injury is adjustable;

Craniotomy required; variability in injury severity

27,42,8

2

highly reproducible between experiments; high mortality rate due to apnea

Repeated mild-TBI Diffuse

rat, mouse, swine


Further characterization required; variability in

56,84,9

3

resemble that of sports TBI

optimal number and frequency of applied injury

Penetrating ballistic- Focal

rat, cat, sheep


Standardization required

46–48 like brain injury

resemble that of penetrating TBI in humans

Cryogenic brain lesion Focal

rat, mouse

Severity of injury is adjustable; highly reproducible;

Replicates human TBI in very specific conditions

94,95

can be easily quantified In vitro Static mechanical injury Mixed

Various cell lines/

Severity of injury is adjustable; inexpensive and user-

Specific mechanical variables at impact cannot be

49–51

cultures (e.g. human,

friendly technique; easily quantifiable; prospect of

easily measured and estimated

mouse, rat, drosophila)

studying secondary injury pathways

(e.g. force, strain); tissue injury may be

difficult to verify (e.g. with Barotrauma injury)

Dynamic mechanical injury Mixed

Various cell lines/

Biomechanics of injury mechanism resemble that of

Tissue deformation cannot be easily measured;


human TBI caused by rapid acceleration/deceleration

potential inconsistency in injury modelling due to

49–51


forces; severity of injury is adjustable; highly reproducible

limited strain rate measurements; some models

can replicate secondary brain injury

are expensive and not user-friendly (e.g. cell -stretch

mechanical injury)

14

Chemical injury Mixed

Various cell lines/

Can be studied in isolation or in conjunction with other No limitations cited


in vitro models (e.g. glutamate with cell-stretch injury model);

50,51


prospect of studying secondary injury pathways

(e.g. excitotoxicity, oxidative stress)

1.3 Deep Brain Stimulation

1.3.1 Overview

The use of electrical stimulation for the treatment of neuropsychiatric disorders was

initially described as early as the late-1940s to early 1950s96. As conducted to date, deep brain

stimulation (DBS) was implemented as an alternative therapeutic technique to conventional

surgical ablation or lesioning of target brain tissue for the treatment of conditions such as

dystonia, essential tremor, and Parkinson’s disease97. Due to its adjustable and reversible

properties, DBS had rapidly superseded the need for lesioning and was found to be an effective

surgical treatment option for movement disorders98, particularly when introduced to subcortical

brain structures, such as the subthalamic nucleus (STN), ventral intermediate nucleus of the

thalamus (Vim), and globus pallidus internus (GPi))99–102. DBS comprises the use of electrodes

implanted at specific brain targets involved in the neurocircuitry of a given disease, from which

electrical stimulation is delivered. These electrodes or leads are typically connected to a pulse

generator (via extension wires), which is placed beneath the skin, near the clavicle to deliver the

desired current. The pulse generator can be programmed with the necessary stimulation

parameters, from a carefully selected combination of a wide array of frequencies, pulse widths,

and currents or voltages98,103. The adjustable property of DBS allows modifications to these

parameters, providing neuromodulatory interventions in accordance with underlying

neuropathological processes98. In addition, the stimulation-induced side effects of DBS can be

attenuated by the adjustment of stimulation parameters or discontinuing the therapy. Upon

reinstatement, therapeutic effects of DBS can frequently be regained19.

15

Following its successful adaption into movement disorders, DBS has since expanded its

application to other neurological and psychiatric disorders, including (but not limited to)

Alzheimer’s disease104–106, epilepsy107, anorexia nervosa108, Tourette’s syndrome19,109,110, chronic

pain111, obsessive-compulsive disorder (OCD)103,112,113, aggressiveness114, treatment-resistant and

major depressive disorder115, post-traumatic stress disorder (PTSD)116, stroke117, as well as

traumatic brain injury118, amongst others. Despite its clinical benefits and success, the

physiological mechanisms by which DBS exerts its effects are still poorly understood. A variety

of hypotheses have been suggested to explain the exact mechanism of action, in addition to

conflicting evidence as to whether DBS results in excitation or inhibition of local axonal

projections and neuronal elements102,119. Further understanding and conflation of ideas to draw

mechanisms for DBS will likely benefit in making precise and improved modifications in order

to efficiently treat other neurological disorders102.

1.3.2 Proposed Mechanisms of DBS

Single pulses of electrical stimulation often depolarize cells and axons near the electrode

site; However, in clinical settings, DBS is often delivered at frequencies above 100Hz (high

frequency stimulation; HFS). As described above in our overview, it was previously thought that

HFS mimicked the effects of targeted ablation120–122, suggesting that DBS behaves as a reversible

lesioning technique123. Stimulation parameters typically employed were initially found to have

an inhibitory effect on target nuclei, where DBS led to a decrease in spontaneous firing of

neuronal populations19. However, the precise mechanism of action for DBS and its therapeutic

impact on a stimulated target is not well understood and is dependent on several factors (e.g.,

duration of stimulation, physiology of the targeted region, composition of neural elements,

frequency, amplitude, pulse width of stimulation). These factors interact with one another to

modulate biological processes at varying subcellular compartments19,98. More recently,

hypotheses beyond the simple excitation/inhibition of target regions and its neuronal elements

have been investigated.

There was an initial belief that DBS has therapeutic benefits resembling that of lesion

therapy due to inhibition of target neuronal activity98,102. Cessation of local firing of neurons has

been induced by STN-, thalamic- and GPi-DBS along with a reduction in abnormal firing

patterns in target sites, leading to a reduction in motor symptoms102. One of the earliest proposed

16

mechanisms was the so-called depolarization block98,102. This occurs when high frequency

stimulation leads to a gradual inactivation of voltage-gated Na+ and Ca2+ channels, and an

increase in extracellular K+ concentrations, likely mediated by glial cells, which further reduces

depolarization of cell membranes124–127. This resultant silencing of neighboring neuronal

populations in the vicinity of DBS electrodes contributed to the early hypothesis of DBS acting

as a reversible surgical lesion in PD patients99,100,123,128,129 and in pre-clinical Parkinsonian

models130–133. Additionally, neurotransmitter release from astrocytes134 following HFS is said to

play a role in suppressing neurotransmitter responses135,136.

Another hypothesis postulated for the local inhibitory effects of DBS at high frequencies is

the disruption of information flow at the stimulation site due to the dissociation of input and

output signals, thereby producing a “jamming” signal102. This may occur when HFS depresses

synaptic function, as stimulation pulses generate action potentials directly onto the efferent axon,

while the soma is silenced. This ‘decoupling’ hypothesis would result in regular spiking output,

and reversal of an otherwise irregular or pathological firing pattern, promoting the abolition of

pathological frequencies137,138. DBS of the subthalamic nucleus in parkinsonian states has

previously demonstrated suppression of motor symptom-affiliated beta-band frequencies during

HFS, as well as shortly after HFS is no longer applied139–141. Subsequently, pathological

oscillatory activity is replaced with more regular rhythms within the STN, which correlate with

improvements in rigidity and bradykinesia142–145. Chiken and Nambu102 postulated that

disruption of information flow through the GPi due to DBS was invoked by spontaneous

discharges due to strong GABAergic inhibition. In addition, STN-DBS was previously found to

block transmission of signals through both hyperdirect and indirect basal ganglia pathways, but

not the direct pathway. This was evident by significantly reduced or diminishing of excitatory

signals but retention of cortically evoked inhibitory signals146. Overall, parkinsonian motor

symptoms can be suppressed through STN- or GPi-DBS, by disrupting abnormal firing rates and

patterns in major basal ganglia pathways, prior to this information flow being transmitted to the

thalamus and finally the motor cortex, from which motor symptoms can be expressed102. This

hypothesis of DBS disrupting pathological oscillatory patterns and signal transmission may

consolidate a more plausible explanation for its bimodal, yet therapeutic effects; DBS regulates

and potentially corrects for inappropriate firing patterns and rates, as well as the dynamic activity

that are responsible for motor symptoms in PD102.

17

In contrast to the inactivation of neuronal cell bodies, axons can still be activated following

high frequency DBS. This may lead to the excitation or inhibition of structures at a distance from

the target, depending on firing patterns, rates and whether the stimulation of axonal projections

releases GABA or glutamate102,147. In addition, with a complex interplay of principal neurons

and interneurons receiving projections from stimulating targets, the consequences of delivering

HFS in structures at a distance are difficult to predict. For example, despite receiving excitatory

glutamatergic inputs from the STN, GABAergic inhibition in the GPi likely overrides these

excitatory drive, thus inducing the activation of inhibitory afferents133,148. Additionally, through

the activation of both glutamatergic and GABAergic afferent projections149, both excitatory and

inhibitory postsynaptic potentials may be generated in neural populations of the STN, suggesting

that DBS can not only generate both excitation signals that are distal from the target region, but

locally inhibit neuronal soma within populations surrounding the electrode102,147. Another

example that is very relevant to this thesis is the inhibitory effects of high frequency, high

current stimulation of the anterior thalamic nucleus (ANT) over dentate gyrus hippocampal cells.

In a series of studies in which ANT-DBS was used to mitigate seizure activity or modulate

memory performance, stimulation was found to reduce the firing rate of dentate gyrus granule

cells150 as well as hippocampal excitability151. This is of interest because the ANT is an

important hub in the limbic circuitry152–154and has direct and indirect connections with the

hippocampal formation155,156. Finally, of great importance are the chronic consequences of DBS,

since this therapy is applied to humans continuously for years. Plastic changes such as long-term

potentiation and increases in neurotrophin levels and neurogenesis have all been described after

electrical stimulation and may underlie some of the behavioral consequences of DBS19.

In summary, DBS at high frequencies in known neuropathological substrates, particularly

within the basal ganglia, has demonstrated inhibition of neuronal activity near the electrodes

through mechanisms that potentially involve a reduced glutamate release and the release of local

GABA via the excitation of GABAergic terminals, and adenosine to promote synaptic inhibition

of afferents, as well as alterations within voltage-gated Na+/K+ channels. On the other hand,

through synaptic modulation, DBS of target structures is also shown to be capable of increasing

axonal firing in efferent projections and axonal fibers within the vicinity of the implanted DBS

electrodes, while concurrently inhibiting or decoupling somatic activity157,158. Ultimately, the

resultant stimulation of any target neuronal population will vary due to the cumulative effects of

18

DBS parameters, and the composition of excitatory and inhibitory neural elements in any given

neuropathological target site98. Therefore, further investigating is required to explain how DBS

can have a similar mechanistic approach when extrapolated to other neurological or

neuropsychiatric disorders, where a diverse range of neural circuitries are engaged and affected

to generate their respective pathologies.

1.3.3 Deep Brain Stimulation in Traumatic Brain Injury

Depending on the severity and condition of brain injury, TBI is notorious for initiating a

cascade of secondary damage, involving neuropathological processes that may lead to cognitive

dysfunction, changes in consciousness as well as a considerable amount of sensorimotor

damage22. Regulation of secondary damage is one of the key means of intervention, following

the onset of injury to help restore loss of physical and cognitive function; to date, therapeutic

strategies to manage this cascade of neurological events is still under development, with widely

varying reported outcomes118. In cases of motor dysfunction, a combination of neuromodulation

and rehabilitative training has been implemented to help promote recovery of motor circuits.

DBS has become a conventional treatment option for addressing movement disorders, especially

when affiliated motor symptoms no longer respond to pharmacotherapy. As mentioned, DBS of

the GPi and STN was shown to effectively treat motor symptoms, dyskinesias and fluctuations in

patients with PD98. However, the limited knowledge on the relationship between behavioural

changes following TBI and the corresponding impacted neuroanatomical structures and neural

circuitry has restrained our ability to propose effective treatment options for addressing TBI

recovery159. Chan et al118 had previously demonstrated improved behavioural and motor recovery

when DBS was delivered in a post-stroke rodent model, and further extended these findings to

support the anti-inflammatory and neuroprotective properties of this treatment modality.

Stimulation of the lateral cerebellar nucleus (LCN), along with motor training in rats promoted

long term potentiation, and functional cortical reorganization, following contralateral unilateral

FPI118; findings of this study further support DBS of the cerebellar dentate nucleus (DN) at

frequencies that mimic neuronal firing rates to activate glutamatergic efferents, thus broadly

influencing thalamocortical activity in patients with acquired brain injury.

Deficits in executive functioning in both pre-clinical models and clinical populations may

be significantly improved through stimulation of the thalamus, regions of the limbic (Papez)

19

circuit as well as frontal cortical areas and subcortical structures119,160. Distorted and

desynchronized hippocampal oscillations following TBI in rodent models resulted in cognitive

dysfunction, including learning, memory, and spatial navigation160; subsequently, DBS following

TBI may assist in realignment of oscillatory activity. In particular, targeting regions such as the

medial septal nucleus (MSN) to improve hippocampal function, had previously demonstrated

normalized object recognition, and shorter latency periods in spatial navigation during Barnes

maze in TBI rats161. Furthermore, acute HFS (either 6 hours, or 7 days after injury) of the median

and dorsal raphe nuclei in the midbrain of a rodent FPI model had improved memory in

behavioural tasks, likely due to the suggested neuroprotective effects of serotonin160; however

one caveat that study had noted was the lack of significant differences between treatment groups

following day 3 of a Morris water maze task, likely due to underlying spontaneous recovery162.

Other targets of stimulation to improve memory include the fornix, and central thalamus which

in turn was shown to increase activity in the dentate gyrus to encourage better regulation of

emotions, and executive functioning in patients with TBI, as displayed by greater performance in

cognitive and functional scores160.

Additionally, a small population of TBI patients with varying levels of consciousness,

including of minimally conscious states (MCS) have reported differing benefits in behaviour and

environmental awareness following DBS163. Unfortunately, a lack of control for targeted

stimulation and the overall heterogeneous nature of head injury acquired by patients create

additional complications in finding efficient and novel treatment options to approach the

challenges faced by patients with MCS163. Optimizing for patient-specific symptoms, the

temporal properties of administering DBS in TBI, neuromodulation, as well as rehabilitation

training conditions may improve therapeutic efficacy in reducing injury-induced motor and

cognitive deficits164. Finally, there is growing evidence to suggest that DBS confers

neuroprotection from excitotoxicity and progressive neuronal loss, thereby improving the

likelihood of recovering from neurodegeneration119. Criteria for considering a therapeutic

intervention to have neuroprotective effects include a decrease in progression of neurological

symptoms, a slowing of neuropathogenesis, as well as attenuation of neuronal cell death119. For

example, evidence from pre-clinical AD models using DBS propose neuroprotective effects by

increasing hippocampal neurogenesis, promotion of synaptic plasticity as well as neurotrophic

factors, and by facilitating clearance of misfolded proteins117,165. However, McKinnon et al119

20

note that acute stimulation parameters are used in these pre-clinical studies, hence not directly

translatable in clinical settings, where chronic stimulation parameters are employed in AD

patients.

More relevant to this thesis, DBS of the ANT resulted in an inhibition of GPi activity, and

a rescuing of neurons in the dorsal striatum, leading to an anti-epileptic effect in a pre-clinical

model of drug-resistant temporal lobe epilepsy166. Excitotoxicity reduction thus normalizing

function in the basal ganglia-limbic circuitry may be a mechanism by which ANT-DBS exerts

neuroprotection166. In terms of memory, ANT-DBS in rats has been shown to improve memory

performance in the Morris Water Maze, possibly via the induction of hippocampal

neurogenesis167–169. ANT-DBS was also found to have a far-reaching neuroprotective effect in

hippocampal regions170,171, through regulating inflammation and promoting adenosine

production166,172. In addition to its anticonvulsant effect in pre-clinical models, data acquired

from human studies, notably in the multicenter, randomized, double-blind, bilateral Stimulation

of the ANT for Epilepsy (SANTE) trial (n = 110) demonstrated the efficacy and safety of DBS in

the treatment of refractory epilepsy, and has since been granted approval as an adjunctive

therapy by the U.S. Food and Drug Administration173,174. Towards the end of the blinded phase,

patients in the treatment group experienced 40.4% reduction in median seizure frequency relative

to baseline, while the control group experienced 14.5%173. A 2-year follow-up by Fisher et al173

noted a 56% seizure reduction rate in patients, while Salanova et al175 reported a median 69%

reduction in seizure frequency in 74 patients, 5 years after the initial SANTE trial. In spite of

these findings, significant differences in neuropsychological test scores for mood and cognition

were not observed between patients in treatment and control groups173, although some patients

reported impairments in memory and depression, following ANT-DBS (majority of these

findings were reported by patients with a previous history of memory impairment and/or

depression)175. These responses were not deemed to be serious and resolved spontaneously.

Long-term follow-up studies of the treatment group demonstrated improvements in executive

function, attention, and subjective cognitive function174–176.

Despite converging evidence for the efficacy of ANT-DBS, it is still difficult to evaluate

the mechanism of action by which ANT-DBS confers neuroprotective effects in patients, notably

due to timing in which DBS is considered an option. Thus, disease progression may have

advanced to irreparable stages119. This is particularly relevant in TBI patients, as numerous

21

factors, including severity, place of injury as well as the stage of neurodegeneration in which

DBS is deployed. Continuous and follow-up studies of DBS in validated animal models are still

necessary to corroborate stimulation parameters and target regions in which the most favourable

neuroprotective effects may be achieved119.

1.4 Rationale, objectives, and hypothesis

While DBS is now a widely accepted treatment option for the management of motor

symptoms in Parkinson’s disease, tremor and dystonia177, there is currently no conclusive

evidence suggesting that DBS will lead to significant outcomes in a moderate-to-severe acute

TBI model. A study using c-Fos and behavioural analyses was able to demonstrate that rats

undergoing stimulation of the ANT (i.e., 130 Hz, 400 µA, 90 µs) experienced decreased activity

in the hippocampus, with a reduction in glutamate release and therefore, less excitotoxicity107.

Animals that underwent contextual fear conditioning tests following high current stimulation of

the ANT displayed impaired memory likely related to a decreased hippocampal recruitment150.

In addition, acute stimulation of the ANT for 6 hours during status epilepticus in the rat

pilocarpine seizure model was shown to have an anticonvulsant effect, while reducing

hippocampal neuroinflammation and apoptosis107. Overall, the ANT and its relayed projections

toward the hippocampus are critical in facilitating contextual and spatial memory150. Given that

neuroprotective effects were demonstrated after stimulation, the ANT will be our target for

improving memory deficits in moderate-to-severe TBI in rats.

To study the effects of acute ANT-DBS in providing neuroprotection and improving

memory performance following moderate-to-severe TBI, this thesis will consist of 3 aims:

Aim 1: To characterize behavioural deficits in the moderate-to-severe TBI fluid percussion

injury (FPI) model in rats.

Aim 2: To investigate the effects of acute ANT-DBS in the FPI model through a battery of

behavioural tests associated with learning, memory and anxiety-like behaviour.

Aim 3: To test whether ANT-DBS reduces hippocampal apoptosis in FPI-exposed rats.

22

Given the DBS effects described above and the relevance of the ANT in memory

processes, this project will test the following hypotheses:

1. Moderate-to-severe TBI will induce memory deficits and anxiety-type responses in rats,

as measured by poorer outcome of performance in a battery of behavioural tests,

compared to non-TBI groups.

2. Conversely, increased performance outcomes in our rodent behavioural paradigm

following ANT-DBS will demonstrate improved memory function, and reduced anxiety-

type behaviour in rats exposed to acute moderate-to-severe TBI.

3. ANT-DBS will decrease levels of the biomarker caspase-3, thus demonstrating reduced

apoptosis signaling in the hippocampus of rats in a moderate-to-severe TBI model.

23

Chapter 2 – General methods & materials

2.1 Animals and Surgical procedures

2.1.1 Animals

The following animal use protocol (AUP) was approved by the research ethics board

(REB) at Sunnybrook Research Institute (SRI). Animals were cared for and used in accordance

with the guidelines outlined by the Canadian Council on Animal Care. Adult, male outbred

Sprague-Dawley rats (225-250g, Charles River Laboratories, Quebec), were individually housed

within the animal care facility at SRI and were acclimatized to 12-hour light-dark cycles (7:00

a.m. – 7:00 p.m.) upon arrival to the facility. The animals were fed in their home cages with a

standard laboratory rat diet and given water on an ad libitum basis. All surgical procedures took

place within operating rooms (OR1, OR2, OR3) located in the SRI animal care facility. All tools

and equipment were sterilized prior to commencement. Handling took place after all surgical

procedures were completed, due to a short acclimatization period prior to surgery.

New cohorts of animals were utilized for each study. These cohorts were applied for

different objectives (i.e., study 1 for validating lateral FPI TBI model using behavioural testing

and future histological analyses; study 2 for ANT-DBS effects in behaviour and histology; study

3 for neurochemical measurements of caspase-3 24 hr post-TBI). Table 2 outlines the number of

animals used per group, across all 3 studies described in this thesis.

24

Table 2: Animal cohorts and corresponding number of animals across experiments 1-3. Brain

recovery for the animals in experiment 3 was performed 24hr post-TBI for neurochemical

analysis:

2.1.2. DBS electrode implantation

A fully assembled stereotaxic frame (Model 900, David Kopf instruments, Tujunga, CA)

with appropriate ear and bite bars was set up. Each animal was anesthetized in a closed induction

chamber with 4% isofluorane (500 ml/min room air) for up to 3-5 minutes. Once the animal was

fully anesthetized, an electric hair clipper was used to shave their head. The animal was then

inserted into a pre-assembled stereotaxic frame. Ear bar distance and nose cone height was

adjusted such that their head was firmly centered and positioned flat in the antero-posterior

plane. Prior to making an incision, eye lubricant (BNP ophthalmic ointment) was applied to the

animal’s eyes. The animal’s body was covered with extra drapes or towels to retain body

warmth. The exposed scalp was sanitized with betadine and 70% ethanol. Ketoprofen was

Naïve TBISham TBI (burr

holes only)DBS-TBI Sham DBS-TBI Control (burr holes-TBI)

2

Effects of acute

ANT-DBS in the

FPI model

through

behavioural

testing

2 - - - 10 9 13 32 Y

3

Testing whether

ANT-DBS

reduces

hippocampal

apoptosis in FPI-

exposed rats.

3 - - - 8 7 8 23 N

-

Y

Total no. of

animals/co

hort

1

13 10 12 - -

No. of animals/group

35

Cohort #

Characterizing

behavioural

deficits in the

moderate-to-

severe TBI (FPI)

model in rats

1

Behavioural

testing?

(Y/N)

Experiment

#Study objective

25

subcutaneously administered (ANAFEN, 10mg/ml). A midline sagittal skin incision from rostral

to caudal was made with a 15 blade so that the anterior region of the skull was exposed. Excess

bleeding and debris were controlled using sterile saline solution (0.9% NaCl) compression with

gauze and/or cotton swabs. At specific stereotaxic coordinates (obtained from Paxinos and

Watson’s rat brain atlas178) to locate the ANT bilaterally (antero-posterior -1.50; medio-lateral

+/- 1.50), holes of the necessary diameters were made to the skull using a drill and the

appropriate drill bits. The dura mater was exposed with a fine tip 25G needle. Two additional

holes were made over anterior skull regions for the insertion of stainless-steel screws used to

anchors the cap. Insulated stainless steel electrodes (250 µm in diameter; Plastics One model

E363) covered with polyamide with a partially exposed tip (~0.5-0.75 mm) were then lowered

bilaterally to a depth of 6.00mm into the ANT and later used as cathodes. The anode was a

stainless-steel electrode (125 µm in diameter; Plastics One model E363) peeled from the varnish

and wrapped around anchoring screws. Both ANT electrodes and the anode were connected to a

plastic pedestal (Plastics One model MS363) using acrylic cement. Acrylic dental cement paste

(dental acrylic powder and liquid curing solvent, Lang) was also used to further seal and secure

the implanted electrode, as well as creating a skull cap. Sham-DBS animals underwent the same

electrode implantation, without receiving stimulation. Another control group (control surgery)

was included that had burr holes drilled into their skull at the above coordinates but did not have

electrodes implanted. Following the procedure, animals were returned to their home facility,

where they would recover for one week, during which they were closely monitored.

2.1.3 Fluid percussion injury (FPI) and acute Deep Brain Stimulation

One week after electrode implantation, animals were retrieved from their home facility and

brought to an impromptu TBI surgery room, where the fluid percussion injury device was

located. Each animal was anesthetized with 4% isoflurane, prepared, and positioned to the

stereotaxic frame, as described for DBS implantation. An incision extending from the DBS cap

to the occipital region was then made such that the sagittal suture is exposed. Once the exposed

surface was cleaned of connective tissue and blood, a craniotomy (5mm in diameter) was drilled

using a trephine drill bit (Meisinger 227RF-050-HP trephine, stainless steel, ID 4.0mm) near the

26

sagittal sutures, between the lambda and bregma near the right parietal cortex. Care was exerted

so that the dura mater was not penetrated. An injury ‘hub’ was temporarily secured over the

craniotomy using acrylic dental cement (Lang) in preparation for lateral fluid percussion injury.

Injury hubs were made by cutting the plastic ends of 25-gauge disposable needles (BD

PrecisionGlide, #2538-CABD305175) at a height and elevated angle suitable for the given

craniotomy. Once the cement solidified, the temporary seal was checked for leaks by injecting a

small pool of saline into the secure hub and observing whether it decreases in volume. This was

to prevent dampening of the seal formed between the liquid in the hub and high-pressure liquid

from the FPI device. Rats in the TBI group were connected to the lateral FPI device (Model

FP302; Amscien Instruments, Richmond, Virginia, USA)27 via the hub, to induce moderate

traumatic brain injuries, with impact pressures of 2.5-3.0 atm. These values were defined based

on pilot experiments conducted in our laboratory demonstrating injury severity. In addition,

sham control animals were also included, where animals underwent the same procedure, but no

FPI is induced. Following FPI, the injury hub with the temporary seal was removed, and the

initial scalp incision was sutured before placing the animals back in their home cages. Sham-TBI

animals were drilled with shallow burr holes as a control group undergoing all surgical

procedures, but without lateral FPI.

Immediately after undergoing TBI, animals were transferred to the ante room of the TBI

behavioural suite, where plexi-glass boxes (Toronto Plastics, 25 cm L x 25 cm W x 45 cm H)

padded with clean bedding were set up for acute stimulation. Before the animal regains

consciousness from the anesthesia, electrodes were connected to a 6-channel SL6C commutator

(Plastics One), via connection cables. Using extended 6-channel cables (Plastics One; 363-340/6,

50 cm) the commutator was connected to a stimulator (ANS, model# 3510). Once connected,

animals were given DBS bilaterally for 6 hours at parameters in the range of those associated

with antiapoptotic and anti-inflammatory effects in our previous studies (130 Hz, 90

microseconds, 300 µA)150. All stimulated animals were periodically monitored to observe any

changes in behaviour or discomfort, and to ensure that DBS electrode caps had not broken off.

Following acute stimulation, animals were disconnected from their stimulation boxes, returned to

their respective cages, and taken back to their home facility to rest and recover.

27

2.2. Behavioural assessments

Prior to testing, animals were closely monitored for 4 days post-surgery for any major

motor impairments (e.g., gait or balancing issues) or abnormal behaviour (e.g., excess grooming,

lethargy, loss of appetite). During the 4-day rest time, animals were consistently handled for up

to 2 minutes each, within their housing facility. Behavioural testing for all groups took place

over the course of 5 days (including 1 rest day) for spatial learning and memory, including a

novel location and object recognition test (NLR/NOR)179,180, marble burying 181 and Barnes maze

182. The following behavioural protocol was designed and optimized, based on literature where

these tasks were previously proposed and described (Figure 2).

Figure 2: Timeline for experiments performed for both studies 1 (TBI groups; no electrode

implantation surgery was performed) and 2 (DBS-TBI groups). Following habituation and

routine handling of Sprague-Dawley rats, DBS electrodes were implanted and FPI was

implemented approximately 7 days later at ~2.5-3.0 atm. DBS-TBI animals received acute

stimulation for 6 h following TBI (day 1). After a fixed interval, the animals underwent a series

of behavioural tests to measure anxiety type behaviour (Marble burying; MB) and memory

(Novel location recognition; NLR, novel object recognition; NOR, and Barnes maze). The

animals were sacrificed, and brains were recovered 24 h after the last day of behavioural testing.

28

2.2.1. Novel location and novel object recognition Habituation: Open field testing

On Day 1 (five days after TBI), animals underwent the habituation phase of the

NLR/NOR tasks where each freely explored an open field arena for 5 minutes (Figure 3). Each

open field consisted of a large, transparent plexi-glass box (Toronto Plastics, 45cm L x 45cm W

x 40cm H), with no other objects or bedding within the field. Each trial was recorded using a

camcorder (Sony Handycam® HDR-CX455) mounted to the ceiling and controlled via

smartphone wi-fi. The animals were then removed from the arena and kept in their respective

home cages, placed in the anteroom of the behavioural suite, until the next behavioural task

commenced.

Figure 3: Open field arrangement for DBS-TBI animals. Each animal was placed in individual

plexi-glass boxes for 5 minutes. No other objects or bedding was included in the open field.

After 5 minutes of exploration, the animals were returned to their respective home cages. Opaque

barriers were placed between open field arenas to prevent animals from seeing each other during

recording sessions.

2.2.2. Marble Burying

Open field habituation was followed approximately 60 minutes later by marble burying

(MB). This test was conducted in rat cages set up with clean bedding, evenly levelled and

29

pressed flat with ~5 cm in depth. 15 brightly coloured glass marbles were placed parallelly on

the surface of the bedding and spaced evenly, in a 3x5 arrangement (Figure 4a). The animals

were then retrieved from their home cages in the anteroom and each animal was positioned at the

center of each MB cage. As soon as all the animals were placed in their MB cages, they

proceeded to roam freely for 30-minute sessions, while these cages were covered with lids.

Throughout the session, behavioural changes were noted for each animal. At the end of the

session, animals were removed and taken back to their respective home cages. Without

disrupting the arrangement of the marbles or the bedding, bird’s-eye and side-view images (e.g.,

Figure 4b, c) were captured for analysis. The number of marbles buried above 50% of their

volume was blindly counted from the images. Used bedding was then discarded and the animals

in their home cages were returned to their housing facility. In this test, a high number of buried

marbles represent anxiety-type behaviour.

Figure 4: (A) Typical setup of marble burying (MB) testing cages before placing animal for

behavioural protocol. (B) MB cage after 30 minutes of behavioural testing. (C) Side-view of

buried marbles for animal ID’d as ‘Fz042’. 15 brightly coloured marbles were lined up in a 3x5

arrangement over clean bedding. ID’d animals were placed in their corresponding test cages and

observed for 30 minutes before returning to their respective home cages.

30

2.2.3. NLR/NOR: Familiarization, novel location and novel object recognition

testing

Day 2 (6 days after TBI) involved the following phases – familiarization, novel location

test, and novel object test phases, each of which was performed in 5-minute sessions per animal.

During familiarization, each animal was placed in an arena containing two identical objects

(objects A + A; Figure 5a). Open field boxes from the previous day were used as the arena for

the above-mentioned phases. Animals were then removed after familiarization and temporarily

placed in a holding cage. They were then replaced in the arena, where one of the identical objects

was relocated to a different corner to establish a novel location for said object. Finally, the same

animals were each tested in 5-minute NOR sessions, where the newly relocated object A was

replaced with a novel object B (i.e., A + B; Figure 5b). The objects utilized included 2 identical

towers made of Lego bricks (i.e., object A), and 2 large falcon cell culture media flasks filled

with clean bedding (i.e., object B). The arena and the objects were regularly cleaned and

sterilized with Clidox®, as well as 70% ethanol, such that the next animal did not detect the

odour of the previously tested animal. At the end of these sessions, animals were returned to their

home cages and allowed to rest for at least 24 hours before proceeding to the final behavioural

task. Apart from the familiarization test, all test sessions were recorded using the overhead video

recording device, as was previously used for the open field sessions. Scoring was conducted

blindly. Since rodents have an innate preference for novelty, animals that recognize the familiar

object and location will prefer to spend more time at the novel object and location183. Variables

measured were the time exploring in the novel or old location or with the novel and old object.

The percent time exploring the novel object or location was calculated as the time exploring the

novel object or location/total exploration time. The novel object or location index was calculated

as time exploring the novel object or location – time exploring the old object or location/ total

exploration time.

31

Figure 5: (A) Novel location recognition (NLR). Each animal was placed in individual plexi-

glass boxes for 5 minutes with two identical objects, one of which was moved to a new location.

While not pictured, the animals underwent familiarization sessions prior to exploring newly

located objects. Each animal was randomly assigned to either objects A (flasks) or objects B

(Lego tower) placed diagonally from each other (white square = previous location) and allowed

to explore for 5 minutes. (B) Novel object recognition (NOR). Similar to NLR, animals were

replaced in their randomly assigned arena, only the newly located object was replaced with a

novel object, unfamiliar to the animal.

2.2.4. Barnes maze

On the 4th and 5th days of testing (8 and 9 days after TBI), visuo-spatial learning and memory

were evaluated using the Barnes maze paradigm182,184. The maze consisted of an elevated

circular arena, with 20 circular holes at equal diameter and spacing (122cm platform diameter,

20 holes, 10cm hole diameter; Figure 6). To encourage animals to quickly locate the escape box,

aversive cues were added, including an overhead centre lightbulb of 100W-equivalent wattage,

and a high-powered table fan to induce high-speed winds across the maze. All other light sources

32

were blocked with dark covers, such that the overhead 100W lightbulb was the only source of

light. Visual cues were made up of cross, square, circular and triangular shapes, cut out from

black construction paper and placed on each wall surrounding the arena, within the animal’s eye

level. The escape box (35.0cm L x 11.5cm W x 10.0cm D) was placed below one of these

circular holes, near a visual cue to indicate the location for each animal to escape, while all other

holes were concealed with false bottoms. The entire arena was surrounded by dark curtains,

behind which the experimenter would observe each trial, and to prevent distracting them during

testing. All animals were kept in the anteroom of the behavioural suite, and one at a time, each

animal was brought in for testing. Each trial had a duration of 3 minutes, after which the animal

was removed, should they fail to locate the escape route. All animals completed a given set of

trials before proceeding to subsequent trials for Barnes maze. The first day of behavioural testing

in the Barnes maze was assigned for acquisition and habituation trials, during which the animals

were trained to familiarize themselves with the task. On the second day, the animals were

exposed once again to the test and scored for their visuospatial memory, by following the same

steps as described above. The following variables were recorded: Time to locate the escape

route, time to enter the escape route, error count (number of times the animals erroneously select

the escape box), latency to the first nose poke, total latency to find the escape route, and the

search strategy. The search strategy is ranked based on a cognitive score scale from the Barnes

maze unbiased strategy (BUNS) classification algorithm, as previously described by Illouz et al

(2016): The animals' ability to locate the escape box was ranked on a scale of 0 to 1, with 0.25

increments, where 0 is considered random (little to no searching), 0.25 = serial (animals search

each consecutive hole around the circumference of the maze), 0.5 = focused search/long

correction (animals search within one quadrant nearest to or away from the escape box,

respectively), 0.75 = correction (animals make 1-2 errors within the quadrant nearest to the

escape box before locating it) and 1 = direct (no errors when locating escape box). A higher

average value on the cognitive scale implies a relatively more direct navigational approach in

locating the escape route of the maze. We note that the Barnes maze in our protocol was

purposely made more difficult with animals undergoing only a few training sessions. This was

because we noticed that TBI animals exposed to more prolonged Barnes maze testing (two or

three days of training followed by probe testing) do not show performance deficits.

33

Figure 6: Barnes maze. 8 days after the initial injury, animals were allowed 3 minutes (180 sec)

to explore, locate and enter the correct escape box within a circular Barnes maze. Aversive cues

were implemented to motivate the need to escape the maze. These included one 100-W light

source affixed above the maze, as well as a high-speed fan. Visual cues (not pictured) consisting

of black geometric shapes made of construction paper were glued onto the walls of the testing

room. Experimenter observed each trial behind dark curtains and animals were retrieved from the

maze if they fail to enter the escape box within the 3-minute duration. Animals who successfully

entered the escape box could remain in there for at least 30 seconds before returning them to

their home cages. Animals were tested for two trials per day, for 2 days, as per a modified

untraining protocol. All parameters were scored manually through video recordings.

2.2.5 Cresyl violet staining

Following the last day of behavioural testing, animals were anesthetized with 5%

isoflurane and sacrificed for brain recovery. Fresh, non-perfused brains were removed from the

skull and stored at -80° C for histology. To assess electrode location, 20-µm thick coronal

sections covering the injury, were obtained using a cryostat (Leica biosystems) and mounted on

labelled glass slides (Fisher Scientific) which were then prepared for cresyl violet staining. For

34

tissue fixation, slides mounted with fresh tissue slices were placed in an incubating chamber

overnight, that was heated at 60°C, and containing a large dish with 500 ml 10% formalin, made

with 37% formaldehyde solution (Sigma Aldrich, cat#F8775). 0.2% cresyl violet stain was

prepared by mixing 2g cresyl violet acetate (Sigma Aldrich, cat#C5042) and 1000ml deionized

water, and filtered overnight. Four drops of glacial acetic acid (Sigma Aldrich, cat#695092) were

added to every 100ml of cresyl violet stain used for the fixed slides.

Within the next 24 hours, fixed slides were removed from incubation, and initially placed

into staining dishes containing deionized water for up to 3 minutes, to wash off any excess

formalin vapour. Subsequent steps involved dehydrating the fixed tissue in incremental

concentrations of ethanol solution (70%, 95%, 100% ethanol) and xylenes (MedStore, #534056),

followed by rehydration of tissue slices by placing the slides in a reversed sequence of the series

of ethanol concentrations (i.e., 100%, 95%, 70%) and deionized water, to allow more efficient

diffusion of the cresyl violet stain within the fresh tissue slices. After approximately 2-3 minutes

of staining, the slides were rinsed with deionized water, subsequently followed by further

dehydration in 70%, 95%, and 100% ethanol solutions and xylenes. Glacial acetic acid was

added to the 95% ethanol step to aid in cellular differentiation within each stained coronal tissue

slice. Finally, each slide was cover slipped using 50x24mm cover glasses (Fisher Scientific, #12-

548-5M) and Eukitt® quick-hardening mounting medium (Sigma Aldrich, cat#03989) and

allowed to dry overnight before viewing under a microscope to confirm the location of our DBS

electrodes.

2.3. Neurochemical experiments

A new cohort of animals (n = 23) prepared for neurochemistry, underwent electrode

implantation/control surgery along with TBI protocols, as described above. Twenty-four hours

following TBI (and subsequent acute stimulation for DBS-TBI groups), DBS/sham DBS animals

were anesthetized with 5% isoflurane and sacrificed through decapitation. Brains were removed

from the skull, divided in two hemispheres, and stored at -80°C for neurochemical analyses. TBI

exposed animals had one TBI and one Non-TBI hemisphere with no electrodes implanted. Sham

DBS-TBI exposed animals had bilateral electrodes implanted but received no stimulation in one

TBI and one Non-TBI hemisphere. DBS-TBI exposed animals had bilateral electrodes implanted

and received stimulation in one TBI and one non-TBI hemisphere.

35

2.3.1. Caspase-3 Enzyme-linked immunosorbent assay (ELISA)

Tissue samples were prepared by first dissecting out the hippocampus and the pericortical

regions from both, the ipsilateral (TBI-induced) and the contralateral hemispheres of the rat brain

using the 1.5mm and 1.0mm WPI biopsy punch tools (#WPKP1018) respectively. The

hippocampus dissected was the region directly below the TBI-induced cortex. The pericortical

region chosen was 1.0mm medial, lateral, anterior and posterior of the TBI-induced cortex. The

dissected tissue was collected in a 2.0mL pre-weighed SARSTED cryotube and homogenized in

250μL of RIPA lysis buffer that included one protease-inhibitor cocktail tablet (ROCHE) per

10mL of buffer solution. Lysates were then sonicated once for 15 sec and centrifuged at 11,000

G for 15 min at 4°C to remove cell debris. The supernatant was collected in a separate tube and

stored at -80°C until further processing.

Total protein concentration was quantified using the bicinchoninic acid assay (BCA,

Thermo Scientific, USA). Rat Casp-3 ELISA kits were purchased from Biomatik (#EKC40528,

Cambridge, Ontario) to measure caspase-3 protein levels in the separate frozen sample aliquots

from each subject. This assay employs a quantitative sandwich enzyme immunoassay technique

that quantifies the target antigen between two layers of antibodies using a pre-coated 96-well

microplate provided by the manufacturer.

Standard and samples (in duplicates) were added to the appropriate microplate wells that

were pre-coated with antibody specific to Casp-3. Any Casp-3 present would bind to the

immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody

specific to Casp-3 was added to all samples, followed by multiple washings, and the addition of

avidin conjugated Horseradish Peroxidase (HRP). Following a wash to remove any unbound

avidin-enzyme reagent, a substrate solution was added to the wells that would result in colour

development in proportion to the amount of Casp-3 bound in the initial step. The colour

development was terminated by addition of sulphuric acid solution and the intensity of the colour

was measured spectrophotometrically at a wavelength of 450nm, with a correction wavelength

set to 570nm. The plates were read on a Synergy H1 microplate reader (Biotek, Winooski,

Vermont, US) and a four-parameter logistic regression curve was used for plotting the standard

curve and data extrapolation. The concentration of Casp-3 in the samples was determined by

36

comparing the optical density of the samples to the standard curve. The coefficient of variance

among the duplicates was less than 15%.

2.4. Statistical Analyses

One-way ANOVA was performed for the comparison of behavioural data in

experiments 1 (naïve, sham-TBI and TBI groups) and 2 (TBI, Sham DBS-TBI and TBI groups),

followed by Tukey’s post-hoc analysis for multiple comparisons. Caspase-3 results were

analyzed with 2-way ANOVA with TBI (TBI, non- TBI) and DBS (no electrode, sham DBS,

DBS) as independent factors. All statistical analyses were completed using Prism 5.0 for

windows (GraphPad Software, San Diego California). Statistical significance was set at p ≤

0.05. In the graphs, results are presented as mean ± standard error.

37

Chapter 3 – Results

3.1 Study 1: To characterize behavioural deficits in the moderate-to-severe TBI

fluid percussion injury (FPI) model in rats

We first conducted experiments to validate our TBI model and behavioural tests. Three

groups were considered: TBI (n=10), Sham-TBI (n=12), and naïve animals not exposed to TBI

(n=13). Our hypothesis was that the group of animals exposed to TBI would present increased

anxiety in the marble burying test and memory deficits in the NOR/NLR and Barnes maze tests.

All rats fed normally, gained weight, and did not present any gross locomotion abnormalities

after lateral FPI.

In the defensive burying test, no significant differences were found across groups (F=1.52;

p=0.23), though animals exposed to sham-TBI and TBI had a 25 and a 32% reduction in the

number of buried marbles compared to the naïve group, respectively (Figure 7).

38

Figure 7: Defensive marble burying test for TBI, sham-TBI (craniotomy, no FPI) and naïve

(no surgical procedure) groups. Animals do not show a significant difference in performance

based on number of marbles buried across groups.

In the novel object recognition test, we found a significant effect for percent time spent

with the novel object (F=3.3, p=0.05) and a trend towards significance for the index to explore

the novel object (F=3.0, p=0.06). Compared to the naïve group, the percent time exploring the

novel object was 21% lower in sham-TBI rats (p=0.04) and 17% lower after TBI exposure.

Similarly, the index exploring the novel object was 63% lower in the sham-TBI group and 50%

lower in TBI exposed animals compared to the naïve group (Figure 8A, B).

Naiv

e

Sham T

BI

TBI

0

2

4

6

8

Marble Burying

Bu

ried

Marb

les

39

Figure 8: Novel object recognition (NOR) in TBI, sham-TBI and naïve groups. The animals’

preference for the novel vs. familiar object was determined by the % time spent with the novel

object (A) and the discriminatory index (B), which utilizes the differences in exploration time for

the familiar object, divided by the total exploration time of the novel and familiar objects.

Rodents have a spontaneous tendency to explore novel objects, and the general trend shows that

TBI animals have less preference for the novel object compared to naïve animals. Significant

differences denoted by ‘*’. One-way ANOVA was applied to compare behavioural data,

followed by Tukey’s post-hoc. Lines over the bars indicate groups in which differences were

found to be significant.

As for the novel location recognition test, we found a trend towards significance for the

percent time spent in the novel location (F=2.6, p=0.08), and significant differences across

groups in the novel location index (F=3.78, p=0.03). Compared to naïve rats, the percent time

exploring the novel location was 12% and 31% lower in sham-TBI and TBI exposed groups,

respectively. The index exploring the novel location was 64% lower in sham-TBI rats and 145%

lower in TBI exposed animals (p=0.03) compared to naïve controls (Figure 9A, B).

40

Figure 9: Memory deficits observed in TBI animals compared to other groups in the novel

location recognition (NLR) test. Like NOR, the animals’ preference for the newly located

object vs. familiar location was determined by the % time spent in the novel location (A) and the

discriminatory index (B), which utilizes the differences in exploration time for the familiar

location, divided by the total amount of exploration of the novel and familiar locations. Values

≥1 demonstrate a preference for the novel location, while a negative value ≤ -1 shows preference

for the old/familiar location. The innate tendency for rodents to prefer exploring and recognizing

a novelly-located object more than the familiar object is equated to better performance in spatial

memory and cognition179. Significant differences denoted by ‘*’. One-way ANOVA was applied

to compare behavioural data, followed by Tukey’s post-hoc. Lines over the bars indicate groups

in which differences were found to be significant.

Barnes maze was conducted for two days, a shorter version of the 4 days test usually used

in preclinical work. As described in the methods section, undertraining was explicitly desired in

our experiments so that TBI animals would present a more striking deficit that could be rescued

by DBS in the second aim of our thesis. In the first day of training (average of two sessions), no

41

significant differences were observed in the most commonly scored Barnes maze variables

across groups, that is the total latency period (F=0.54; p=0.60) and error count (F=1.46; p= 0.25).

Most importantly, during the day of testing naïve animals had a decrease in the time to locate and

enter the escape route, error count, latency to the first nose poke, total latency and an increase in

the search strategy compared to the training sessions (Figure 10). This suggested that animals

learned the task and performed as expected. In contrast, animals exposed to TBI had a worse

performance in every variable scored, suggesting a learning deficit with the established

conditions (Figure 11). The Sham-TBI group performed in between naïve and TBI exposed

animals, but never reached the deficits observed in the latter group.

In the testing day, significant differences across groups were found for the time to locate

the escape route (F=9.45, p=0.0006), time to enter the escape route (F=8.34, p=0.001), primary

latency to the first nose poke (F=10.08, p=0.0004), and the total latency (F=8.56, p=0.01). A

trend towards significance was found for the error count (F=3.05, p=0.06), while no-significant

differences were observed for search strategy (F=1.56, p=0.22). Figure 11 and Table 2 show that

both TBI and Sham-TBI exposed rats had a worse performance than naïve animals in almost all

variables. In most measures, scores in TBI animals were worse than in Sham-TBI rats.

Table 3: Barnes maze results in Aim 1- statistical data comparing Naïve, Sham-TBI and TBI

exposed animals.

Variable ANOVA Naïve vs Sham-TBI

Naïve vs TBI

TBI vs Sham-TBI

Time to locate the escape route

F=9.45, p=0.0006* p=0.26 p=0.0004* p=0.02*

Time to enter the escape route F=8.34, p=0.001* p=0.02* p=0.001* p=0.44

Error count F=3.05, p=0.06 p=0.51 p=0.05* p=0.36

Latency to the first nose poke F=10.08, p=0.0004* p=0.23 p=0.0003* p=0.02*

Total latency F=8.56, p=0.01* p=0.02* p=0.001* p=0.44

Search strategy F=1.56, p=0.22 p=0.92 p=0.22 p=0.39

42

Figure 10: Barnes maze data for TBI treatment and control groups displaying changes in

performance across all variables, between train and test days. TBI animals tend to make

more errors and take longer durations to explore and locate the escape box, compared to naïve

animals. Search strategies were scored on a scale of 0-1 where values closer to 0 = more random

exploration, while values closer to 1 = more direct navigation to escape the maze.

43

Figure 11: Average Barnes maze performance in all variables (latencies, search strategy

and error count) for TBI treatment and control groups. TBI animals generally had longer

primary and total latencies to explore and locate the escape box, respectively; naïve animals tend

to outperform the TBI groups all variables (even when no significant difference is observed, as

for the search strategy). Significant differences denoted by ‘*’. One-way ANOVA was applied to

compare behavioural data, followed by Tukey’s post-hoc. Lines over the bars indicate groups in

which differences were found to be significant.

One-way ANOVA was performed for the comparison of behavioural data in experiments

1 (naïve, sham-TBI and TBI groups) and 2 (TBI, Sham DBS-TBI and TBI groups), followed by

Tukey’s post-hoc analysis for multiple comparisons.

44

Figure 12: Rat brains recovered 24hr post-Barnes maze testing. Contusion on TBI (right)

animals observed where fluid percussion injury (FPI) was exerted on the right hemisphere, in

comparison to Sham-TBI (left).

3.2 Study 2: To investigate the effects of acute ANT-DBS in the FPI model

through a battery of behavioural tests associated with learning, memory and

anxiety-like behaviour.

After defining TBI-induced memory deficits, we have treated groups of TBI exposed rats

with DBS (n=10), Sham-DBS (n=9), or control surgery (no electrodes implanted; n=13). All

animals in the DBS and Sham-DBS groups had electrodes implanted within the ANT.

No significant differences were found across groups in the defensive burying test (F=0.79,

p=0.46; Figure 13). Similarly, no significant differences across groups were observed for both

the percent time (F=2.50, p=0.1) and index (F=2.50, p=0.1) in the novel object recognition test

(Figure 14A, B). Differences across groups were also not found to be significant for the percent

time (F=0.38, p=0.69) or index (F=0.38, p=0.69) in the novel location recognition test (Figure

15A, B).

45

Figure 13: Average number of buried marbles in the DBS treatment group demonstrate no

significant improvement in anxiety type behaviour, compared to sham DBS-TBI, and TBI

only groups. Animals did not show a significant difference in performance based on number of

marbles buried across groups after acute ANT-DBS. This suggests that acute ANT-DBS does not

improve anxiety-type behaviour in animals with moderate-to-severe TBI. One-way ANOVA was

applied to compare behavioural data, followed by Tukey’s post-hoc.

TBI

Sham D

BS T

BI

DBS T

BI

0

2

4

6

Marble Burying

Bu

ried

Mar

ble

s

46

Figure 14: NOR performance in DBS-TBI groups demonstrate no significant difference in novel

object exploration (A) or preference (B). This suggests that acute stimulation of the ANT has no

significant therpaeutic effect on non-spatial or working memory following moderate-to-severe

TBI. One-way ANOVA was applied to compare behavioural data, followed by Tukey’s post-

hoc.

47

Figure 15: NLR performance in DBS-TBI groups demonstrate no significant difference in novel

location exploration (A) or preference (B), in comparison to sham DBS-TBI, or TBI only groups.

One-way ANOVA was applied to compare behavioural data, followed by Tukey’s post-hoc.

In the Barnes maze, no significant differences were observed in the first day of training

for the total latency period (F=2.57; p=0.09) and error count (F=0.53; p= 0.59). Similar to

experiments in aim 1, TBI-exposed rats had worse or unaltered memory performance in every

variable scored compared in testing sessions, relative to the training sessions, suggesting a

learning deficit in this group of animals (Figure 16). TBI-exposed animals given DBS had a

decrease in the time to locate and enter the escape route, error count, latency to the first nose

poke, total latency and an increase in the search strategy compared to the training sessions,

suggesting an expected pattern of learning. Sham DBS-TBI animals implanted with electrodes

that received no DBS showed signs of memory improvement compared to TBI, though less

pronounced than those recorded in the DBS-TBI group.

48

Figure 16: Barnes maze data for DBS-TBI, Sham DBS-TBI and TBI groups displaying

changes in performance across all variables, between train and test days. TBI animals

tended to make more errors and take longer durations to explore and locate the escape box, while

TBI animals receiving acute ANT-DBS had an improvement in performance by making fewer

errors, with a shorter duration in exploring and escaping the maze, which suggest an

improvement in spatial memory in moderate-to-severe TBI.

DBS was largely found to improve memory performance in the Barnes maze. Significant

differences across groups were noted in the time to enter the escape route (F=8.54, p=0.001),

error count (F=3.97, p=0.03), total latency (F=8.75, p=0.001), and search strategy (F=5.75,

p=0.008). In contrast, results were not found to be significant for the time to locate the escape

route (F=2.43, p=0.11) and the latency to first nose-poke (F=2.44, p=0.10). As can be

appreciated in Figure 17 and Table 3, we found significant improvements in TBI rats given DBS

compared to those exposed to TBI alone. In some variables (e.g., time to enter the escape route,

total latency), TBI animals given DBS also fared better than those receiving sham stimulation. In

others (time to locate the escape route, latency to the first nose poke), sham stimulation exerted a

modest effect, with a mild improvement in memory performance. Finally, the mere insertion of

electrodes in the absence of stimulation was found to be part of the therapy, as observed in the

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number of errors and search strategy. When these variables were considered, the magnitude of

stimulation effects was similar to that of non-stimulated animals, suggesting that electrode

insertion played a role in the therapeutic response of DBS.

Figure 17: Average Barnes maze performance across all variables (latencies, search

strategy and error count) in DBS-TBI, Sham DBS-TBI and TBI only groups. In general,

DBS animals demonstrated an improvement in performance across all variables measured,

suggesting that ANT-DBS may facilitate recovery from spatial memory deficits. Significant

differences denoted by ‘*’. One-way ANOVA was applied to compare behavioural data,

followed by Tukey’s post-hoc. Lines over the bars indicate groups in which differences were

found to be significant.

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Table 4: Barnes maze results in Aim 2 - statistical data comparing controls (TBI Ctl), Sham

DBS and DBS-treated animals, all of which were exposed to TBI.

Variable ANOVA TBI Ctl vs TBI Sham DBS

TBI Ctl vs TBI DBS

TBI Sham DBS vs TBI DBS

Time to locate the escape route

F=2.43, p=0.11 p=0.79 p=0.09 p=0.36

Time to enter the escape route F=8.54, p=0.001* p=0.97 p=0.002* p=0.007*

Error count F=3.97, p=0.03* p=0.07 p=0.05* p=0.99

Latency to the first nose poke F=2.44, p=0.10 p=0.84 p=0.09 p=0.33

Total latency F=8.75, p=0.001* p=0.99 p=0.002* p=0.005*

Search strategy F=5.75, p=0.008* p=0.04* p=0.01* p=0.93

Additionally, histological sections stained with cresyl violet revealed that electrodes in DBS and

sham treated animals were all within the boundaries of the anterior thalamic nucleus (Figure 18).

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Figure 18: Electrode location. Schematic representation of atlas plates showing the tip of the

electrodes in TBI animals undergoing DBS (black circles; n=10) or sham-treatment (grey circles;

n=9). Note that electrodes were located within the boundaries of the anterior nuclear complex.

While electrodes were bilaterally implanted, they were represented unilaterally in this schematic

figure. Numbers below the plates denote distance from bregma. Plates adapted from Paxinos and

Watson’s rat brain atlas178.

3.3 Study 3: To test whether ANT-DBS reduces hippocampal apoptosis in FPI-

exposed rats.

Once we have defined that DBS induced significant memory improvements in TBI

exposed animals, we prepared new batches of rats for neurochemical analysis. Animals

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undergoing TBI (n=8), DBS-TBI (n=8) or Sham DBS-TBI (n=7) were sacrificed 24 hours after

lateral FPI, and their brains processed with ELISA for the measurement of caspase-3 in the

perilesional cortex and dorsal hippocampus (Figure 19). In the perilesional cortex, 2-way

ANOVA revealed no significant TBI effect (F(1,40) = 0.18; p=0.67), DBS effect (F(2,40) =

0.39; p= 0.68) or TBI vs DBS interaction (F(2,40) = 0.02; p=0.98). Similarly, 2-way ANOVA

revealed no significant TBI effect (F(1,40) = 0.11; p=0.73), DBS effect (F(2,40) = 0.39; p= 0.68)

or TBI vs DBS interaction (F(2,40) = 0.01; p=0.99) in the hippocampus beneath the lesion.

Figure 19: Caspase-3 measurements obtained using an enzyme immunosorbent assay

(ELISA) for TBI and non-TBI hemispheres. Animals were sacrificed 24hrs after their

respective interventions. Levels of caspase-3 were measured relative to protein content in the

hippocampus and perilesional cortex (tissue samples obtained within 1.0mm radius of TBI-

induced cortex). We report no significant differences across groups, notably, no substantial

increase in caspase levels within TBI animals. 2-way ANOVA applied with TBI (TBI, non- TBI)

and DBS (no electrode, sham DBS, DBS) as independent factors.

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Chapter 4 – Discussion

The overall outcome of our paradigm generated a reduction in memory deficits for

animals with DBS compared to non-stimulated treatment groups, when tested with the Barnes

maze, on most behavioural metrics. However, DBS groups had not gained a significant change in

behaviour, or memory deficits when taking results of the NOR/NLR or the defensive marble

burying test into account. Marble burying performances also led to non-significant differences

between sham-TBI and TBI groups (i.e., 25% sham-TBI vs. 32% TBI reduction in marble

burying). In both novel object and novel location tests, TBI animals demonstrated worse

performance compared to naïve animals, as indicated by their exploration time and

discrimination of the novel stimuli. However, behavioural deficits in sham-TBI animals were

comparable to TBI groups (i.e., 21% sham-TBI vs. 17% TBI in NOR) during NOR, which may

likely be attributed to surgical methodology, such as heat induced while drilling a sham

craniotomy or the volume of anesthesia implemented. Despite observing similar results in novel

object discrimination and exploration for both sham-TBI and TBI animals, the latter fared worse

in performance overall.

Another aspect of our paradigm to note is the undertraining protocol for Barnes maze,

which had involved two days in total. In a pilot study, we had employed a standard Barnes maze

protocol, consisting of a habituation phase 24 hours prior to training to reduce the likelihood of

anxiety behaviour185, and allow the animals to habituate to the maze as well as the escape box.

This was followed by a 3-day acquisition/training phase (2 trials, each 180s per animal). Finally,

an acquisition probe phase was implemented 48 hours after the last training session to assess

memory retention, ascertain the search strategies of the animals and whether or not distal cues

were also involved186. The different stages of Barnes maze phases evaluate spatial reference

memory, working memory, retrieval of memory, and in some cases, (where a reversal learning

phase is included) cognitive flexibility182,186,187. Training and probe trials evaluate spatial

learning and memory, and hippocampal functioning182,185, while cognitive flexibility associated

with reversal learning, involves the frontal cortex188. However, during our pilot studies involving

the TBI treatment and naïve control groups, the standard Barnes maze protocol did not present

any notable deficits in behaviour or spatial memory for TBI animals compared to naïve groups,

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after the first 1-2 days of acquisition. Therefore, we had modified the standard protocol, such that

the animals had undergone 2 training trials in total, over the span of 1 day, followed by testing on

the next day. This is similar to previous studies in different preclinical models. For example, one

study by Attar et al. (2013) assessing spatial memory in a strain of AD mice had reported

significant cognitive deficits during the probe trial, when a similar, shorter (5 training trials)

Barnes maze paradigm was implemented, in comparison to a longer protocol189. By doing so,

they had successfully evaluated spatial memory in a more difficult, yet cost-effective approach

within AD transgenic mice189. Undertraining was also used as a strategy to show the behavioural

effects of entorhinal cortex DBS on memory performance in rodents exposed to the Morris water

maze190.

Our decision of undertraining for Barnes maze demonstrated significant differences

across groups for both primary and total latencies, as well as the number of errors made prior to

locating the escape box. Therefore, we believed that this variant of the Barnes maze protocol best

fitted our experimental aims. Furthermore, unlike a spatial reference memory and learning test

like the Morris water maze, the Barnes maze allows for memory and cognitive assessment

without the added anxiety associated with its aquatic aspect189. However, the Barnes maze still

consists of modest aversive stimuli, which makes this test relatively less motivating compared to

the Morris water maze185. In our case, we found that animals demonstrated high exploration,

where, despite knowing the location of the escape box, they would not reach or enter to safety as

quickly as during the initial trials. This prolonged exploring resulted in an increase in primary

latency times, which is not uncommon for this behavioural test185. Additional weak aversive

stimuli were also introduced to our paradigm (e.g. overhead bright light source, high speed fan),

so that the animals respond to their innate agoraphobia191 and are compelled to reach the escape

box without additional delays. Conversely, taking caution in implementing too many aversive

stimuli in the Barnes maze is important, as they can potentially distract animals from focusing on

their task of seeking immediate shelter, but acclimatizing to the environment would reduce this

confounding variable185. O’ Leary & Brown (2012)192 had concluded that the overall learning

and memory performance in rodents was not affected by the number of habituation/shaping

trials, but the authors had recommended a habituation phase when there is no knowledge of any

motor deficiencies or anxiety level in our test subjects192,193. We had extensively handled the rats

prior to testing and motor abilities were also assessed to reduce the effects of confounding

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anxiety levels or motor deficits. Overall, TBI, followed by sham-TBI animals consistently

performed worse than naïve animals.

Our TBI model was intended to replicate a scenario in which stimulation would be

delivered to patients affected by moderate-to-severe TBI shortly after the impact. In addition to

our selected TBI model, our modified behavioural protocol was more likely to reveal significant

deficits in learning and memory. Subsequently, our main goal was to rescue any possible

memory deficits with acute DBS, hypothesized to occur by providing some degree of

neuroprotection within a short recovery window, as examined in Aim 2. Stimulation parameters

for DBS in our animal model were derived from work in animal models of epilepsy107 and

approximated to reflect clinically relevant settings meant for therapeutic intervention in humans.

4.1. ANT-DBS

Reports of DBS of subcortical targets are far and few between, especially in the case of

TBI studies160. Direct structural and secondary damage from TBI that follows the initial insult

and nearby distal regions, have previously been targeted with DBS to alleviate disorders of

consciousness160. In particular, thalamic nuclei and associated white matter tracts necessary for

attention and arousal are targeted in patients with moderate-to-severe TBI; however, because of

the heterogeneity of structural and functional damage caused by TBI, patients are also likely to

develop comorbidities of movement disorders, depression and memory deficits160. Studies testing

for memory and cognitive function in TBI models or patients are limited, but cumulative

findings all have directed to components of the Papez circuit, anterior thalamus and frontal

cortical regions as primary targets for attenuating memory deficits and improving higher

executive function in the TBI subpopulation160

This study was designed to evaluate the efficacy of DBS in the anterior nucleus of the

thalamus, testing whether reductions in anxiety-like behaviour and recovery from memory

deficits could be observed. Known for its significance in learning and memory processes, the

ANT is a key component of the limbic circuit of Papez194. Specific subnuclei of the ANT make

up three parallel hippocampal-ANT circuits195,196; crucial for relaying emotional and visceral

information to the pre-frontal cortex, thus being involved in higher executive function and

cognition (anteromedial; AM)197; firing rhythmic theta activity to the hippocampal formation, in

a return-loop system (anteroventral; AV)197; and finally, the anterodorsal nucleus (AD), which is

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involved in both spatial navigation (via head direction cells198) and non-spatial forms of

memory195,196. Several behavioural experiments in animals have alluded to performance

disruption in a wide range of spatial learning tasks, when lesioning the ANT195,199,208,200–207,

particularly those involved in spatial alternation150,195, as well as disruption in temporal order

discrimination during non-spatial tasks209. In addition, clinical studies have previously

demonstrated a disturbance in mnemonic processes, contributing to diencephalic amnesia196.

Furthermore, while a series of reciprocal and interconnected brain regions support different types

of spatial learning207, lesions in the ANT may disrupt a range of fundamental body signals

necessary for effectively processing spatial information210.

During our behavioural paradigm following acute DBS, the defensive marble burying

trials demonstrated no significant differences. The high variability across treatment groups may

have occurred due to chance, where some marbles may not be considered buried if the animal

had clearly stepped on them during exploration. Though defensive burying paradigms have been

proposed to measure anxiety-type responses, we note this is an innate behaviour, where rodents

tend to bury objects that are unfamiliar or may be perceived as dangerous. That said, defensive

burying is well validated and routinely used. As no changes in buried marbles were found

between TBI and sham-TBI groups, it is not unexpected that ANT-DBS did not rescue this

behaviour. With negative data, however, whether ANT-DBS delivered immediately after TBI

improves anxiety-type behaviour remains elusive.

Treatment and control groups during both novel object and novel location recognition

tests garnered no significant differences in both percent time spent with novel stimuli, and

discrimination between familiar and novel stimuli. Readily-learned spatial alternation tests (e.g.

T-maze, radial arm maze) are known to be particularly sensitive to ANT lesioning in

rodents183,206; however, not only is working memory affected by ANT lesions, but tasks

involving the need for reference memory are also affected (e.g. finding the location of the escape

box on the Barnes maze, or locating the submerged platform of a Morris water maze (e.g. Wolff

et al. (2008)211). Deficits in place learning were also found following ANT lesions where, for

instance, behavioural tasks with rats placed in a Morris water maze arena show an increase in

latencies to locate the hidden escape platform, as well as greater inaccuracies in search strategy

on removal of the escape platform211,212. In addition, proactive interference and temporal

discrimination are likely to be impaired and play a role in alternation deficits, as demonstrated by

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behavioural studies where objects or odours are presented in a continuous sequence of

information183,209, suggesting that spatial/non-spatial tasks with a highly demanding memory

requirement would be challenging for rats with ANT lesions. As theta activity in the AV nucleus

contributes to the processing and distinction of temporal information via synchronous oscillation

of AV neurons with hippocampal theta rhythms, lesions in the ANT could interfere with this

synchronous activity183,213. Consequently, this theta disruption would affect a rodent’s ability to

temporally organize consecutive stimuli on exposure to a continuous stream of information

during memory tasks. However, when testing for memory during object-recognition tasks in

discretely parsed trials, ANT lesion studies have demonstrated no direct effect on the ability to

recognize a novel stimulus in between-block trials205,210. Furthermore, significant damage to the

hippocampus in rats would suggest loss of both parsing/chunking of information, as well as

making finer temporal distinctions when presented with continuous sets of stimuli183. Overall,

complexities in spatial alternation deficits affiliated with ANT damage can be explained by one

or both of the following factors: (i) Damage to the three major nuclei of ANT would lead to

impairments in spatial processing, as these nuclei comprise a diverse range of spatial functions;

and (ii) unintentional damage to sites adjacent or relatively close to the ANT during surgical

procedures may disrupt neural plasticity and activity, thus amassing behavioural deficits in both

spatial and non-spatial memory210. Resultant combined lesions of all three nuclei would increase

severity of behavioural impairments during highly complex spatial learning tasks, compared to

confined lesioning of selective ANT subnuclei210,214. As moderate-to-severe TBI can be a

combination of both focal and diffuse injuries to the brain, it is not surprising to find that our

lateral FPI near the hippocampus would prompt deficits for animals in a wide range of spatial

and non-spatial behavioural tasks, with each deficit ascribed to one or several components of the

limbic circuitry. Like the Morris water maze, successful escaping of the Barnes maze also

involves effectively acquiring both navigational skills and spatial cues. While our protocol was

relatively shorter than the regular Barnes maze paradigm, consecutive and repetitive trials on

both days for TBI animals did not necessarily improve their latencies to locate the escape box.

Contrary to our findings in NOR and Marble burying, DBS had a significant impact on

TBI rats during the Barnes maze trials, particularly in the number of errors prior to entering the

escape box, total latency period and search strategy; however, primary latencies to locate the

escape box were not significant across groups. Of the subcomponents of the anterior thalamus,

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AM is responsible for processing temporal information and response flexibility, AD provides

directional signals while AV allows for place learning and discrimination195. Animals with a

lesioned ANT may fail to achieve regular patterns of accuracy during behavioural tasks, as

observed during Barnes maze trials, compared to their naïve counterparts. DBS used at high, but

still clinically relevant stimulation current has led to a decrease in spontaneous neuronal firing,

as well as excitation of axonal projections around the electrodes placed at our target region19.

Conversely, the frequency range of stimulation may also have an effect on overall behaviour and

cognition, as suggested by theta frequency-dependent restorative effects on cognitive

performance of DBS in TBI rats215. Our Barnes maze data may suggest overall improvement in

memory following DBS in TBI rats; however, whether this was an immediate consequence of

stimulation on oscillatory activity, or changes in cell firing in the ANT or structures at a distance

from target remain to be demonstrated, as DBS was applied several days prior to behavioural

testing. Based on our previous findings of significant decrease in hippocampal caspase activity in

a rodent model of status epilepticus107, we hypothesized that one possible explanation for the

improvement in spatial memory performance observed after DBS could be that of a reduced

hippocampal apoptosis following TBI.

4.2. Caspase-3

A large family of proteins, known as caspases is the primary mediator for the regulated

destruction of cells, or apoptosis216. Initiation of apoptosis directs a sequence of biochemical

changes (e.g. nuclear fragmentation, cell shrinkage)217, gradually leading to programmed cell

death. Axonal injury following head trauma, neuronal and glial cell deaths all contribute to the

overall neuropathology of TBI, and these cells observed within contusions tend to display

apoptotic morphologies, within the first 24 hours and up to one week post-injury218. Furthermore,

apoptotic oligodendrocytes and neurons exhibiting nuclear DNA strand breaks have previously

been identified in situ using the terminal deoxynucleotidyltransferase-mediated dUTP nick end-

labelling (TUNEL) technique. These TUNEL+ cells thus confirmed the presence of apoptotic

neuronal cell death in human white matter tracts following TBI218. In addition, not only have

apoptotic and necrotic cells been detected in TBI models at the site of injury post-trauma, but

regions that are remote to the site of injury are also affected up to weeks following head

trauma.218

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Caspase-3 is the most abundant cysteine protease in the brain and is crucial for DNA

fragmentation and chromatin condensation processes during programmed cell death218. It is also

an essential protein for brain development219,220. Numerous studies have consolidated neuronal

cell death as a common event occurring in neurological disorders, including ischemic

stroke117,220 and epilepsy107,221. An upregulation in caspase-3 had been reported in both clinical222

and pre-clinical CCI TBI studies223,224. The caspase-3 pathway contributes to the accumulation of

proteolytic activity leading to the production of cleaved substrates, such as isoforms of tau, a

notable biomarker of neurodegenerative disorders224. Conversely, inhibiting this caspase-3-

mediated pathway following moderate insults may be a potential therapeutic approach to

TBI220,224. In pre-clinical rodent models, an upregulation of caspase-3 activity is usually observed

24-72 hours following TBI, with maximal expression 7 days following the initial insult107.

Furthermore, one study has previously demonstrated chronic, prolonged upregulation of caspase-

3 for a least 3 months post-TBI224. DBS has been proposed to reduce neuronal apoptosis through

the upregulation of anti-apoptotic and down-regulation of pro-apoptotic genes and inflammatory

responses, thus providing a neuroprotective effect; however, finding a direct causal relationship

between DBS and the proposed neuroprotective processes is still poorly understood225,226.

Our current study measured caspase-3 levels from rat hippocampal and perilesional tissue

recovered 24 hours post-TBI, with treatment groups receiving acute ANT-DBS or sham

stimulation one week prior to experimental TBI. Our reasoning for this timeframe was to

determine whether we could observe a remarkable difference in caspase-3 activity, with acute

ANT-DBS leading to a significant reduction in caspase-3 levels compared to TBI-only groups, as

its neuroprotective effects could be facilitated. Our decision for collecting brain samples 24

hours post-TBI, as opposed to 3-7 days (optimal time interval to detect caspase-3 changes) was

based on previous work suggesting that at this shorter timeframe, caspase-3 concentrations are

several-fold higher compared to control brain tissue227. Moreover, the 24-hour window would

allow us to collect samples for measuring other substrates, including glutamate transporter

proteins, cytokines, and microglial markers to be analyzed in future studies. While caspase-3 is

altered at 24 hours post-TBI, longer time intervals might have not generated measurable

differences for the other substrates mentioned above. However, our findings demonstrated that

acute stimulation of ANT led to no significant DBS effects on caspase-3 activity within tissue

obtained after the 24-hour post-TBI period and compared to animals in control groups. There are

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a few limitations to this study that should be noted: ELISA did not discriminate the cell types

responsible for the detection of caspase levels. We have only measured caspase 3 and not its

cleaved products. Given that caspase-3 and its cleaved bi-products have prolonged damaging

effects to neuronal cell survival beyond 24 hours post-TBI107,224, samples obtained at longer

timeframes (e.g. 72-hours post-TBI, 7 days post-TBI) for each group would likely have provided

more compelling evidence for neuroprotection by acute DBS. Future work remains to be

conducted measuring additional markers of apoptosis (TUNEL, electron microscopy studies) at

the optimal timeframe to detect the actual effects of ANT-DBS on apoptosis in TBI models.

4.3. Limitations and future perspectives

In our first study, we have investigated a reasonable number of animals but some of our

findings only yielded trends towards significance. Sham-TBI involves drilling the bone, which

may have generated heat damage in cortical areas, thus may have influenced behaviour. As TBI

rats were significantly different from naïve groups, we have decided to pursue experiments in

aim 2. Another potential control would have been animals exposed to anesthesia, opening of

superficial skin/subcutaneous planes but without bone drilling.

Whether acute ANT-DBS is sufficient in providing significant neuroprotective effects in a

clinical setting is still unclear, especially given that TBI can affect several brain regions, and lead

to long-term neuropathology that may not be as obvious shortly after the initial insult. One

criticism for our DBS-TBI model was the sequence in which we had performed the surgeries,

where implantation of DBS electrodes occurred prior to utilizing our FPI model, as opposed to

the converse sequence of events. Translation of this model into clinical settings may be

misleading, suggesting a pre-treatment paradigm, where TBI had occurred in a patient with

previously implanted electrodes. We had chosen our sequence of DBS implantation followed by

trauma, to reduce the likelihood of electrodes missing target regions during implantation, due to

heterogeneous anatomical distortion caused by lateral FPI. While TBI was exerted some distance

away from our DBS targets, the FPI device may cause diffuse injury, extensively disrupting the

anatomical position of target sites. Moreover, our arrangement of surgical procedures was

suitable for our behavioural paradigm. If the electrodes were implanted immediately after FPI,

the electrodes themselves would have a potential effect on the animals receiving stimulation,

during behavioural testing (e.g., insertional effect). Stereotaxic surgeries already require

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prolonged anesthesia protocols to locate anatomical landmarks for implanting electrodes.

Consequently, to reduce prolonged sedation, post-anesthetic neurological impact on behaviour,

we chose not to anesthetize animals for extended periods228. Should we have chosen to stimulate

the animals with transiently implanted electrodes that could later be removed, animals would

have needed to be under anesthesia for approximately 7 hours (surgery + 6h of DBS). Another

limitation for our experiments is that we did not have sufficient data from the literature to

estimate an effect size and calculate statistical power before the study. This did not allow us to

determine the appropriate number of animals per treatment group prior to the study. All ‘n’

values were estimated based on previous work conducted in epilepsy animals (e.g., Amorim et

al, 2015107). As behavioral analyses were found to be significant, our sample size was sufficient

to demonstrate an improvement in memory performance after DBS.

In our model, acute stimulation was advantageous in isolating TBI effects on spatial

memory in behavioural testing. Chronic intermittent stimulation extending for several days or

weeks, typically used in clinical settings, may further elucidate neuroprotection via ANT-DBS.

Future pre-clinical studies may consider chronic DBS before determining its efficacy in

moderate-to-severe TBI. Previous clinical findings have suggested that long-term DBS in PD

patients demonstrated significant structural and global functional changes in connectivity and

subsequently have an influence on neuroplasticity229; this would likely add to the rationale of

chronic stimulation in moderate-to-severe TBI patients for longer sessions for its therapeutic

effects to apply. Unfortunately, highly variable, and conflicting results have been demonstrated

for DBS implemented on TBI, whether on a well-characterized rodent model, or in human

studies160. Case studies of utilizing electrical stimulation on memory tend to report conflicting

results, for which cautionary interpretation is required19. Rather than facilitating memory and

cognitive function, high-current ANT stimulation led to impairments in rats150; these memory

deficits were also consistent in epilepsy patients receiving ANT stimulation173. Epileptic activity

has been shown to increase aberrant adult hippocampal neurogenesis, of which the newborn

granular cells display abnormal morphology and irregular patterns of integration into

hippocampal circuits230; however, a low-current acute stimulation did not promote aberrant

neurogenesis190. While the ANT is a crucial part of the limbic circuit, it is also possible that acute

stimulation of another limbic region, such as the entorhinal cortex (EC) would have yielded a

different outcome. In contrast, Encinas et al. (2011)168 had demonstrated high frequency

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stimulation of the ANT promoted adult neurogenesis within the subgranular zone, leading to

proliferation and survival of in vivo adult-generated granular cells of the dentate gyrus (DGC);

however, at the time, its effect on memory and cognitive function was yet to be determined. In

addition, Stone et al. (2011)190 for instance, demonstrated acute DBS supported the proliferation,

differentiation, survival and maturation of new adult-generated DGCs, while subsequently

integrating and contributing to functions of hippocampal circuitry. This was confirmed by the

formation of spatial memory in mice during a Morris water maze task within 6 weeks of EC

bilateral acute stimulation190. Conversely, the authors also noted that facilitating spatial memory

formation occurred several weeks after DBS, as opposed to treatment groups receiving

stimulation within 1 week before or after behavioural training190. This finding may inform our

own behavioural testing results, particularly in the variability of primary latencies across groups

during Barnes maze trials. Although our behavioural data suggest an improvement in spatial

memory in DBS groups acutely stimulated ~ 2 weeks before training, it does not clarify whether

acute stimulation preceding the behavioural task by several weeks would have maximized the

functional integration of new DGCs, thus establishing activity-dependent hippocampal

connectivity. Training an additional group of DBS-TBI animals in a similar behavioural

paradigm, albeit occurring after a prolonged period following acute stimulation (e.g., 5-6 weeks

post-acute DBS), may have provided further information on hippocampal-dependent spatial

memory following Barnes maze. Moreover, detailed analyses of primary path lengths before the

animal locates the escape box may have provided additional evidence of whether a higher

frequency of spatially precise search strategies were being used by stimulated rats, suggesting

improved spatial memory. Further investigation of the neurocircuitry modulated by DBS

(whether acute or chronic) is likely necessary, for example via immediate early genes, as well as

additional imaging (e.g., through retroviral labelling of newly generated adult neurons;

fluorescent anterograde tracing).

In our neurochemical study, only caspase-3 levels were measured. Our decision for

quantifying capase-3 activity using ELISA, as opposed to opting for other assays (e.g., western

blot, immunohistochemistry) was made due to its high sensitivity and reliability231 in detecting

rodent caspase-3 in a mixed cell population within homogenized brain tissue. It is important to

note that all caspase-3 sample concentrations were well within the detection range of our kit (i.e.,

0.312 ng/ml-20 ng/ml). We were disappointed to find no notable difference in measurements of

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caspase-3 in brain regions affected by TBI, in relation to total protein concentration in our

homogenized brain tissue, and compared to control samples (i.e., tissue from hemisphere

contralateral to TBI region). Despite the high sensitivity rate (i.e., 0.078 ng/ml) of our ELISA kit,

as well as accuracy within the affiliated standard curve, future studies using other assays more

suitable for measuring apoptotic activity, should be considered. Other measurements of apoptotic

markers using the TUNEL assay, followed by electron microscopy could yield different results;

however, cautionary measurement of neurons exhibiting caspase-3 activation using TUNEL is

recommended. This is due to its non-specific aspect, in that TUNEL will label all forms of DNA

fragments with free 3’-hydroxyl termini, regardless of what molecular mechanisms led to these

free termini232. Hence, careful interpretation is required when differentiating apoptotic cells from

non-apoptotic cells (e.g., necrotic cells, cells undergoing gene transcription or DNA repair).

Another limitation is that we did not quantify cleaved caspase-3, a well-studied biomarker of

apoptosis and a known effector of caspase-mediated pathways. This is due to notable factors,

including the lack of reliable and widely available ELISA kits for measuring rodent-specific

cleaved caspase-3. Furthermore, significant upregulation of cleaved caspase-3-immunopositive

cells was previously observed at chronic time intervals, beginning from one month after

experimental TBI was performed in rats, persisting for up to 3 months after TBI233. Given that

our timeline of experimentation occurred within acute timepoints (i.e., caspase-3 measurement

24h post-FPI), it is likely that we would not detect measurable concentrations of cleaved

caspase-3 in our paradigm. In conjunction with chronic stimulation, performing

immunohistochemical analyses of coronal brain sections affected by TBI, using cleaved caspase-

3 specific antibodies may render further insights of whether DBS confers neuroprotection in

moderate-to-severe TBI models. Necrotic processes may contribute to changes in caspase levels

within 24 hours post-TBI227. Furthermore, we have yet to measure concentrations of other

contributing substrates (e.g., cytokines, microglial markers, glutamate transporter proteins) to

neuroinflammation. The neurotrophic factor BDNF is known to be highly active in regions of the

hippocampus, and functions in growth and differentiation of newly developed synapses and

neurons, as well as supporting existing neurons. Survival of hippocampal cells following TBI

may be facilitated by neuroprotection induced by BDNF, through the active suppression of

caspase-3 expression234. DBS has previously been shown to augment levels of BDNF235,236, in

regions such as the pedunculopontine tegmental nucleus (PPTg-DBS), with the hippocampus

also involved in neuroprotection following rat PPTg-DBS234. BDNF may also contribute to anti-

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anhedonic-like effects following chronic stimulation of the ventromedial prefrontal cortex237.

Thus, it is possible that increases in trophic factors rather than a reduction in neuroinflammation

are involved in DBS effects on improving memory.

Finally, whether ANT-DBS follows a similar mechanism of increased BDNF levels to

inhibit caspase-3 activity is yet to be investigated. Quantitative measurements of BDNF may be

considered in parallel to caspase-3 activity, which could potentially provide a coherent

understanding of how effectively neuroprotection is facilitated by acute DBS in TBI rats.

4.4. Conclusions

In summary, this thesis presents validation of a moderate-to-severe TBI rodent model,

which demonstrated impaired cognition and memory, within which acute ANT-DBS stimulation

improved some aspects of memory, in particular, spatial navigation in TBI rats. Our findings

remain inconclusive for determining mechanism of memory improvement from moderate-to-

severe TBI using acute ANT-DBS. Although further evidence of neuroprotective properties of

DBS (whether we consider acute or chronic stimulation) may be resolved from investigating

additional neuroinflammatory markers, findings of this thesis reiterate that neuromodulation of

the ANT may improve deficits in memory and cognition, when implemented shortly after

moderate-to-severe TBI.

65

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