TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE, … · ii TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE,...

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TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE, ATTENUATES BEHAVIOUR DEFICITS FOLLOWING TRAUMATIC BRAIN INJURY IN RATS by Wen-Jia Zhang A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright by Wen-Jia Zhang (2011)

Transcript of TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE, … · ii TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE,...

TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE, ATTENUATES BEHAVIOUR DEFICITS FOLLOWING

TRAUMATIC BRAIN INJURY IN RATS

by

Wen-Jia Zhang

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

Department of Physiology University of Toronto

© Copyright by Wen-Jia Zhang (2011)

ii

TAT-9C, A TAT-FUSION CYSTEINE-RICH PEPTIDE,

ATTENUATES BEHAVIOUR DEFICITS FOLLOWING

TRAUMATIC BRAIN INJURY IN RATS

Wen-Jia Zhang

Master of Science

Department of Physiology

University of Toronto

2011

Abstract

Peroxynitrite, a highly oxidative molecule, plays a role in neuronal cell death following traumatic

brain injury (TBI). A peptide comprised of the HIV-1 tat transduction domain fused to nine

cysteine residues (Tat-9c) was previously designed to act as an exogenous target for nitrosylation

by peroxynitrite. The present study’s aim was to explore the efficacy of Tat-9c in maintaining

neurological function following TBI. Rats treated with Tat-9c exhibited significant improvement

in performance compared to controls 24 hrs following TBI in the Beam-Walk task but not in the

Rota-Rod task. Injured animals, given the drug, show a recovery as indicated by similar

performance on the Morris Water Maze task compared to sham controls. These findings suggest

Tat-9c may constitute a potential therapy for improving motor and cognitive function following

TBI.

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Acknowledgments

This thesis would not have been possible without the assistance of a few individuals who

believed in me and kept me going along this adventure to the very end. Firstly, I would like to

thank Dr. Michael Tymianski for giving me this opportunity to experience and contribute to this

exiting field in neuroscience. It was also a pleasure to work alongside other esteemed scientists

and scientists-in-training in Dr. Tymianski’s lab: Ishraq Alim, Andrew Barszczyk, Douglas J

Cook, Hong Cui, Zhanxin Ji, Rongwen Li, Hong Sun, Xuijun Sun, Kinga Szydlowska, and Lucy

Teves. I would also like to thank the members of my supervisory committee, Dr. Andrew Baker,

Dr. James Eubanks, and Dr. Martin Wojtowicz for their advice and constructive criticism. For

the unconditional love and limitless hearty meals, I would like to thank my family for their

encouragements, including my vociferous parrot for keeping me awake. I am also grateful to my

friends for making sure I still had a life outside the laboratory. Finally, I owe my deepest

gratitude to Jeff Dason for his generous assistance and friendship over the years, to Rene Persaud

for destroying me on my practice dissertation so I didn’t feel as bad during the real one, and

especially to Dr. Martin Wojtowicz who has provided me with superb mentorship and excellent

teaching all the way since my naïve undergraduate years – I would not be where I am today

without the exceptional patience, support, and guidance from these extraordinary people.

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Table of Contents

ABSTRACT ................................................................................................................................... ii

ACKNOWLEDGMENTS ........................................................................................................... iii

TABLE OF CONTENTS ............................................................................................................ iv

LIST OF TABLES ...................................................................................................................... vii

LIST OF FIGURES ................................................................................................................... viii

LIST OF ABBREVIATIONS ..................................................................................................... ix

INTRODUCTION ......................................................................................................................... 1

1.1 Traumatic Brain Injury ......................................................................................................... 1

1.1.1 Epidemiology of Traumatic Brain Injury .................................................................. 1

1.1.2 Definition and Severity Index of Traumatic Brain Injury ......................................... 2

1.1.3 Neuropathological Classification of TBI .......................................................................... 2

1.1.4 Biomechanical Classification of TBI ......................................................................... 3

1.2 Pathophysiology of Traumatic Brain Injury ........................................................................ 4

1.2.1 Cerebral Blood Flow .................................................................................................. 4

1.2.2 Neuroinflammation .................................................................................................... 4

1.2.3 Excitotoxicity and Oxidative Stress ........................................................................... 5

1.2.3.1 Glutamate Excitotoxicity ................................................................................. 5

1.2.3.2 NMDAR-Mediated Glutamate Excitotoxicity ................................................. 6

1.2.4 Protease-Mediated Cell Death ................................................................................... 9

1.2.5 Cell Death Pathways of Secondary Injury in Summary ............................................ 9

1.3 INJURY MODELS OF TRAUMATIC BRAIN INJURY................................................. 11

1.3.1 Weight Drop Model ................................................................................................. 11

1.3.2 Cortical Control Impact (CCI) Model ...................................................................... 13

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1.3.3 Fluid Percussion Injury (FPI) Model ....................................................................... 15

1.4 Current Research for Treatment of Traumatic Brain Injury .............................................. 17

1.4.1 Management of TBI ................................................................................................. 17

1.4.2 Neuroprotection Drugs for TBI ............................................................................... 17

1.4.3 Neuroprotection Through the Scavenging of Radical Species ................................ 20

1.5 Design of Tat-9c and Tat-9a .............................................................................................. 21

1.6 Neurobehaviour Models for Assessing TBI Severity ........................................................ 22

1.6.1 Beam-Walk .............................................................................................................. 22

1.6.2 Rota-Rod .................................................................................................................. 23

1.6.3 Morris Water Maze .................................................................................................. 24

HYPOTHESIS AND GOALS .................................................................................................... 26

2.1 Research Goals ................................................................................................................... 26

2.2 Hypothesis I ....................................................................................................................... 27

2.3 Hypothesis II ...................................................................................................................... 27

2.4 Hypothesis III ..................................................................................................................... 27

MATERIALS AND METHODS ............................................................................................... 28

3.1 Lateral Fluid Percussion Injury Model .............................................................................. 28

3.1.1 Surgical Preparation ................................................................................................. 28

3.1.2 Drug Injection .......................................................................................................... 28

3.1.3 Fluid Percussion Injury ............................................................................................ 29

3.2 Treatment Preparation ........................................................................................................ 31

3.2.1 Preparation of Tat-9c for Animal Injection ............................................................. 31

3.2.2 Preparation of Tat-9a for Animal Injection ............................................................. 31

3.2.3 Preparation of Saline for Animal Injection .............................................................. 31

3.2.4 Preparation of Sham Animals .................................................................................. 31

3.3 Implication of Various Behavioural Paradigms ................................................................. 32

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3.3.1 Beam-Walk Model ................................................................................................... 32

3.3.2 Rota-Rod Model ....................................................................................................... 32

3.3.3 Morris Water Maze Model ....................................................................................... 33

3.4 Statistical Analysis ............................................................................................................. 33

RESULTS .................................................................................................................................... 34

4.1 Validation of Behaviour Assays Utilized to Assess Severity of TBI................................. 34

4.1.1 Beam-Walk Model ................................................................................................... 34

4.1.2 Rota-Rod Model ....................................................................................................... 36

4.1.3 Morris Water Maze Model ....................................................................................... 38

4.2 Effects of Tat-9c on Motor Behaviour Function Following TBI ....................................... 43

4.3 Effects of Tat-9c on Memory Function Following TBI ..................................................... 47

DISCUSSION .............................................................................................................................. 50

5.1 Behaviour Assays Utilized to Assess Severity of TBI ....................................................... 50

5.2 Tat-9c Protects Motor Behaviour Function Following TBI ............................................... 51

5.3 Effects of Tat-9c on Memory Function Following TBI ..................................................... 52

5.4 Limitations of the Study ..................................................................................................... 52

5.4.1 Disadvantages of the FPI Model .............................................................................. 52

5.4.2 Limitations of Tat-9c ............................................................................................... 53

5.4.3 The Complex Nature of TBI .................................................................................... 53

5.4.3.1 Cell Death Pathway Targets ........................................................................... 53

5.4.3.2 Limitations and Variability Amongst Subjects .............................................. 53

5.5 Future Directions ................................................................................................................ 55

CONCLUSIONS ......................................................................................................................... 57

REFERENCES AND LINKS..................................................................................................... 58

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List of Tables

Table 1-1 Neuroprotective strategies for traumatic brain injury………………..…………..19

viii

List of Figures

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 1-6

Figure 1-7

Figure 1-8

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 4-3

Figure 4-4

Figure 4-5

Figure 4-6

Figure 4-7

Figure 4-8

Figure 4-9

Figure 4-10

Figure 4-11

NMDAR-mediated glutamate excitotoxicity

The major pathways associated with secondary injury following TBI

Representation of the weight drop model of TBI

Representation of the controlled cortical impact (CCI) model of TBI

Representation of the fluid percussion injury (FPI) model of TBI

Representation of the beam walk device

Representation of the rota-rod device

Representation of the Morris Water Maze (MWM) assay

Dorsal representation of a rat skull

Representation of the magnitude of injury

Beam-walk motor function is impaired after TBI

Rota-rod motor function is impaired after TBI

Learning ability was not affected following TBI

Swim speed was not affected following TBI

Total distance traveled was not affected following TBI

Spatial memory is impaired after TBI

Beam-walk motor deficits are reduced in Tat-9c treated animals

Number of falls is reduced in Tat-9c treated rats following TBI

No improvement in rota-rod performance by Tat-9c treated rats

Rate of learning in Tat-9c rats was no different to any other group

Effects of Tat-9c on spatial memory

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List of Abbreviations

AIF

AMPA

atm

BBB

CBF

CCI

CNS

CsA

DNA

EPO

FPI

HBO

HIV-1

MPTP

MWM

NAC

NMDA

NMDAR

nNOS

NO

ONOO-

PSD-95

TAT-9A

TAT-9C

TBI

apoptosis inducing factor

-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

standard atmospheric pressure

blood brain barrier

cerebral blood flow

controlled cortical impact

central nervous system

cyclosporin A

deoxyribonucleic acid

erythropoietin

fluid percussion injury

hyperbaric oxygen

human immunodeficiency virus – type 1

mitochondrial permeability transition pore

morris water maze

N-acetylcysteine

N-methyl-D-aspartate

NMDA receptor

neuronal nitric oxide synthase

nitric oxide

peroxynitrite

postsynaptic density protein of 95kDa

tat transduction domain fused to 9 alanine residues

tat transduction domain fused to 9 cysteine residues

traumatic brain injury

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Chapter 1 INTRODUCTION

1.1 Traumatic Brain Injury

1.1.1 Epidemiology of Traumatic Brain Injury

Traumatic brain injury (TBI), also entitled the “Silent Epidemic,” presents severe health and

economic burden to the global community. It is known as the “Silent Epidemic” by doctors

because the uninformed public has been rattled by its devastating repercussions (Abelson-

Mitchell, 2008; Headway, 2001; The Brain Injury Association of Canada). According to the

World Health Organization, TBI will surpass many diseases and become the major cause of

death and disability worldwide by the year 2020 (Hyder et al., 2007). The Centers for Disease

Control and Prevention estimates that 1.4 million people sustain a TBI each year in the United

States alone and of those 1.4 million people, 50,000 individuals will die from their injuries and

another 235,000 will be hospitalized (Summers et al., 2009). In 2000, the Centers for Disease

Control and Prevention estimates that at least 5.3 million Americans are currently suffering from

long-term disabilities caused by TBI. As a result of TBIs, direct costs (medical costs,

rehabilitation, and treatment) and indirect costs (loss of wages and productivity) total an

estimated $60 billion in the United States in 2000. The leading causes of TBI are falls (28%),

motor vehicle-traffic crashes (20%), other accidental events (19%), and assaults (11%) while a

further 9% of all TBI cases do not have a known cause (Summers et al., 2009).

TBIs can also vary in severity ranging from mild to severe. A survey from 1995 showed that

most TBIs are classified as mild, 51%, with 21% as moderate, and 19% as severe. The rest were

unknown (Thurman et al., 1999). Overall, due to its tremendous prevalence in society, TBI

represents a major socioeconomic crisis in the world.

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1.1.2 Definition and Severity Index of Traumatic Brain Injury

Traumatic brain injury can be characterized as a non-congenital insult to the brain from an

external mechanical force with possible permanent or temporary impairment of cognitive,

physical, and psychosocial functions (Dawodu, 2003). The severity of TBI is broken down into

three categories: mild, moderate, and severe.

A mild head injury is defined as a brief period of unconsciousness but can also have long-lasting

sequelae (Guerrero et al., 2000). People with mild head injury may suffer from memory deficits,

thought processing, and concentration (Abelson-Mitchell, 2006).

A moderate head injury is described as a loss of consciousness between 15 minutes and six hours

(Headway, 2001). The symptoms of moderate head injury include physical, psychological,

cognitive, and behavioural deficits (Abelson-Mitchell, 2006).

A severe head injury occurs when a person has been in a coma for at least six hours and

generally, the longer a patient stays in a coma, the poorer the outcome will be for physical and

cognitive function (Annoni et al., 1992).

1.1.3 Neuropathological Classification of TBI

The highly destructive characteristics of TBI occur at two distinct levels. First of all, there is an

immediate CNS tissue disruption categorized as primary injury. Primary injury is a result of the

initial mechanical impact where shearing, compression, and stretching of the brain results in

rapid neuronal death of the affected area (Nolan, 2005; Wong et al., 2005). Following primary

injury, a secondary injury mediated by several complex biochemical signaling pathways become

activated. This process may arise hours following primary injury and can continue for several

days thereafter leading to apoptosis of neurons (Nolan, 2005; Wong et al., 2005).

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1.1.4 Biomechanical Classification of TBI

Upon impact, the mechanical load on the brain causes the brain to move either in normal but

excessively accelerated or anatomically abnormal ways, depending on the site and direction of

the load. Due to the varying ways a mechanical load can be applied to the cranium, three types of

mechanical loading responsible for TBI have been classified as impact, impulsive, and static

loading (Ommaya et al., 2002).

Impact loading involves direct contact of a solid object to the head at a given speed. Its duration

is defined as < 50 s. Due to the collision of an object with a skull, movement and deformation of

tissue occurs and deformation of the skull at the site of impact is common. Impact loading can

occur whether the head was stationary (inert) or moving (accelerated) (LaPlaca et al., 2007).

Impulse loading, on the other hand, does not involve any contact towards the head and is chiefly

a consequence of excessive acceleration or deceleration of the head. Because there is no impact,

usually it does not result in any skull deformation. The duration of impulse loading ranges

between 50 – 200 ms (Davis, 2000). An example of impulse loading occurs when a sudden brake

is applied to a motor vehicle previously driving at high speeds. The seatbelt will stop the body

from moving but also cause the head and neck to snap forwards and backwards, thereby causing

damage to the brain.

Finally, static loading is caused by excessive force applied to the head for over 200 ms. Static

loading is the least common type (Ommaya et al., 2002) and it often results in deformation of the

skull since static loading happens usually in the context of a head injury caused by compression

(Davis, 2000).

In all types of mechanical loading, a sudden change in head motion will occur. This acceleration

can elevate intracranial pressure in certain areas of the skull, create axonal injury or bleeding if

head movement ruptures neuronal or vascular tissue, and cause tissue damage as a result of stress

waves produced by the brain hitting the skull (Davis, 2000). All types of mechanical loading are

associated with primary injury.

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1.2 Pathophysiology of Traumatic Brain Injury

The pathways involved in secondary injury leading to apoptosis of neurons can include altered

cerebral blood flow, inflammation, increased intracellular calcium and extracellular glutamate

levels as well as oxidative damage to various molecules of the cell (Liu et al., 2002; Moro et al.,

2004; Werner et al., 2007). As the secondary injury does not manifest immediately following the

primary injury, it is thought that there is potential for neuroprotection for TBI at the secondary

injury level by manipulating the signaling pathways involved (Beauchamp et al., 2008).

1.2.1 Cerebral Blood Flow

In order for the brain to function, it requires a constant supply of oxygen and nutrients; hence,

cerebral blood flow (CBF) is tightly coupled to neuronal activity and cerebral glucose

metabolism (Giza et al., 2001). The blood-brain barrier (BBB) is created by specialized vascular

endothelial cells and tight junctions to prevent the passive diffusion of electrolytes and large

proteins. The BBB helps to maintain the integrity of the cerebral environment (Greve et al.,

2009). Following TBI, the BBB can fail leading to a collapse of the cerebral environment and the

disruption of the autoregulation for CBF (Greve et al., 2009). CBF can be decreased up to 80%

after a TBI (Giza et al., 2001) as a result of various secondary injury conditions such as cerebral

edema, hemorrhage, hypoxia, inflammation, and increased intracranial pressure (Nolan, 2005).

Furthermore, disrupted CBF may also play a role in exacerbating secondary injury by providing

less oxygen and nutrients to the brain (thus compounding the stress that neurons undergo

following TBI) (Bramlett et al., 2004) as well as stimulate the release of glutamate, free radicals,

and nitric oxide (Greve et al., 2009).

1.2.2 Neuroinflammation

Neuroinflammation is the activation of the brain’s innate immune system in response to an

inflammatory challenge and is characterized by many cellular and molecular changes within the

brain such as activation of glial cells, synthesis of pro-inflammatory molecules such as

cytokines, chemokines, and prostanoids, and up-regulation of infiltrating leukocytes (Hein et al.,

2009). Both primary and secondary insults can induce a neuroinflammatory response leading to

an increase in BBB permeability, synthesis of scar tissue, and release of neurotoxic mediators

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such as nitric oxide and thus, aggravate secondary brain damage (Hein et al, 2009; Werner et al.,

2007).

1.2.3 Excitotoxicity and Oxidative Stress

1.2.3.1 Glutamate Excitotoxicity

Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS).

Excitatory neurotransmitters cause ion channels on the cell membrane to open resulting in a net

influx of positively charged ions (cations). This net influx of cations causes the neuron to

depolarize and if sufficient levels of excitation are provided, action potentials are fired.

Glutamate toxicity, neuronal death due to overstimulation of the CNS by high concentrations of

glutamate, was first observed in the retinal layers of neonatal mice fed monosodium glutamate

(Lucas et al., 1957), but the term “glutamate excitotoxicity” was not coined until 1969 by Olney.

Glutamate acts on both ionotropic and metabotropic receptors. The ionotropic glutamate

receptors are divided into three types according to their agonists: N-methyl-D-aspartate

(NMDA), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate. All

three channels possess various sites by which their biochemical properties can be altered. AMPA

and kainate receptors are ligand-gated ion channels that are calcium impermeable and by

triggering downstream cell death pathways, they can play a minor role in excitotoxicity

(Waxman et al., 2005). However, the NMDA receptor (NMDAR), which is activated by binding

of glutamate and glycine, is highly permeable to calcium and is the primary receptor responsible

for excitotoxicity (Olney, 1969). Following TBI, the NMDARs are over-activated by the

excessive release of glutamate and thus, the NMDARs depolarize and disrupt the membrane

potential leading to neuronal overexcitation and death through excitotoxicity (Giza et al., 2001;

Kermer et al., 1999). Glutamate also acts on metabotropic receptors to release calcium from

stores but it is its involvement with ionotropic receptors that plays an important role following

TBI (Arundine et al., 2004).

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1.2.3.2 NMDAR-Mediated Glutamate Excitotoxicity

It was first thought in 1985 that calcium entry into neurons was the essential determinate for

glutamate excitotoxicity (Choi, 1985). However, subsequent studies confirmed that it is

specifically the path of calcium entry (mediated by NMDARs) and not the calcium load itself

that is central to the neurodegenerative process (Choi, 1987; Sattler et al., 1998; Tymianski et al.,

1993).

The neurodegenerative process initiated by calcium entry through the NMDAR is associated

with nitric oxide synthase (NOS), which generates nitric oxide (NO) leading to cell death

(Dawson et al., 1991, 1993, 1994, and 1996). Thus, the current model of NMDA-mediated

glutamate excitotoxicity was formed (Figure 1-1).

NO is a gaseous free radical that is found in all tissues, especially in high concentrations in the

brain where it plays a large role in various physiological and pathological pathways (Cherian et

al., 2004). Physiologically, NO is involved in mediating CBF as a potent vasodilator, memory

formation and synaptic plasticity, release of neurotransmitters, and intracellular signaling

(Cherian et al., 2004; Moncada et al., 2006). Pathologically, on its own, NO is a weak toxin

however it can easily react with other species containing an unpaired electron (Gibson et al.,

2005) such as superoxide. When NO and superoxide react with one another, it produces the

reactive nitrogen species known as peroxynitrite (ONOO-) through the chemical reaction: O2-

+

NO ONOO-. ONOO- was introduced 20 years ago as the principal reactive oxygen species

(ROS) involved in producing tissue injury in a variety of neurological disorders (Beckman,

1991). The lethality of ONOO- is actually induced through the potent free radicals derived from

its decomposition (Gibson et al., 2005; Singh et al., 2006) and at physiological pH, ONOO- is

highly reactive (Aulak et al., 2004). ONOO- can decompose in two ways. The first is when

ONOO- is protonated to form peroxynitrous acid (ONOOH) which then undergoes hemolytic

decomposition to form the highly reactive nitrogen dioxide radical (NO2) and OH through the

chemical reaction ONOOH NO2 + OH (Hall et al., 2010). The second reaction is when

ONOO- reacts with carbon dioxide (CO2) to form nitrosoperoxocarbonate (ONOOCO2) which

then decomposes into NO2 and the carbonate radical (CO3): (ONOOCO2 NO2 + CO3

(Hall et al., 2010). Also ONOO- can interact with lipids to form lipid peroxyl radicals (LOO)

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(Krishnaswamy and Chi, 2006). All of the radicals derived from ONOO- (OH, NO2, CO3,

LOO) can in turn either peroxidize lipids (Radi et al., 1991), nitrate proteins (Beckham et al.,

1992; Ischiropoulos et al., 1992) or fragment DNA to cause harm (Fiskum et al., 2004; Salgo et

al., 1995a; Salgo et al., 1995b). All of the above radicals are shown to be up regulated in the first

24 hrs post TBI in rodents (Cobbs et al., 1997; Graham et al., 2000; Rao et al., 1999) and the

damaging effects of ONOO- is further validated in a fluid percussion injury (FPI) model of TBI

showing that ONOO- formation results in neuronal protein nitration and cell death (Lau et al.,

2006).

Postsynaptic density protein of 95kDa (PSD-95) has been shown to provide a structural link

between neuronal nitric oxide synthase (nNOS) and NMDARs (Sattler et al., 1999). Hence by

uncoupling the interaction between PSD-95 and the NMDAR, neuroprotection can be achieved

through the inhibition of NO production as demonstrated by Arundine et al., 2004.

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Figure 1-1. The relationship between NMDAR-mediated glutamate excitotoxicity and

increased nitric oxide production. Glutamate binding to the NMDAR causes the NMDAR

receptor to open allowing an influx of calcium into the cell. This influx of calcium binds to

calmodulin which then binds and activates nNOS and causes it to increase production of NO.

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1.2.4 Protease-Mediated Cell Death: Caspases and Calpains

Caspases have been shown to be active following TBI in both cell cultures and whole animal

models (Clark et al., 2000; Keane et al., 2001; Qiu, et al., 2002; Yakovlev et al., 1997) and

calpains are a family of enzymes which have been shown to be another important mediator of

cellular injury following TBI (Greve et al., 2009).

Caspases represent a family of cysteine proteases, which fragment DNA (Wong et al., 2005). It

was first discovered to be involved in apoptosis, or programmed cell death, in the nematode C.

elegans (Wong et al., 2005). Caspases are translated as an inactive proenzyme, which can only

be activated by proteolytic digestion of the proenzyme into two subunits, which then form

together to make an active holoenzyme. Active caspases can then activate other caspases and

cause cellular cytoskeleton and genomic DNA to degrade.

Calpains are calcium-dependent cysteine proteases that, under normal physiological conditions,

have a variety of cellular regulatory functions such as cytoskeletal maintenance; however,

following TBI, they have been shown to be key enzymes targeting axonal proteins leading to

cytoskeletal breakdown and disruption of axonal transport (Greve et al., 2009). Calpains have

also been implicated in the release of endonuclease apoptosis-inducing factor (AIF) from the

mitrochondria, which causes the degradation of genomic DNA (Polster et al., 2005).

1.2.5 Cell Death Pathways of Secondary Injury in Summary

Despite the importance of CBF, neuroinflammation, excitotoxicity, reactive nitrogen species,

caspases, and calpains in TBI (Figure 1-2), there has yet to be any successful pharmacological

intervention developed for the treatment of secondary neuronal injury. One reason for this is that

various studies involving potential therapies have demonstrated histological improvement

without any behavioural protection (Clark et al., 2000). Although the histological aspect of TBI

is unquestionably valuable, in the clinical setting, the end-goal is to rescue the behavioural

deficits following TBI and therefore this study will focus on the behavioural outcomes following

an injury. Furthermore, although it is acknowledged that the cell death pathways associated with

secondary injury are extensive and complex, this study will focus on affecting the excitotoxicity

outcomes of TBI and the resulting behavioural consequences.

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Figure 1-2. The major pathways associated with secondary injury following TBI. The

complexity of the cell death pathway illustrating the important role played by the alteration of

CBF, neuroinflammation, excitotoxicity, reactive nitrogen species, caspases, and calpains

following a TBI. Figure is courtesy of Park et al., 2008.

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1.3 Injury Models of Traumatic Brain Injury

There are several experimental models of TBI that attempts to recreate the pathological

conditions through the application of mechanical forces. Examples of these models include

devices that induce impact to a intact or freely moving cranium (Nilsson et al., 1977), generate

an impact to a fixed, immobilized cranium (Gurdjian et al., 1943), cause TBI by accelerating or

rotating the skull (Gennerelli et al., 1983), and trigger TBI by rapid impact of a fluid bolus

against the intact dural surface of the brain (fluid percussion injury model). Currently the three

most commonly used animal models of TBI are the weight drop model, the controlled cortical

impact model, and the fluid percussion injury model (FPI).

1.3.1 Weight Drop Model

The weight drop model, first characterized by Feeney et al., 1981, is considered by most to be

the original TBI model (Morales et al., 2005). The weight drop model employs the gravitation

forces of a weight to deliver the impact on an anesthetized animal performed under head

restraints (Feeney et al., 1981). As illustrated in Figure 1-3, this can be performed either to the

closed cranium, but most commonly, the weight is dropped through a craniotomy directly onto

the brain (O’Connor et al., 2011). The severity of the injury is controlled by adjusting the height

and mass of the weight. One advantage of this model is that very little preparation of the animal

is required (no trephination of the skull) thus it is a fast and easy technique; however, a clear

disadvantage include the possibility of a rebound injury which can cause chances of inaccuracy

and inability to control the velocity of the injury (Morales et al., 2005).

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Figure 1-3. Representation of the Weight Drop Model of TBI. Developed by Feeney et al. in

1981, a free-falling tube guided weight is dropping onto either a closed animal cranium or

directly onto the brain. The severity of injury was determined by the initial height and mass of

the weight.

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1.3.2 Controlled Cortical Impact (CCI) Model

The controlled cortical impact (CCI) model was first developed in the ferret (Lighthall, 1988)

and later adapted for the rat by Dixon et al. in 1991. This model uses a pneumatic impactor to

generate mechanical energy delivered to the intact dura following trephination of the exposed

skull (Morales et al., 2005). As illustrated in Figure 1-4, while the head of the animal is

restrained, pressurized air is employed as the source of the mechanical energy for loading to the

brain. The severity of the injury is controlled by the depth of cortical deformation, the duration of

the impact, also known as the dwell time, as well as the velocity. The CCI model improves upon

the weight drop model as it is able to control the velocity and depth of impact and there is also no

risk for a rebound injury to occur (O’Connor et al., 2011). It is well documented that the cerebral

hemodynamic responses in the animal such as elevated intracranial pressure, decreased blood

and cerebral perfusion pressures, histological and cellular alterations, as well as functional

deficits are all directly related to the depth and velocity parameters of the CCI making it arguably

the best model of head trauma in terms of having the best control over mechanical factors

(Cernak, 2005). Furthermore, the CCI model has been shown to reproduce the wide spectrum of

focal-type damage in TBI observed in the clinic setting. These include observed changes in brain

edema, elevated cerebral blood flow, neuroendocrine and metabolic changes, and coma as well

as skull deformation and related cortical compression (Cernak, 2005). However, one

disadvantage of the CCI model is the lack of brain-stem deformation, resulting in minimal

mortality, an aspect that does not mimic the real-life setting of TBI (Morales et al., 2005).

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Figure 1-4. Representation of the Controlled Cortical Impact (CCI) Model of TBI.

Developed by Lighthall in 1988, an anaesthetized animal is restrained to a device consisting of a

pneumatic cylinder, a piston and a piston tip, mounted on an adjustable horizontal crossbar. In

order to induce a TBI, the pressurized air drives the impactor downward to the intact dura. The

severity of the injury is controlled by the depth of cortical deformation, the duration of the

impact, as well as the velocity of impact.

15

1.3.3 Fluid Percussion Injury (FPI) Model

The FPI model has since become the primary model for experimental TBI (Laurer et al., 2002).

It was initially developed for use in the cat and rabbit animal model (Hayes et al., 1987);

however in 1987, it was adapted to the rat model by McIntosh et al., 1987. Levels of injury

severity can be adjusted by changing the pendulum height, which defines the force of the fluid

pressure pulse transmitted through the saline tube and ultimately towards the exposed intact dura

of a restrained and anesthetized animal. This is illustrated in Figure 1-5. There are three severity

levels measured in standard atmospheric pressure (atm): mild (about 1.4 atm), moderate (about

2.7 atm), and severe (about 3.7 atm) (Prins et al., 1996). The neuropathological sequelae caused

by the FPI device transpires in the hippocampus, thalamus, and cortex of the brain with limited

involvement of the brainstem and contralateral hemisphere (Hicks et al., 1996; Smith et al.,

1997). The FPI model reproduces several aspects of human TBI such as focal contusion,

hemorrhages, traumatic axonal injury (Graham et al., 2000; McIntosh et al., 1989) as well as

BBB disruption, neuronal loss, and alterations in CBF (Narayan et al., 1996). It also causes acute

and chronic behavioural abnormalities similar to human TBI (Faden et al., 2001; Hamm, 2001;

Pierce et al., 1998; Sanders et al., 1999). The extensive use and productivity of the FPI model

over the past 20 years has helped refine clinical trials in human head injury and vice versa

(Thompson et al, 2005) and thus, the FPI model was chosen for this study.

16

Figure 1-5 Representation of the Fluid Percussion Injury (FPI) Model of TBI.

Developed by Hayes et al., in 1987, a pendulum from a certain height impacts the piston

containing saline, which in turn forces a fluid bolus into the sealed cranial cavity of the rat

causing a TBI. The severity of the injury is controlled by changing the pendulum height, which

defines the force of the fluid pressure.

17

1.4 Current Research for Treatment of Traumatic Brain Injury

There have been many experimental studies showing a reduction of secondary injury is possible;

however, the management of TBI is challenging due to its complexity of sequelae (Jain, 2008).

These studies include different management strategies for TBI and various drugs for

neuroprotection. However, many successes in the laboratory have yet to be translated into the

clinic setting (Zhang, 2005).

1.4.1 Management of TBI

There are many different ways to manage TBI to help reduce its physical and cognitive deficits.

The most important aspect of TBI management is the control of cerebral edema and raised

intracranial pressure (Jain, 2008). Current treatments for cerebral edema include osmotherapy

(administration of hypertonic mannitol or hypertonic saline) however they are not often

beneficial since they withdraw water from healthy brain areas as well as damaged areas (Jain,

2008). Hyperbaric oxygen (HBO) therapy (administration of oxygen at pressures greater than the

atmospheric pressure at sea level) has shown signs of being an effective treatment for cerebral

edema and assists in reducing intracranial pressure; however, this method is limited to the

availability of hyperbaric chambers (Jain, 2008).

1.4.2 Neuroprotection Drugs for TBI

There are many different neuroprotective strategies being investigated (Table 1-1). As of June

2008, approximately 150 clinical trials for TBI are listed; however, the failure rate in clinical

trials has been high (Jain, 2008). Hence, continued research efforts are required to identify and

test new neuroprotective agents as well as examine combined therapies for treating TBI. Three of

the most well known drugs designed for TBI are Citicoline, Cyclosporin A (CsA), and

Erythropoietin (Jain, 2008).

Citicoline is endogenously used for getting more choline into the brain. In a rat model of TBI,

citicoline was shown to prevent neuronal loss in the hippocampus and helped decrease cortical

contusion volume. Citicoline also improved neurological recovery in rats (Dempsey et al, 2003).

Cyclosporin A, an immunosuppressant, has been shown to improve brain function, memory, and

learning in animals models of TBI and the neuroprotective properties of CsA are found to be

18

mediated through modulation of the mitochondrial permeability transition pore (MPTP). By

inhibiting the opening of MPTP with CsA, the mitochondrial membrane potential and calcium

homeostasis is maintained and isolated in the mitochondrial, thereby preventing cell death

(Sullivan et al., 1999).

Erythropoietin (EPO) has also demonstrated neuroprotective potential in cell culture and animal

models of TBI however the exact mechanism of EPO is unclear. It has been hypothesized that

EPO protects neurons by inhibiting TBI-induced neuronal apoptosis (Liao et al., 2008) and that

EPO protects motor deficits by reducing cerebral edema (Grasso et al., 2007). Currently, it is in

phase II/III clinical trials for TBI (Jain, 2008).

19

Table 1-1. Neuroprotective strategies for traumatic brain injury. Table of many different

neuroprotective strategies currently being investigated for the treatment of TBI.

20

1.4.3 NeuroprotectionThrough the Scavenging of Radical Species

One approach to neuroprotection through the pathway illustrated in Figure 1-1 is to design a

compound that chemically scavenges for the radical species produced by ONOO-. By offering a

more attractive compound as bait, one hopes to limit the destructive actions of the free radicals.

Two compounds have been previously designed to accomplish this: polyethylene glycol-

conjugated superoxide dismutase (PEG-SOD) and Tirilazad; however, both failed to provide any

therapeutic benefit following TBI (Hall et al., 2010). The reason for PEG-SOD’s failure lies

within the compound’s large size. Being a large protein, it was found to be unlikely to penetrate

the BBB and therefore its radical scavenging effects would have been only limited to the

microvasculature (Hall et al., 2010). Tirilazad, on the other hand, fared better than PEG-SOD. In

the laboratory, it was demonstrated to be effective in multiple animal models of TBI (Dimlich et

al., 1990; Hall et al., 1988; McIntosh et al., 1992) but once it progressed into the clinical

development phase, the results of Tirilazad failed to show a significant positive effect in

moderate or severe TBI patients (Marshall et al., 1998). It is unknown why Tirilazad’s

theurapeutic effects did not translate from the laboratory to the clinical setting. Perhaps its failure

lies in its extremely specific mechanism of action - preventing lipid peroxidation by targeting

only the lipid peroxyl radical (LOO) (Hall et al., 2010). As described earlier, the free radicals

derived from ONOO- not only peroxidize lipids (Radi et al., 1991), but can also nitrate proteins

(Beckham et al., 1992; Ischiropoulos et al., 1992) and fragment DNA (Fiskum et al., 2004; Salgo

et al., 1995a; Salgo et al., 1995b). Therefore, a more effective compound would have attributes

such as having a smaller size in order to cross the BBB and to target not one but many different

free radicals resulting from ONOO-.

21

1.5 Design of Tat-9c and Tat-9a

Peptide-based therapies have been steadily gaining in popularity. Peptides can be made cell-

permeant by fusing them to the cell-membrane transduction domain of the human

immunodeficiency virus-type 1 (HIV-1) Tat protein (Fawell et al., 1994). This approach has been

used to show neuroprotective effects of disrupting the nNOS/PSD-95 interaction in in vivo

models of acute cerebral ischemia (Aarts et al., 2002). Furthermore, it has been demonstrated

that tyrosine-containing peptides can successfully scavenge peroxynitrite-derived radicals and

protect motor neuron cultures from peroxynitrite-mediated cell death (Ye et al., 2007). However,

Alvarez et al. (1999) illustrated that cysteine residues are more reactive when compared to

tyrosine. When a derivative of cysteine, N-acetylcysteine (NAC), was examined in in vivo

studies, no gross histological benefits following TBI were observed (Thomale et al., 2006); yet,

NAC was able to protect against oxidative insults in vitro (Gow et al., 2000). Based on these

findings, Tat-9c, a cysteine-rich, cell-permeant, polypeptide composed of the tat protein

conjugated to nine cysteines was developed.

The Tat-9a peptide was also synthesized as a negative control for Tat-9c. Tat-9a is composed of

nine alanines attached to the tat protein.

22

1.6 Neurobehaviour Models of Assessing TBI Severity

Many neuromotor deficits such as difficulties with coordination, posture, and steadiness of

movement following TBI have been reported in humans (Schalen et al., 1994). The Beam-Walk

and Rota-Rod tests are often used to measure neuromotor deficits following TBI in animal

models.

1.6.1 Beam-Walk

The Beam-Walk is one of the most common techniques used to assess motor function following

FPI (Dixon et al., 1991). It is used to assess the more complex components of vestibulomotor

function and coordination that a simple beam-balance test cannot detect (Hamm et al., 1994). In

the Beam-Walk test, animals are required to traverse a suspended narrow beam and enter a goal

box at the end of the beam as illustrated in Figure 1-6. Motor function is assessed by measuring

the animal’s latency to enter the goal box at the end of the beam.

Figure 1-6 Representation of the Beam Walk Device. For the Beam-Walk test, animals are

required to traverse a suspended narrow beam and enter a goal box at the end of the beam. Motor

function is assessed by measuring the animal’s latency to enter the goal box at the end of the

beam.

23

1.6.2 Rota-Rod

The Rota-Rod task was first developed by Dunham et al. in 1957. The procedure required a rat to

maintain its balance on a rod rotating at a constant speed as illustrated in Figure 1-7. Jones et al.

increased the sensitivity of the task by gradually increasing the speed of the rod while it rotated

in 1968. Hamm et al. in 1994 was the first to apply it as a test for motor deficits following TBI.

Hamm et al., also found that the Rota-Rod test is a more sensitive and efficient test at detecting

injury severity produced by a TBI than the Beam-Walk and Beam-Balance tests.

Figure 1-7 Representation of the Rota-Rod Device. For the Rota-Rod test, animals are

required to maintain its balance on a rod rotating at a constant or gradually accelerating speed.

Motor function is assessed by measuring the animal’s latency to stay balanced on top of the

rotating rod.

24

1.6.3 Morris Water Maze

Cognitive performance in the laboratory is tested through a variety of hippocampal-dependant

spatial mazes. The Morris Water Maze (MWM) was designed to assess the cognitive processes

of spatial learning and working memory (D’Hooge et al., 2001; Morris et al., 1982). As

illustrated in Figure 1-8, the MWM consists of a large circular pool filled with opaque water. A

small escape platform is hidden beneath the water and during a number of training trials, animals

learn to find the location of the platform to escape from the water (Morris et al., 1982). Basic

MWM protocols include hidden-platform acquisition training and probe trial testing (D’Hooge et

al., 2001). Hidden-platform acquisition records how long it takes for an animal to find the hidden

platform however during a probe trial, the platform is removed and the trained animal is allowed

to swim for a fixed amount of time. A probe trial tests the spatial accuracy, or memory, of the

animal (D’Hooge et al., 2001). The MWM was the first cognitive test used with the FPI model

(Smith et al., 1991) and still remains the most frequent test to assess cognitive function after FPI.

25

Figure 1-8 Representation of the Morris Water Maze (MWM) testing room and apparatus.

The MWM consists of a large circular pool filled with opaque water. A small escape platform is

hidden beneath the water and during a number of training trials, animals learn to find the location

of the platform to escape from the water. Hidden-platform acquisition records how long it takes

for an animal to find the hidden platform however, in Day 1 of training, often the animal’s swim

path and duration is longer as shown above. If the animal is capable of learning following a

number of training trials, the swim path and duration taken to the platform should be lessened as

shown above in Day 6. During a probe trial, the platform is removed and the trained animal is

allowed to swim for a fixed amount of time. A probe trial tests the spatial accuracy, or memory,

of the animal to remember where the platform is located. If the animal has memory, it should

spend most of its time swimming in the correct quadrant as shown above in Day 7. The above

illustration is courtesy of Buccafusco, 2009.

26

Chapter 2 HYPOTHESIS AND GOALS

2.1 Research Goals

Despite significant research detailing various cell death pathways involved in secondary injury

following a TBI, no successful pharmacological therapies have been fashioned that consistently

attenuate neurological deficits caused by a TBI and/or in manner where the mechanism of drug

action is clear and defined (Jain, 2008).

However, past research has determined that peroxynitrite plays a key part in neuronal cell death.

Hence, our lab has developed a peptide, Tat-9c, which we believe will be a potent nytrosylation

target for peroxynitrite and potentially prevent neuronal cell death caused by peroxynitrite.

One way of testing new drugs is to examine TBI injured animals as a whole unit instead of

fragments of their neuronal structures. Thus, the aim of this study was to explore the efficacy of

Tat-9c by observing an animal’s gain or loss of function following TBI.

In this thesis, the following hypotheses were tested:

27

2.2 Hypothesis I

The Beam-Walk, Rota-Rod, and Morris Water Maze behaviour paradigms can

discriminate the severity of a TBI implemented through a FPI device.

Since the aim of this study was to explore the efficacy of Tat-9c by observing an animal’s gain or

loss of function following FPI, various neurobehaviour paradigms needed to be validated in

order to investigate their robustness at assessing injury severity and recovery. This hypothesis

was derived from previously published data supporting the Beam-Walk, Rota-Rod, and Morris

Water Maze as effective tests for TBI (Hallam et al., 2004; Hamm et al., 1994).

2.3 Hypothesis II

There will be significant increase in motor function following FPI in rats treated with Tat-

9c compared to Tat-9a or saline treated animals.

This hypothesis was postulated based on the findings of Lau et al., 2006 who showed that

ONOO- formation results in neuronal protein nitration and cell death and the following theory

that Tat-9c will be a potent nitrosylation target for peroxynitrite; thus, Tat-9c can potentially

prevent neuronal cell death leading to a decrease in motor performance.

2.4 Hypothesis III

There will be significant improvement in memory performance following FPI in rats

administered with Tat-9c compared to Tat-9a or saline treated animals.

This hypothesis was based on our findings from our second hypothesis. It is conceived that if

Tat-9c is acting in the same manner to restore motor function, it may play the same protective

role in the hippocampus where spatial learning and memory occurs.

28

Chapter 3 MATERIALS AND METHODS

All of the following animal experiments were approved by the University Health Network

Animal Resource Center, which is fully accredited by the Canadian Council on Animal Care.

3.1 Lateral Fluid Percussion Injury Model

The lateral FPI model was performed as described in Lau et al (2006).

3.1.1 Surgical Preparation

Male Sprague-Dawley rats weighing 350g – 375g were used in this study. 24 hours prior to

injury, rats were anesthetized using 2% isofluorane. The scalp of the rat was shaved and the

incision site was swabbed with alcohol to minimize infection. A sagittal incision was made from

the bregma to the lambda. Curved hemostats were placed to retract the overlying skin. Cotton

swabs were then used to separate the connective tissue as well as to clean the area of the exposed

skull. Using a dental drill, a small indent was made in the middle of the cranium flanked by the

bregma, the lambda, the sagittal suture, and the right temporal ridge (Figure 3-1). With the indent

as a guide, a 4.8mm craniotomy was made using a trephine. A modified Leur-loc was then

attached over the craniotomy and sealed into place using Acron MC/R dental acrylic (GC

America, Alsip, IL) and Krazy glue. Once the dental acrylic cement has hardened, the Leur-loc

was then filled with saline and a small piece of gelfoam was placed inside the Leur-loc to reduce

infection. The skin was sutured over the entire setup and the animal was returned to its cage

overnight.

3.1.2 Drug Injection

The following day, animals were anesthetized using 2% isofluorane and animal weights were

recorded. To substantiate a functional effect, we first tested Tat-9c as a pretreatment. This is a

common approach for testing therapies for injury as demonstrated by Shein et al, 2009; Di

Giorgio et al, 2008; Czeiter et al, 2009). Therefore 30 minutes prior to injury, either saline or

5nmol/g of Tat-9c or Tat-9a was administered via tail vein injection over 10 minutes. Once the

injection was complete, the animals were returned to their home cages to await another 20

29

minutes before TBI was induced. All injections were performed with the experimenter blind to

the drug injected. An assistant prepared the drugs in advance to the drug injection.

3.1.3 Fluid Percussion Injury

Before a TBI was produced, animals were again anesthetized with 2% isofluorane and the

incision site from the previous day was reopened. The piece of gelfoam inside the Leur-loc was

removed and the entire area was cleansed with saline. Prior to attaching the animal to the FPI

device (Custom Design and Fabrication, Richmond, VA), the Leur-loc was again filled with

saline. Once the animal was securely attached to the device via the Leur-loc (Figure 1-5), they

were subjected to a 2.4 atm moderate injury as described previously (McIntosh et al., 1989). A

pressure transducer attached to an oscilloscope recorded the magnitude of each injury (Figure 3-

2). Following injury, sometimes it was necessary to resuscitate an animal by chest compressions.

If the animal survived, the entire Leur-loc setup with dental acrylic was removed and the wound

was again sutured.

Figure 3-1 Dorsal Representation of a Rat Skull. A small indent was made in the

middle of the cranium flanked by the bregma, the lambda, the sagittal suture, and the right

temporal ridge. This indent is where the FPI device will be connected to the rat’s skull.

30

Figure 3-2. Representation of the magnitude of injury. A pressure transducer

attached to an oscilloscope records the magnitude of each injury. There are three severity levels

measured in standard atmospheric pressure (atm): mild (about 1.4 atm), moderate (about 2.7

atm), and severe (about 3.7 atm).

31

3.2 Treatment Preparation

3.2.1 Preparation of Tat-9c for Animal Injection

The Tat-9c peptide (YGRKKRRQRRRCCCCCCCCC) was synthesized at the Advanced Protein

Technology Centre (Hospital for Sick Children, Toronto, Ontario, Canada) by Dr. Nam-Chiang

Wang. The lyophilized form of the peptide was resuspended in deoxygenated normal saline

solution and the completely soluble solution was aliquoted under anoxic conditions. Aliquots

were stored at 4oC until use (5nmol/g).

3.2.2 Preparation of Tat-9a for Animal Injection

The Tat-9a peptide (YGRKKRRQRRRAAAAAAAAA) was also synthesized at the Advanced

Protein Technology Centre (Hospital for Sick Children, Toronto, Ontario, Canada) by Dr. Nam-

Chiang Wang. The lyophilized form of the peptide was resuspended in deoxygenated normal

saline solution and the completely soluble solution was aliquoted under anoxic conditions to

mirror the preparation of Tat-9c as much as possible. Tat-9a was designed to be a negative

control for Tat-9c. Aliquots were stored at 4oC until use (5nmol/g).

3.2.3 Preparation of Saline for Animal Injection

Saline solution was purchased from Baxter DIN 00786160.

3.2.4 Preparation of Sham Animals

Sham animals underwent surgical preparation for the FPI in the same manner as treatment

animals however they were not subjected to the injury. Sham animals were included in this study

to ensure that treatments such as anaesthesia or surgery will not affect the outcome parameters.

This is especially important as isoflurane, the anaesthetic approved in our protocol, has been

shown to exert some neuroprotection on the injured brain (Statler et al., 2006). Animals

remained under anesthetic during suturing and returned to their home cage to await behavioural

trials.

32

3.3 Implication of Various Behavioural Paradigms

Animals were evaluated on motor or cognitive function immediately after injury and for up to 15

days post-injury depending on the various behaviour models utilized. All the following

behaviour experiments were performed with the experimenter blinded to the treatment that the

animals received.

3.3.1 Beam-Walk Model

The Beam-Walk model consists of a 90cm x 2.5cm wooden beam suspended 60cm above a

padded table in a dark room. At one end of the beam, negative stimulus was provided through

white noise and bright light (60W light bulb). At the other end of the beam, a positive

environment was supplied by a dark cardboard box. Prior to injury, animals were trained on the

Beam-Walk by placing them at the end with the negative stimulus. Once the animals

successfully traversed the beam within 10 seconds over 3 consecutive trials, they were deemed

trained and returned to their cages. Following surgical preparation and before injury, animals

were tested on the Beam-Walk to determine their baseline values. The average latency of 3 trials

was recorded. Any animal that had difficulty crossing the beam was discarded from the

experiment as it would signify that an unintentional injury has occurred during the surgical

preparation before a FPI was given. Once an animal has been subjected to a FPI, the average

latency of 3 trials was recorded for various time points following the injury. If an animal could

not traverse the beam and fell off, they were given an automatic 60 seconds.

3.3.2 Rota-Rod Model

The Rota-Rod model consists of a rotating rod suspended 50cm over weight sensitive planks that

stop recording time if a rat falls on it. Before injury, rats were acclimatized to the setup and

trained to stay on the rotating rod for at least 2 minutes. The speed of the rod started at 2rpm and

was steadily increased by 3rpm in 10 second intervals until the animal fell completely off the

rungs of the rod. Following surgical preparation and before injury, animals were tested on the

Rota-Rod to determine their baseline values. Any animal that had difficulty staying on the

rotating beam for less than 30 seconds was discarded from the experiment as it would signify

33

that an unintentional injury has occurred during the surgical preparation before a FPI was given.

Once an animal has been subjected to a FPI, the mean duration of 3 trials per day each rat spent

on the rotating rod was recorded for various time points following the injury.

3.3.3 Morris Water Maze Model

This test was performed as described previously (Hallam et al., 2004). The water maze task was

used to measure spatial reference memory 11-15 days post-injury allowing animals who were

given a TBI enough time to recover their motor deficits in order to swim. Water temperature

levels were kept between 23-25C. Non-toxic blue paint (made by Gothic Tempera and

purchased at various art stores) was diluted into the pool until the platform was not visible.

Visual cues in different shapes (circles, rectangles, symbols, etc.) were cut out of cardboard

paper and foam material and placed around the pool in plain sight of the animal. Animals were

tested 4 trials each day for 5 consecutive days to find a hidden platform in 120 seconds. As soon

as the animal has found the platform, time was stopped. The swim path, swim speed, and latency

to find the hidden platform were measured using a computer controlled tracking system

(SMART, San Diego Instruments, San Diego, CA). If an animal was unable to find the platform

within 120 seconds, the experimenter would lead the animal towards it and allow it to stay on the

platform for an extra 30 seconds before returning it to its cage where they were thoroughly dried.

On the last day, 15 days post-injury, a final probe trial was also conducted where the platform

was removed. The rat was placed inside the water maze and allowed to swim freely for a total of

120 seconds. From the probe test, the percent of time the animal spent in the correct quadrant

was measured and recorded.

3.4 Statistical Analysis

All data were expressed as mean ± SD, except where noted. Data analysis was performed using

SigmaStat3.0; SPSS, Chicago, IL. Behaviour data for each dependent variable were analyzed

with repeated measures ANOVAs. Statistical significance was considered at the p < 0.05 level.

34

Chapter 4 HYPOTHESIS AND GOALS

4.1 Validation of Behaviour Assays Utilized to Assess Severity of TBI

To assess the impact of Tat-9c on neurobehavioural outcome following TBI, we used the Beam-

Walk, Rota-Rod, and the Morris Water Maze, which have all been previously demonstrated to

effectively measure TBI severity as mentioned above. Although the success of these behaviour

assays is well documented, it was still essential to verify whether these assays were able to

reflect the impairments caused by the TBI in this study.

4.1.1 Beam-Walk Model

The Beam-Walk model was first assessed using male Sprague-Dawley rats. All animals were

trained 24 hours before surgical preparation and tested for baseline results between the surgical

preparation and the injury time point. TBI rats were subjected to a moderate 2.4atm FPI. At 1, 2,

3, and 7 days post-injury, Beam-Walk performance was assessed (Figure 4-1). Experiment data

was collected up to one week following TBI since the Beam-Walk becomes insensitive to motor

deficits after one week. Results showed a significant difference between TBI and sham groups at

all time points post-injury which is consistent with previous literature (Hamm et al., 1994). TBI

injured animals required on average seven times longer than sham animals to traverse the beam

24 hours following TBI and also demonstrated a gradual trend towards behavioural improvement

throughout the experiment. These results suggest that the Beam-Walk is an effective model to

assess severity of TBI but only up to one week post-injury.

35

Figure 4-1 Beam-walk motor function is impaired after TBI. Rats were trained 24

hours before surgical preparation and tested for baseline results between the surgical preparation

and the injury time point. A moderate 2.4atm FPI was used to induce TBI. There was a

significant difference between TBI and sham groups at all time points post-injury. Data are

expressed as mean ± SD and analyzed using repeated measures ANOVA (Sham vs TBI group)

(SigmaStat3.0; SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

36

4.1.2 Rota-Rod Model

The Rota-Rod model was tested in conjunction with the Beam-Walk model using the same group

of 10 male Sprague-Dawley rats. All animals were trained 24 hours before surgical preparation

and tested for baseline results between the surgical preparation and the injury time point. TBI

rats were subjected to a moderate 2.4atm FPI. At 1, 2, 3, and 7 days post-injury, Rota-Rod

performance was assessed (Figure 4-2). These time points were used to coincide with the Beam-

Walk experiments. Results showed a significant difference between TBI and sham groups at all

time points post-injury. TBI injured animals showed greatest motor deficits (about 70%

decrease) 24 hours following TBI which was then preceded by a gradual trend towards

behavioural improvement throughout the experiment. These findings also suggest that the Rota-

Rod is a more sensitive index for assessing motor impairment as the rate of motor recovery is

slower than the Beam-Walk. This also is consistent with previous research comparing the Beam-

Walk and Rota-Rod models (Hamm et al., 1994). The greatest differences between TBI and

sham animals were seen in the first three days post-injury. Hence, it was decided that future

experiments involving the Beam-Walk and Rota-Rod would only be tested up to 72 hours post-

injury.

37

Figure 4-2 Rota-Rod motor function is impaired after TBI. All animals were

trained 24 hours prior to surgical preparation. TBI rats were subjected to a moderate 2.4atm FPI.

There was a significant difference between TBI and sham groups at all time points post-injury.

Data are expressed as mean ± SD and analyzed using repeated measures ANOVA (Sham vs TBI

group) (SigmaStat3.0; SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05

level.

38

4.1.3 Morris Water Maze Model

The Morris Water Maze Model was used to assess cognitive defects caused by TBI. The TBI

group was subjected to a moderate 2.4atm FPI and 11 days post-injury, all animals were

introduced to the Morris Water Maze. Results showed there was no significant difference

between TBI and sham groups at all time points when measuring latency to platform (Figure 4-3)

which is consistent with previous literature (Hallam et al., 2004; Reid et. al. 2008). After just

four days, all animals were able to learn the location of the hidden platform. The average swim

speed (Figure 4-4) and total distance traveled (Figure 4-5) were also not significant between the

two groups indicating that 11 days post-injury is an adequate time point to start the behaviour

experiment as the injured animals have recovered their motor function sufficiently enough to

swim. Furthermore, a final probe test was performed 15 days post-injury. Results indicate that

TBI injured animals performed significantly lower than sham animals in the memory test (Figure

4-6). In all, the results confirm that the Morris Water Maze is a practical test for showing

deficiencies in memory impairment following TBI.

39

Figure 4-3 Learning ability was not affected following TBI. TBI rats were

subjected to a moderate 2.4atm FPI while sham rats underwent identical procedure without the

injury given. Animals were tested using the Morris Water Maze at day 11 post-injury. There was

no significant difference in latency to platform between TBI and sham groups. Data are

expressed as mean ± SD and analyzed using repeated measures ANOVA (Sham vs TBI group)

(SigmaStat3.0; SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

40

Figure 4-4 Swim speed was not affected following TBI. TBI rats were subjected to a

moderate 2.4atm FPI while sham rats underwent identical procedure without the injury given.

Animals were tested using the Morris Water Maze at day 11 post-injury. There was no

significant difference in the average swim speed between the two groups demonstrating that the

TBI rats have recovered their motor function sufficiently enough to swim. Data are expressed as

mean ± SD and analyzed using repeated measures ANOVA (Sham vs TBI group) (SigmaStat3.0;

SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

41

Figure 4-5 Total distance traveled was not affected following TBI. TBI rats were

subjected to a moderate 2.4atm FPI while sham rats underwent identical procedure without the

injury given. Animals were tested using the Morris Water Maze at day 11 post-injury. There was

no significant difference in total distance traveled between TBI rats and sham rats, which further

signify that the TBI rats have recovered their motor function sufficiently enough to swim. Data

are expressed as mean ± SD and analyzed using repeated measures ANOVA (Sham vs TBI

group) (SigmaStat3.0; SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05

level.d’h

42

Figure 4-6 Spatial memory is impaired after TBI. The Morris Water Maze was used

to assess spatial memory. TBI rats were subjected to a moderate 2.4atm FPI while sham rats

underwent identical procedure without the injury given. A probe test was performed 15 days

post-injury. There was a significant reduction in the percent time spent in the correct quadrant by

TBI injured animals compared to sham animals. Data are expressed as mean ± SD and analyzed

using T-test. Statistical significance was considered at the P < 0.05 level.

43

4.2 Effects of Tat-9c on Motor Behaviour Function Following TBI

To examine Tat-9c’s properties, a larger study using 20 male Sprague-Dawley rats was

conducted to further characterize behavioural differences between the treatment groups. Rats

were treated with either 5nmol/g Tat-9c, Tat-9a, or saline 30min prior to FPI. At 1, 2, and 3 days

post-injury, Beam-Walk and Rota-Rod performance was assessed. The Beam-Walk results

showed a trend towards behavioural improvement with Tat-9c but only demonstrated statistical

significance at the 24 hours post-injury time point (Figure 4-7). However, overall, Tat-9c greatly

reduced the number of falls on the Beam-Walk when compared to other treatments (Figure 4-8).

On the other hand, the Rota-Rod assay surprisingly did not find any statistical differences

between Tat-9c and the other treatment groups at all time points following TBI (Figure 4-9).

Possible explanations for this are considered in the Discussion Section: 5.2 on pg 67.

44

Figure 4-7 Beam-walk motor deficits are reduced in Tat-9c treated animals.

Rats were treated with 5nmol/g Tat-9c, Tat-9a, or saline 30min prior to induction of TBI. There

was a significant reduction in time to cross beam after 24 hours for Tat-9c. However, no

significant differences were found at later time points. Data are expressed as mean ± SD and

analyzed using repeated measures ANOVA (Tat-9c vs Tat-9a; Tat-9c vs Saline) (SigmaStat3.0;

SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

45

Figure 4-8 Number of falls is reduced in Tat-9c treated rats following TBI.

Rats treated with Tat-9c did not fall during the beam walk test whereas Tat-9a and saline treated

rats did fall.

46

Figure 4-9 No improvement in Rota-Rod performance by Tat-9c treated rats.

There were no significant differences in time spent on rod between Tat-9c and the other

treatment groups at all time points following TBI. Data are expressed as mean ± SD and

analyzed using repeated measures ANOVA (Tat-9c vs Tat-9a; Tat-9c vs Saline) (SigmaStat3.0;

SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

47

4.3 Effects of Tat-9c on Memory Function Following TBI

A further study involving 89 male Sprague-Dawley rats was performed to examine cognitive

behavioural differences between the treatment groups: sham control, saline control, Tat-9a, and

Tat-9c. Results show no significant differences in rate of learning for Tat-9c treated animals

compared to all other groups during testing days 11-15 post injury (Figure 4-10). According to

Fig. 4-11, there was also no statistical differences between Tat-9c treated animals and sham

animals in the memory probe test while Tat-9a and saline treated animals performed significantly

poorer than sham in the probe test (Figure 4-11). However, by comparing Tat-9c, Tat-9a, and

saline to each other, there were also no statistical differences. These ambiguous results are

reviewed in the Discussion Section: 5.4 on pg 70-72.

48

Figure 4-10 Rate of learning in Tat-9c rats was no different to any other

group. There were no significant differences in the time to platform for Tat-9c treated animals

compared to all other groups at all days tested. Data are expressed as mean ± SD and analyzed

using repeated measures ANOVA (Tat-9c vs Tat-9a; Tat-9c vs Saline; Tat-9c vs Sham)

(SigmaStat3.0; SPSS, Chicago, IL). Statistical significance was considered at the P < 0.05 level.

49

Figure 4-11 Effects of Tat-9c on spatial memory. The Morris Water Maze was used to

assess spatial memory. There was no significant differences in time spent in correct quadrant

between Tat-9c treated animals and sham animals. However, Tat-9c rats did not perform better

than Tat-9a or saline treated animals as well. Data are expressed as mean ± SD and analyzed

using T-test (Sham vs Tat-9c; Sham vs Saline; Sham vs Tat-9a) (SigmaStat3.0; SPSS, Chicago,

IL). Statistical significance was considered at the P < 0.05 level.

50

Chapter 5 DISCUSSION

5.1 Behaviour Assays Utilized to Assess Severity of TBI

In order to assess severity of TBI, three assays were tested: Beam-Walk, Rota-Rod, and Morris

Water Maze. All three assays were able to significantly discriminate injured animals from sham.

It was important to validate the Beam-Walk task as it was modified and consists of components

based from the original model created by Feeney et al., 1982 and the one used by Hallam et al.,

2004 who designed the Beam-Walk with four pegs as barriers. Another reason why it was

important to test the Beam-Walk and Rota-Rod test was that the methods of this study requires

the same animals to be trained and tested on the Beam-Walk and Rota-Rod together. This has not

been done previously and it was necessary to observe if there was any interference of the training

of one task with another present. The process of forgetting has long been observed and studied

and the standard school of thought is that it is caused by interference. Interference can be

proactive (forgetting caused by prior learning) or retroactive (forgetting caused by subsequent

learning) (Wixted, 2004). Because of the interference phenomenon, we needed to verify that

failure to perform any of the behavior tasks was due to the injury and not to interference.

Luckily, Figures 4-1 and 4-2 were consistent with other findings which verify that interference

did not play a role in our experiments. Another important feature in our Beam-Walk and Rota-

Rod tests was the addition of baseline testing for all animals. This allowed for the use of each

animal to be their own control and also assisted in detecting any animals that were

unintentionally injured prior to TBI. The Beam-Walk and Rota-Rod results were consistent with

finding by Hamm et al., 1994 where data demonstrated a significant difference in motor function

between TBI and sham injured animals. It was also similar in that performance of injured

animals improved gradually to baseline values throughout the time period confirming that the

Beam-Walk and Rota-Rod tasks are only effective at assessing TBI severity one week post-

injury.

The Morris Water Maze test was included in this study to investigate the cognitive function of

animals following TBI. The Morris Water Maze specifically measures new acquisition of a

spatial reference memory task. The extensive memory deficits observed by the injured group is

similar to previous studies (Smith et al., 1991; and Hallam et al., 2004), which found that TBI

51

can cause deficits in spatial memory. TBI animals appeared to be able to retain the ability to

learn. This is also consistent with reports from Reid et. al. 2008 who showed that the learning

curve for TBI and sham animals was not significantly different.

5.2 Tat-9c Protects Motor Behaviour Function Following TBI

Tat-9c appears to improve motor function in rats following TBI in the Beam-Walk study but

only at the 24 hours post-injury time point. Changes were most pronounced on the Beam-Walk

task where complex coordination with cognitive components was required to successfully

traverse the beam. Not only did the time latency improve in the Tat-9c treated group at the 24

hours time point but there was also a decreased incidence of falls from the beam at all time

points. However, it is difficult to judge the effects of Tat-9c beyond 24 hours post-injury since

the animals administered with negative controls, Tat-9a and saline, had also quickly recovered

their motor function to the point where Tat-9a animals performed identically to their baseline

values after the 48 hours post-injury time point. It was surprising to see that Tat-9a animals

showed such quick signs of recovery as Tat-9a was meant to be a negative control and should

have no positive effects. This phenomenon may be due to the tat sequence alone as it was first

discovered by Vaslin et al in 2009 that unconjugated tat demonstrated a neuroprotective effect

against mild NMDA excitotoxicity however the underlying mechanisms of Tat’s interference

with the NMDAR signaling is still unknown.

Furthermore, it was interesting to find no significant differences in the Rota-Rod test between

treatment groups. As the Rota-Rod test is described to be more sensitive and proficient at

assessing motor deficits following FPI (Hamm et al., 1994), it can be interpreted that Tat-9c was

unable to recover finer motor function required to perform the Rota-Rod test and not the Beam-

Walk test. Thus, while Tat-9c was able to rescue Beam-Walk deficits, subtler motor impairments

likely persisted and these were demonstrated using the more sensitive Rota-Rod assay. Another

explanation for the negative results could be that Tat-9c’s therapeutic effects were not distributed

evenly in the brain however no histology was done to examine this. A future experiment to

explore this would be to tag tat-9c with a fluorescent tag in order to determine its distribution

within the brain. It has been reported that locomotion induces activation of the sensorimotor

cortex however when accelerating, the prefrontal cortex and premotor cortex also becomes active

(Suzuki et al., 2004). Since the Rota-Rod test involves an accelerating rotating rod, different

52

areas of the brain were required compared to the Beam-Walk; therefore, the results could suggest

that Tat-9c plays a stronger role in different areas of the brain.

5.3 Effects of Tat-9c on Memory Function Following TBI

Tat-9c also appears to improve cognitive function in rats following TBI in the Morris Water

Maze study but only in the probe test for memory. Tat-9c had no effect on learning which is

what we expected since Tat-9c was not designed to improve existing cognitive function but to

protect against functional decline due to injury. Since TBI has already been shown to have no

effect on learning (Reid et al, 2008), we did not expect Tat-9c to play any effect either. The

results from the probe test revealed that there was no statistical differences between Tat-9c

treated animals and sham animals while Tat-9a and saline treated animals performed

significantly poorer than sham in the probe test. There were no significant differences between

the Tat-9c and Tat-9a or saline groups. Explanations for this are described below under

Limitations of the Study. Nonetheless, these results show that Tat-9c can protect memory

function following TBI but more subjects are required to be assured.

5.4 Limitations of the Study

5.4.1 Disadvantages of the FPI Model

One concern is the variability of injury presented by the lateral fluid percussion model. The FPI

model has been demonstrated to replicate many of the features associated with human TBI

(McIntosh et al., 1989, Pierce et al., 1998, Statler et al., 2001, Morales et al., 2005, Thompson et

al., 2005) and it is currently the most commonly used experimental model of TBI (Frey et al.,

2009). However, investigators utilizing this model are often confronted with two main

disadvantages: the model’s sensitivity to individual operators and its sensitivity to slight

differences in the piston’s o-rings and cylinder (Frey et al., 2009). It is absolutely necessary for

the operator to skillfully position and release the pendulum consistently throughout an

experiment.

53

5.4.2 Limitations of Tat-9c

The mechanism of action of Tat-9c provides the drug with a very limited and short therapeutic

window. The drug would need to be administered rapidly following TBI to provide even a

chance of interfering with the initial up regulation of free radical production which occurs in the

first 60 minutes post trauma (Hall et al., 2010). The attractiveness of Tat-9c as a potential

therapy for TBI is diminished due to its clinical impracticality however, when there are no

alternative therapies at present time, limited is still better than nothing at all.

5.4.3 The Complex nature of TBI

5.4.3.1 Cell Death Pathway Targets

Another reason for the lack of successful pharmacological intervention developed for TBI may

be that many proposed therapies have a mechanism of only individual specific targets on the cell

death pathway. It is becoming increasingly evident that various cell death pathways do not exist

independently of each other, and two or more of these pathways are likely active following TBI.

These interactions may either be synergistic where they assist each other in exacerbating cell

death or competitive, where they slow the progression of cell death. It is difficult to observe

these interactions in in vivo experiments however there is evidence from in-vitro studies

suggesting that these interactions may play a large role in determining the efficacy of

neuroprotective drugs (Pike et al., 2000; Mohr et al., 1997; Guttmann et al., 2001). Therefore,

designing a drug that only targets a small portion of the cell death pathways following TBI such

as Tat-9c may be limiting its therapeutic abilities and hence the observed efficacy of the drug

was not as robust as expected.

5.4.3.2 Limitations and Variability Amongst Subjects

Rodents have become the most widely experimented subject for examining TBI within the

laboratory. They are easy to handle individually while carrying out the surgery and also simple to

utilize in large numbers. The costs are relatively low compared to larger mammals and with the

availability of knockout animals, desired genes can be genetically ablated (or overexpressed with

the use of transgenic mice), making the study of a certain gene or protein of interest possible in

rodents (Morganti-Kossmann et al. 2010). However, no matter how severe a TBI can be induced

54

in rodents, seldom will they enter a state of coma that is often seen with TBI patients clinically

(Morganti-Kossmann et al. 2010). This represents one limitation to the use of rodent models as

severe disabilities of TBI in humans will not display in the rodent and any potential therapies

discovered to alleviate severe symptoms in the rodent will not translate to humans.

Another limitation is that responses to TBI varies from species to species and even within the

same strain of rodent, individual rodents have been shown to behave differently in neurological

motor and cognitive testing (Morganti-Kossmann et al. 2010) and thereby skewing any

measurements of potential therapies.

55

5.5 Future Directions

Although this study provided a crucial first step in the investigation of Tat-9c as a possible drug

therapy for TBI through in vivo models, there is still much more research that needs to be

accomplished.

The biggest question that needs to be resolved is the mechanism by which Tat-9c provides

neuroprotection. The initial hypothesized mechanism of action of Tat-9c is that it acts as an

intracellular scavenger reacting with peroxynitrite derived free radicals. However, there can be

other scenarios in which Tat-9c is attenuating behavioural deficits. For example, since Tat-9c

was designed as a polypeptide, it avoids a number of potential neurotoxic mechanisms attributed

to L-cysteine or homocysteine, which as a single amino acid, can alter NMDARs and inhibit

glutamate uptake in astrocytes (Janaky et. al., 2000).

The present study could also be enhanced by investigating the therapeutic window of Tat-9c.

Although Tat-9c showed promise when injected as a single bolus pre-injury, this application is

not very useful clinically. It is clear from the Beam-Walk results that Tat-9c’s therapeutic

potential can come in effect as early as 24 hours after TBI but it is unknown if Tat-9c can still

provide benefits when given post-injury. For example, the protection provided by Tat-9c may

simply be a result of the very efficient ROS scavenging at the time of impact. An ideal future

experiment would be to test Tat-9c’s effectiveness each hour post TBI and determine the latest

time-point it can be administered and still attenuate behavioural deficits.

In addition, as the pathophysiology of TBI is multi-faceted and complex, the combination of

other potential drug therapies with Tat-9c can also be assessed. For instance, if Tat-9c is used in

combination with anti-apoptotic drugs, the net beneficial effect may be potentiated.

Furthermore, due to the variability of disabilities seen amongst individual animals subjected to

the uniform experimental protocol, perhaps a better way to approach testing for potential drug

therapies is to first categorize the severity of injury AFTER the injury has occurred and then test

and compare the severity groups amongst each other. For example, following application of

injury, animals can then be divided between those who tolerated the infarct relatively well

compared to those who may have required chest compressions. Perhaps the results of Tat-9c

would have been more dramatic in one group over the other but if combined together, as

56

performed in this study, the results would have dampened and this may also help explain the

large error bars.

Overall, initial experiments with Tat-9c show that it appears to be a potential candidate for a TBI

drug but further experiments are required to confirm its mechanism of action and define its

therapeutic limits.

57

Chapter 6 CONCLUSIONS

One of the major findings of this study includes the validation of various behaviour paradigms to

assess severity of TBI. Without this step, further evaluation of potential drug therapies for TBI

would not be as thorough. Three assays were tested: Beam-Walk, Rota-Rod, and Morris Water

Maze. All three models were able to significantly discriminate injured animals from sham. The

Beam-Walk and Rota-Rod assays illustrated that due to innate recovery, these models were best

utilized up to one week post-injury. These findings are consistent with previous research

presented by Hamm et al (1994). The Morris Water Maze results are also consistent with those

found by Reid et al (2008) where it is demonstrated that TBI has no effect on spatial learning but

plays a crucial role in memory. Once the behaviour methodology was established, evaluation of

Tat-9c as a potential drug therapy for TBI was performed. Overall, the results demonstrate that

Tat-9c has the potential to provide early and late behavioural protection following TBI. The

Beam-Walk model highlighted the early beneficial effects of Tat-9c where the motor function of

those animals administered with Tat-9c was significantly superior than those given saline or Tat-

9a. Moreover, in the Morris Water Maze test, there was no statistical difference between Tat-9c

treated animals and sham animals in the memory probe test while Tat-9a and saline treated

animals performed significantly poorer than sham suggesting that Tat-9c can still provide late

behavioural protection as much as 15 days post-injury. These significant findings demonstrate

that TAT-9c may be a potential therapy for improving motor function and memory following

TBI.

58

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