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Increasing functional recovery after a cervical spinal cord injury Plunet, Ward Thomas 2006

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INCREASING F U N C T I O N A L R E C O V E R Y A F T E R A C E R V I C A L SPINAL CORD INJURY by WARD THOMAS PLUNET B.Sc, The University of Victoria, 1997  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Neuroscience)  T H E UNIVERSITY OF BRITISH C O L U M B I A October 2006  © Ward Thomas Plunet, 2006  Abstract Spinal cord injury (SCI) in adult mammals results in a loss of function with minimal spontaneous recovery due to secondary damage, the inhibitory nature of the injured adult CNS, and the weak intrinsic axonal growth response of injured neurons. In an attempt to increase the weak growth response I tested if pretreatment of rubrospinal neurons with brain-derived neurotrophic factor (BDNF) one week prior to axotomy or at time of injury would produce functional improvements. Both treatments reduced cell body atrophy and dieback of rubrospinal axons, indicating a cell body response was elicited. However, neither BDNF pretreatment nor acute treatment promoted rubrospinal tract regeneration. In addition, the two-fold increase in rubrospinal tract sprouting into the gray matter rostral to the lesion site did not reach significance. Despite the lack of rubrospinal regeneration, B D N F pretreatment improved functional recovery while BDNF-acute treatment had no effect. The BDNF-treated animals had reduced weight gain and BDNF infusion into the brain has been shown to inhibit food intake. Reduced food intake (dietary restriction) is known to extend lifespan since the 1930s but was recently found to be neuroprotective. I explored the possibility that the improved recovery in the BDNF pretreatment animals was mediated via dietary restriction by testing whether everyother-day fasting (EODF) started one month prior to SCI and continued in the post-injury testing period would also prove effective. EODF animals displayed improved functional recovery, reduced GFAP immunostaining, smaller lesion sizes, and greater numbers of healthy neurons surrounding the lesion area. To test a more clinically relevant treatment I next examined if E O D F would promote recovery if initiated after a SCI. EODF promoted better recovery on three independent behavioral tests. Lesion size was dramatically reduced and there was an increase of surviving neurons in the EODF animals. Additionally, there was an increase.of corticospinal tract sprouting proximal and distal to the lesion site. The expression of trkB (receptor for BDNF) was changed both at, and distal to, the lesion site, which could play a role in both the neuroprotection and increased plasticity observed.  Table of Contents Abstract Table of Contents List of Tables List of Figures List of Symbols, Nomenclature, and Abbreviations Acknowledgements Statement of Contributions  Chapter 1 General Introduction  ii iiii vi vii viii x xii  1  1.1. Overview 2 1.2. Background 3 1.2. A. When does axonal regeneration and return of function occur? 3 1.3. Three main reasons why adult mammalian CNS fails to functionally recover after spinal cord injury and the need for combinatorial approaches 5 1.3. A. Secondary damage 6 I.3.A./. Lesion size correlates with behavior 8 1.3.A.ZZ. Progressive increase in lesion size 9 1.3.A.m. Current neuroprotective treatments 9 I.3.B. The adult mammalian CNS is inhibitory to axonal growth 10 1.3.B.L Glial scar 11 I.3.B.z'z. Adult mammalian CNS myelin is inhibitory to axonal growth 12 1.3. C. The weak intrinsic axonal growth response of CNS neurons 15 L3.Cz'. Neurotrophins 15 1.3.C.z'z'. Differences in intrinsic cell body response 19 1.4. Overcoming CNS inhibitory molecules by increasing the intrinsic cell body response.... 23 1.4. A. Conditioning lesion 23 I.4.B. Cell body priming of neurons to increase the intrinsic growth response 23 1.4.C Priming supraspinal descending neurons in vivo with growth factors 25 1.4. D. Side effects of priming neurons with neurotrophins 25 1.5. Rationale of pretreatment studies 27 1.5. A. Exercise 27 1.5. B. Dietary restriction 28 1.6. Dietary restriction has diverse effects on the biological system 29 1.6. A. Dietary restrictions increases lifespan 29 1.6.B. How dietary restriction works 30 1.6.C What are the positive effects of dietary restriction on diseases and leading causes of death? 31 I.6.D. Dietary restriction/Every-other-day fasting will not make you weak and frail 33 1.6. E. Effect of dietary restriction on the central nervous system 35 1.7. Dietary restiction may improve the outcome of spinal cord injuries via multiple mechanisms 38 1.7. A. Dietary restriction may reduce secondary damage and replace lost cells 38 I.7.B. Dietary restriction may reduce the inhibitory to axonal growth properties of the adult CNS myelin/glial scar 44 I.7.C. Dietary restriction may stimulate the intrinsic response of injured neurons after SCI 46 iii  1.8. Further considerations how dietary restriction might be beneficial after SCI 1.8.A. Short-term dietary restriction can produce molecular changes I.8.B. Calorie restriction or every-other-day fasting? 1.8. C. Human dietary restriction/every-other-day fasting studies 1.9. Basis of experimental paradigm 1.9. A. What do SCI patients want? I.9.B. What SCI model to study? I.9.C. Lesion Model 1.10. Chapter overviews and hypotheses 1.11. References  49 49 50 50 53 53 53 54 57 62  Chapter 2 BDNF acute or pretreatment of rat rubrospinal neurons does not promote regeneration or sprouting but pretreatment promotes functional recovery after a cervical spinal cord injury 89 2.1. 2.2. 2.3. 2.4. 2.5.  Introduction Material and Methods Results Discussion References  90 92 95 98 112  Chapter 3 Dietary restriction is neuroprotective and promotes functional recovery from cervical spinal cord injury 115 3.1. 3.2. 3.3. 3.4. 3.5.  Introduction Materials and Methods Results Discussion References  Chapter 4 Dietary restriction implemented after cervical spinal cord injury is neuroprotective, promotes plasticity, and improves functional outcome 4.1. 4.2. 4.3. 4.4. 4.5.  Introduction Materials and Methods Results Discussion References  Chapter 5 Conclusions and future directions  116 118 121 124 135  138 139 140 145 149 170  174  5.1. Summary of thesis 175 5.2. Strengths and Weaknesses 177 5.3. How the chapters of this thesis are related 180 5.4. How this thesis relates to the literature 181 5.4. A. Cell body neurotrophin treatment and pretreatment/priming of neurons for increased axonal growth 181 5.4.B. Complicating effects of exogenously applied B D N F in SCI 182 5.4.C. Need for combinatorial treatments and possible complications 183 5.4.D. Problems and complications with current neuroprotective treatments 184 5.4.E. Previous work on dietary manipulation after SCI 185 5.4.F. Previous research on CR/EODF effect on neuroprotection and plasticity 186 5.4.G. Choices of behavioral tests in this thesis and implications of this thesis for neurotrauma behavioral testing 187 iv  5.4. H. Possible role of trkB on CST sprouting in EODF animals 5.5. Possible negative and positive consequences of CR/EODF on SCI patients 5.5. A. Possible negative consequences of CR/EODF on SCI patients 5.5.B. Possible additional positive benefits of CR/EODF for SCI patients 5.6. Future experiments 5.7. Final thoughts 5.8. References  188 191 191 192 198 202 203  v  List of Tables Table 1.1. Effect of CR/EODF on the top 10 leading causes of death Table 4.1. Correlations of morphology with behavior  59 160  vi  List of Figures  Figure 1.1. Cross section of rat cervical spinal cord Figure 2.1. Experimental timeline and weekly weight gain of animals  60 102  Figure 2.2. BDNF pretreatment increased ipsilateral forelimb usage in the Schallert cylinder task  104  Figure 2.3. BDNF-acute and BDNF pretreatment rescues red nucleus cell size  106  Figure 2.4. BDNF-acute and BDNF pretreatment reduced rubrospinal dieback  108  Figure 2.5. BDNF-acute or BDNF pretreatment fails to promote rubrospinal tract sprouting in the gray matter rostral to the lesion site  110  Figure 3.1. Rats on EODF gained weight, but at a slower rate than control  127  Figure 3.2. EODF increased the number of NeuN-positive neurons in the penumbra of the lesion  129  Figure 3.3. EODF reduced lesion size and GFAP immunoreactivity  131  Figure 3.4. EODF improved ipsilateral forelimb usage after a cervical spinal cord injury.... 133 Figure 4.1. EODF increased functional recovery after cervical spinal cord injury  152  Figure 4.2. EODF reduced lesion size and protected neurons after a cervical spinal cord injury 154 Figure 4.3. EODF increased corticospinal axon total length rostral and caudal to the lesion site 156 Figure 4.4. EODF increased the trkB to trkB ratio  158  Figure S4:1. EODF animals continued to gain weight and were not overactive  162  Figure S4.2. Control animals displayed a decline in ipisilateral forelimb usage  164  Figure S4.3. CST branching did not differ between groups at C2  166  Figure S4.4. BDNF protein expression did not differ between groups  168  fl  tc  vii  List of Symbols, Nomenclature, and Abbreviations 3NP AD AMI 0 ATP BAD BDNF ChABC CJDS CNS GAG CR CREB CRP CSPG CST DAG DR DRG DREZ DSPG ECM EGFR eNOS EODF EPO GAP-43 GDI GDI1 GDNF GFAP GRP-78 GSH HSP-70 HSPG IF IGF-1 IP3 KSPG Lingo-1 LPS LTP MAG MCAO-R MODS MP MPTP NGF NT3  -  3-nitropropionic acid A D A M metallopeptidase domain 10 adenosine triphosphate Bcl-xL/Bcl-2-Associated Death Promoter brain-derived neurotrophic factor Chondroitinase A B C CNS injury-induced immune deficiency central nervous system glycosaminoglycan calorie restriction cAMP response element binding protein C-reactive protein chondroitin sulphate proteoglycans corticospinal tract diacylglycerol dietary restriction dorsal root ganglion dorsal root entry zone dermatan sulphate proteoglycan extracellular matrix epidermal growth factor receptor epithelial Nitric Oxide Synthase every-other-day fasting erythropoietin growth associated protein guanine dissociation inhibitor guanosine diphosphate dissociation inhibitor 1 glial derived neurotrophic factor glial fibrillary acidic protein glucose-regulated protein 78 glutathione heat-shock protein-70 heparan sulphate proteoglycan intermittent fasting insulin-like growth factor 1 inositol-l,4,5-triphosphate keratan sulphate proteoglycan LRR and Ig domain containing No go receptor interacting protein lipopolysaccharide long-term potentiation myelin-associated glycoprotein middle cerebral artery occlusion-reperfusion multiple organ dysfunction syndrome methylprednisolone l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine nerve growth factor neurotrophin 3 Vlll  NT4  - neurotrophin 4 - nuclear factor K B - oligodendroycte myelin glycoprotein OMgp p75 - p75 neurotrophin receptor - preconditioning PC - peripheral nerve graft PNG - peripheral nervous system PNS - regeneration associated genes RAGs RGM - repulsive guidance molecule - reactive oxygen species ROS RST - rubrospinal tract - semaphorin 3 a Sema3a - systemic inflammatory response syndrome SIRS SIRT1 - sirtuin 1 SCI - spinal cord injury SOD - superoxide dismutase TACE - tumor necrosis factor-a-converting enzyme Trk - tropomyosin-related kinase trkB - brain-derived neurotrohpic factor receptor trkB - full-length trkB receptor trkB - truncated trkB receptor TNF - tumor necrosis factor TNF-a - tumor necrosis factor alpha XIAP - X-linked inhibitor of apoptosis  NFKB  NTR  fl  tc  ix  Acknowledgements I owe deep gratitude to my mother and father who provided love and constant support as I meandered through life. I would like to thank Dr. Wolfram Tetzlaff for taking in a stray student and guiding me through this scientific experience. His encyclopedic knowledge of the field was always helpful. He could have easily dismissed me a number of times as I wandered lost and aimlessly through the scientific forest without even breadcrumbs to save me. I am equally thankful for him giving me the freedom to explore new ideas and thoughts; and once I found something interesting but unconventional, having such an open mind to embrace it and encourage my work (not to mention actually implementing a self-experiment of the new findings). Hopefully his belief in this work will continue to pay off in scientific dividends. To my supervisory committee (Dr. Lynn Raymond, Dr. John Steeves, Dr. Jane Roskams, and Dr. Wolfram Tetzlaff) I would like to thank you for your patience, and guidance. Thank you Dr. John Steeves for taking me as a student at the start of my PhD. I would like to thank U.B.C. Faculty of Neuroscience and Graduate Studies for granting me leaves of absence and NSERC for their 4 years of financial support. Thanks to the administrative staff of ICORD for all their organizational help and I wish ICORD the best of luck with their new building and future. I would like to thank a number of very fine and interesting people who's intelligence and friendships might have been the only reason I didn't quit my PhD throughout the years which includes Karl Fernandes, Jackie Vanderluit, Chris McBride, Dave Pataky, John McGraw, Gordon Hiebert, Katherine Stilwell, Egido Spinelli, Loren Oschipok, Carmen Chan, Bonnie Tsang, Angel Wong, Jie Liu, Jacqui Hudson, Lowell McPhail, and Jaimie Borisoff. A special thanks goes to Lowell McPhail since we share a similar circuitous path to our current destination, you offered great advice and support not to mention the debt I owe you for the vast amount of work we collaborated on but never resulted in any papers for all your efforts hopefully in the future. To Jacqui Hudson for a hallway conversation that led me to rock climbing (and my girl friend to reach the world championships in climbing) and signing up and finishing Ironman Canada. To Jaimie Borisoff and his wife Carrie Linegar for being kind hearted enough to constantly invite me over for dinner and discussions when I was truly a lost sheep. Jaimie, we should have taped all those philosophical discussions. The best parts of my PhD experience were the various philosophical discussions. Again, for the same dark periods of my life I would like to thank Karl, Jackie, Kim Reid, and Lisa McGladdery for their friendship. I would like to thank Jie Liu for his fine surgical skills, friendship, and constantly smiling face. I am indebted to Jae Lee who first started as a volunteer then as a part-time employer, and x  constantly put in more hours than he needed to, or was paid for, and without him as a naive and blinded behavior scorer I would have not been able to run my E O D F experiments correctly. To the Ramer lab, I would like to thank you all for my education on the sensory system and nuances of behavioral testing. Special thanks to Leanne Ramer and Angela Scott for their leading by example as exemplified by their enthusiasm and dedication to the pursuit of science. I would like to thank Dr. Matt Ramer for his constant supply of knowledge and I am very grateful for his editing skills. To Everlast and Amanda Marshall for mentioning the term PhD in top-40 music and demonstrating the possible worthlessness of such a degree, and to Green Day for keeping me constantly in an anti-establishment mood throughout my PhD years with a series of albums. To George Price, who committed suicide in 1975, for leaving with us his mathematical discovery of how altruism can evolve via group selection but at the same time how it is a mere product of evolution and there is nothing virtues about acting altruistic. To William D. Hamilton (pioneer of the altruism field) whose own form of altruism resulted in his death from malaria while tracking down monkey feces samples to explore a controversial theory of the origin of AIDS. To David Foster Wallace and Neal Stephenson for their books "Infinite Jest" and "Cryptonomicon" and scaring me with their genius level intelligence. The person I owe the most, and for the rest of my life, is Clarrie Lam. Not only would I not have completed my PhD scientifically without her immeasurable bench-work skills but I would have never even pursued the more risky scientific endeavors without her belief in me (or maybe it was her belief in the treatment). Beyond the science, Clarrie has brought happiness to my life through her constant love and friendship. I would truly be lost without her. This thesis is dedicated to her.  xi  Statement of Contributions This thesis contains work that has been previously published or submitted for publication.  Ward T. Plunet, Clarrie K. Lam, Jie Liu, Wolfram Tetzlaff (2006). B D N F acute or pretreatment of rat rubrospinal neurons does not promote regeneration or sprouting but pretreatment promotes functional recovery after a cervical spinal cord injury. Submitted.  Ward T. Plunet, Clarrie K. Lam, Jae H. T. Lee, Jie Liu, Wolfram Tetzlaff (2006) Dietary restriction is neuroprotective and promotes functional recovery from rat cervical spinal cord injury. Submitted.  Ward T. Plunet, Clarrie K. Lam, Jae H. T. Lee, Jie Liu, Wolfram Tetzlaff (2006) Dietary restriction implemented after cervical spinal cord injury is neuroprotective, promotes plasticity, and improves functional outcome. Submitted.  The thesis author Ward Plunet was the primary researcher for all the results presented in the above articles. Clarrie Lam offered assistance with the molecular biology techniques. Technical expertise with surgical techniques was provided by Jie Liu. Jae Lee performed the blinded to treatment group behavorial scoring for chapter 3 and 4.  The above statements and assessment of work performed for this thesis by the author and collaborators are stated as above.  Wolfram Tetzlaff, M D , PhD  Chapter 1 General Introduction  1.1. Overview  Spinal cord injury (SCI) in adult mammals results in a loss of motor and sensory function below the injury site, and recovery is limited. Three major obstacles stand in the way of functional recovery: secondary damage, inhibitory nature of the injured adult central nervous system (CNS), and the weak intrinsic axonal growth response of injured neurons. The initial trauma instigates a cascade leading to secondary damage, resulting in further loss of vital CNS tissue. The combination of the inhibitory nature of the glial scar and adult CNS myelin, along with a weak intrinsic axonal growth response typically results in the regeneration failure of injured CNS axons. One experimental manipulation to increase the intrinsic growth response is cell body treatment with neurotrophins, which I explored in chapter 2. However, a more optimal treatment would be a safe, clinically feasible, combinatorial treatment that addresses all three major obstacles to improve functional recovery. Dietary restriction may reduce secondary damage, decrease the inhibitory environment after injury, and promote better neurotrophin signaling. I examined this clinically feasible combinatorial SCI treatment in chapters 3 and 4. In addition to the loss of motor/sensory function, SCI leads to a number of secondary complications that range from chronic pain to cardiovascular disorders. The loss of function and secondary complications can affect all aspects of the life of SCI patients, from the practical choices of profession and recreation, to the more psychological such as dealing with the changes both within themselves and in how they are perceived in society (DeSanto-Madeya, 2006).  2  1.2. Background 1,400 Canadians and 11,000 Americans are estimated to suffer a SCI each year (Jackson et al., 2004; Furlan and Fehlings, 2006). Before the 1940's SCI patients had short life expectancy, but with the invention of penicillin these patients life expectancy are expected to be close to the normal (Guttman, 1976). Over the subsequent 70 years other than offering SCI patients a closer to normal lifespan, some symptomatic treatments, researchers and clinicians can offer little hope for return of function above what naturally occurs. The healthcare costs for patients/individuals with thoracic SCI is $ 11,000-20,000 per year while the costs for cervical SCI patients/individuals is more expensive at $ 12,000-95,000 (depending on completeness and level) (Dobkin and Ffavton, 2004). According to the Centers for Disease Control America spends an estimated $ 9.7 billion per year on SCI individuals (Ackery et al., 2004) and SCI is reported to be the second most costly medical condition (Winslow et al., 2002). Therefore, purely at an economic level, research that leads to an improvement after SCI could save society a substantial sum of money (on the assumption that the cost of research leading to the treatment and the actual cost of the treatment does not exceed the current and accumulative yearly healthcare costs). While the field of SCI research is addressing the particular problem of returning function after a SCI, knowledge learned from this avenue of research has crossover potential in the diverse fields of neurodegeneration, traumatic brain injury, and stroke. Each of these fields has something to learn from the others, which could provide novel treatments in these complimentary fields of investigations.  1.2.A. When does axonal regeneration and return of function occur? In contrast to the CNS in adult mammals, their peripheral nervous system (PNS) regenerates after injury and function returns. While this blanket statement can be found in many opening paragraphs in regeneration papers, regeneration in the peripheral nervous system is far from complete, structurally or functionally, in experimental animals such as rodents and this situation is even worse in humans. In humans, this clinical problem haunts the surgeons of today (Sumner, 1990) as only 50% of patients who had a surgically repaired peripheral nerve injury experienced any functional recovery at a useful level (Lee and Wolfe, 2000).  3  Unlike adult mammals, injured CNS of other phylogenies do regenerate and show functional recovery. Organisms of older phylogenic origins have a higher capacity for regeneration of injured axons in both the CNS and PNS as compared to more recently evolved species. Adult fish (Lamprey and Teleosts) (Sharma et al., 1993; Davis and McClellan, 1994) along with urodeles (salamander and newts) (Forehand and Farel, 1982) show a high level of regeneration and functional recovery after SCI. Anurans (frogs and toads) can regenerate their optic nerve in adulthood, but their spinal cord is only capable of functional regeneration if the injury occurs before metamorphosis (Forehand and Farel, 1982). Mammalian CNS neurons are also capable of functional regeneration but only if the injury occurs embryonically or peri-embryonically. Once mammals reach adulthood their regenerative capacity of the CNS is severely limited. Therefore, the central questions in the SCI field are why the adult CNS does not regenerate and how to increase axonal regeneration to promote functional recovery.  4  1.3. Three main reasons why adult mammalian CNS fails to functionally recover after spinal cord injury and the need for combinatorial approaches Secondary damage after the initial physical trauma, combined with the inhibitory nature of the injured adult CNS, and the transient and limited intrinsic growth response of the injured neurons are the three main factors responsible for the lack of functional recovery after a spinal cord injury. Traditionally researchers have attacked each problem in isolation. Over the last five years it has been more widely recognized that a combinatorial approach trying to treat all three obstacles might be optimal for improving functional recovery (Ramer et al., 2005). However, the concern is that a combined treatment approach could prove highly invasive and introduce a significant risk factor to the patient. Although the majority of literature in this field treats neuroprotection, CNS inhibitory environment, and cell body response as three separate subjects, they are inter-related. It is well established that neurotrophins promote functional recovery when delivered to the injury site (see section 1.3.C.ii). This recovery is typically attributed to an increased intrinsic cell body response and subsequent enhancement of sprouting/regeneration but the alternative functions and effects of neurotrophins are often neglected. The neurotrophin brain-derived neurotrophic factor (BDNF) is an antioxidant (Joosten and Houweling, 2004); and antioxidant treatment by themselves have demonstrated improvement in behavioral recovery after SCI (Kamencic et al., 2001; Hillard et a l , 2004; Genovese et al., 2005). Therefore, the reported beneficial behavioral effects of B D N F when applied to the lesion site (Liu et a l , 1999; Kim et al., 2001; Ruitenberg et al., 2003; Schwartz et al., 2003) might be primarily, or partially, due to its antioxidant properties rather than its axonal-growth promoting properties. This suggests it might be difficult to separate neuroprotection and intrinsic cell body response in some cases. Additionally, the intrinsic cell body response and the inhibitory nature of adult CNS after injury (CNS myelin and glial scar) are also closely inter-related. The limited intrinsic growth response by the injured neurons is not adequate to promote axonal growth in the hostile environment of adult CNS (see section 1.3.C). The environment in which the injured axons are situated can change gene expression at the cell body level (Fernandes, 2000). Changes in the growth state of the neuronal cell bodies alter the responsiveness of axons to their environment. For example, the weak intrinsic response of injured CNS neurons (see section 1.3.C.ii) is sufficient to allow some growth into a more favorable environment such as a peripheral nerve graft (Kobayashi et al., 1997) but not in the CNS (chapter 2). The addition of BDNF cell body treatment which increases the growth state as 5  measured by increased GAP-43 and promotes greater growth into a peripheral nerve graft (Kobayashi et a l , 1997). The obvious interaction between the three main factors contributing to the lack of functional return after SCI illustrates that a trichotomy approach is too simple for exploring SCI. With these inter-relations in mind, I will further present each of these three main factors separately for the ease of explanation.  I.3.A. Secondary damage After the initial physical trauma to the spinal cord there is cascade of biochemical changes leading to secondary damage in which additional vital tissue is lost due to both necrotic and programmed cell death (apoptosis) (Beattie et al., 2000). Cells that survive the initial physical trauma die both at the epicenter and in the relatively undamaged tissue adjacent to the injury because of the expanding wave of secondary damage. These cells can be potentially rescued from this fate if secondary damage can be reduced, making neuroprotection a prominent target for intervention. A number of mechanisms are believed to participate in secondary damage, which includes hemorrhage, vascular dysregulation, ischemia, edema formation, oxidative stress, and excessive inflammatory response. These mechanisms do not act in isolation and most of them are linked in some fashion, and hence the concept of a cascade of events developed. Due to the physical damage to blood vessels, disruption of the microvasculature, hemorrhage, thrombosis, vasospasm of intact vessels, combined with edema, there is a reduction of blood flow to the injured area leading to ischemia (Tator and Fehlings, 1991). Since the multiple factors leading to ischemia are caused by the initial physical trauma, it appears irreversible (untreatable). What we can do though, is reduce the consequences of ischemia. Physical trauma and ischemia lead to the release of the excitatory amino acid neurotransmitters glutamate into the extracellular space (Benveniste et al., 1984; Strijbos et a l , 1996). The extracellular glutamate causes a large rise of intracellular Ca  levels (through release  of intracellular stores and entry from extracellular sources) which increases the expression of apoptotic genes (Kroemer et al., 1995), leading to cell death. If a cell manages to survive (due to a balance of pro-apoptotic and pro-survival signaling), the rise of calcium results in a host of additional detrimental changes within the cell. These changes include cytoskeletal damage caused by calpains and protein misfolding. Additionally, one of the most damaging aspects of increased calcium level, combined with the loss of adenosine triphosphate (ATP) (see below), is 6  the rapid increase in free radicals including reactive oxygen species (ROS). ROS attack vital lipid bilayers, proteins, and nucleic acids which causes a breakdown of the cell (Werling and Fiskum, 1996). Free radicals oxidize fatty acids in the cellular membrane (lipid peroxidation) which in turn generates more free radicals and hence a vicious membrane-damaging cycle occurs that can devastate the integrity of the cell (Hall, 1994). Lipid peroxidation occurs not only in the cells' outer membrane but also in the mitochondrial lipid bilayer resulting in functional deficits (Bernardi et al., 1998). Damaged components of the electron-transport chain, and ischemia induced decrease of superoxide dismutase (SOD) activity, further produce more free radicals (Grill et al., 1992; Ferrari, 1996; Sadek et al., 2002). In an undamaged state, only a small amount of the electrons (~ 5 %) that passes down the electron transport chain is not stored in the substrate N A D H . These electrons combine with oxygen to produced the superoxide radical 0 '* 2  (Kowaltowski et al., 2001). Usually the superoxide anion is converted by SOD to hydrogen peroxide (H2O2) (Fridovich, 1978; Hassan and Fridovich, 1979) which is then reduced to water (H 0) and oxygen (O ) by catalase and gluatathione peroxidase (GPx) (Emerit, 1988; Siess et al., 2  2  1995). However, in the ischemic state there is a decrease of dismutase activity, which leads to an increase in superoxide anions. The increase of free radicals and lipid peroxidation will cause further damage to the mitochondria (and other cellular components). Another obvious consequence of damage to the mitochondria (specifically the electron-transport chain) is a lower production of ATP. Loss of cellular ATP causes a reduction of ATP-dependent Na+/K+ pump activities. The resulting loss of ion equilibrium causes passive swelling. The lipid peroxidation of the mitochondrial membrane also causes osmotic swelling of the mitochondria and the release of apoptogenic mitochondrial proteins, which increases the probability of cell death (Fiskum, 2001). Additionally, the increased intracellular superoxide anions leak into extracellular space where the levels of superoxide dismutase are low (Emerit, 1988), wreaking more damage both to the already injured cells and the neighboring healthy cells. This cycle outlined above within a cell, and the cycle of cascading damage to neighboring cells is perpetuated (Gogvadze et al., 2003) and hence play a role in the phenomenon of an expanding lesion size (see section 1.3.A.ii). Another complicating factor is that superoxide anions can cause vasoconstriction which would contribute to the ischemic state (Gryglewski et al., 1986). The injured system adapts and ischemia is reduced as reperfusion of the area occurs, but paradoxically this process causes additional damage by further increasing free radical production due to the greater access to oxygen by damaged mitochondria (Lukacova et al., 1996; Basu et al., 2001). The increase of free radicals during the re-perfusion period causes an increased rate of 7  lipid peroxidation (Ambrosio et al., 1993; Kramer et al., 1994). Lipid peroxidation of the polyunsaturated acids in the phospholipids membrane also produces cytotoxic breakdown products such as aldehydes, alkenals, and hydroxyalkenals (Esterbauer et al., 1991). Even beyond this cycle, there is the possibility of additional sources of free radicals from cells invading the injury site. Neutrophils and macrophages migrate to the injury site and surrounding area in the first few days after injury (Anderson, 1992). Neutrophils generate reactive oxygen species when stimulated (Badwey et al., 1980; Hampton et al., 1998). Though more recent research suggest that neutrophils do not significantly contribute to ROS production after SCI (de Castro et al., 2004). Ischemia is more prominent in the grey matter as compared to the white matter. Neurons are particularly vulnerable to ROS due to low levels of antioxidants and neurons' high basal metabolic rate required to maintain their functions. More specifically, spinal cord tissues have higher levels of ROS and lipid peroxidation than cortical tissues (Sullivan et al., 2004). Some treatments that reduce ROS are effective in traumatic brain injury but not SCI; and Sullivan and colleagues argues this is likely due to the higher levels of ROS and lipid peroxidation in the spinal cord. This would suggest the vital importance of treatments that can have a strong effect on reducing ROS in SCI. While we might not be able to stop the initial ischemia that results from the physical trauma, we can try to reduce the downstream devastating effects. Since ischemia plays a major role in the initiating of the cascade, we might be able to use the successful treatments from the stroke field and transfer them into SCI (and treatments that demonstrate increased axonal plasticity in SCI can be used in more traditional ischemic models). This is one example of how research from different fields is mutually beneficial.  1.3.A./. Lesion size correlates with behavior  A large number of groups have found a negative correlation between lesion size and behavioral recovery (Basso et al., 1995; Fehlings and Tator, 1995; Basso et al., 1996; Basso, 2000; Ma et a l , 2001; Cao et al., 2005a; Kloos et al., 2005; Pearse et al., 2005) i.e. animals with smaller lesions show a better outcome. While this correlation is usually examined in contusion injury models it also is demonstrated in crush models. More specifically, in a lesion (crush) similar to the one I used in this thesis (see section 1.9.C), Ruitenberg and colleagues observed a  negative correlation between lesion size and behavioral recovery, while failing to find a correlation between the enhanced sprouting (which was increased in the treated group) and recovery (Ruitenberg et al., 2003). A n additional paper with the same lesion model also noted a negative correlation between lesion size and behavioral function on both the B B B and forelimb usage (Schwartz et al., 2003). This set of data suggests that the reduction of secondary damage (neuroprotection) plays an important role in functional recovery even in crush-lesion models.  1.3.A.M. Progressive increase in lesion size After the initial physical trauma, the lesion expands in size over time (Balentine, 1978). There are a number of competing and complimentary theories behind the growth of lesion that includes ischemia, hemorrhage, macrophage infiltration and inflammation, and mitochondria ROS production (see 1.3.A) (Blight, 1991; Fitch and Silver, 1997). Crush injuries, similar to the one used in the experiments throughout this thesis, produce a continual increase in lesion size over several weeks (Fujiki et al., 2005). Neuroprotective strategies such as minocyline treatment can halt this time dependent growth of lesion size and result in better behavioral recovery (Lee et al., 2003b). Interestingly, lesion sizes in human SCI appear to expand for several months to years after the injury (Hughes, 1991), and apoptosis might continue up to several years (Johnson et al., 1995). This would suggest that neuroprotective intervention even in the semi-chronic state might reduce the ongoing damage that occurs in human SCI.  1.3.A.I7I. Current neuroprotective treatments  There are a number of neuroprotective treatments that have been reported to improve outcome in animal studies and clinical trials after SCI. These include glucocorticoids (dexamethasone and methylprednisolone (MP)) (for review see (Hall and Springer, 2004)), Naloxone (Flamm et al., 1982; Flamm et al., 1985), tirilazad (Hall et al., 1994; Bracken et al., 1997; Bracken et al., 1998) the monosialoganglioside compound GM1 (Bose et al., 1986; Fusco et al., 1986; Geisler et al., 1990), and inhibitors of COX2 (Hains et al., 2001). More recent research has added to this list of neuroprotective agents that might be easily transferred to clinical trials since they are already approved by the F D A to treat other human diseases. Minocycline proved to reduce lesion size, apoptosis, and improve function after a SCI (Lee et al., 2003b; Wells et al., 2003). This was independently confirmed by other groups who examined  further molecular correlates of minocycline on recovery from axonal injury (McPhail et al., 2004; Stirling et al., 2004; Teng et al., 2004). Another clinically used drug erythropoietin (EPO) reduced secondary inflammation, lesion size, and promoted recovery (Gorio et al., 2002; Brines et al., 2004) via a mechanism not linked to its erythropoietic effect (Brines et al., 2004). The main effect of MP appears to be the inhibition of injury-induced lipid peroxidation (Hall, 1993; Hall et al., 1995). This conclusion is supported by the findings that tirilazad, a steroid lacking glucocorticoid receptor activity, which inhibits lipid peroxidation, was also reported effective in clinical trials. However, there is considerable controversy on the actual clinical effectiveness of MP and tirilazard treatment (Hall and Springer, 2004). Interestingly, EPO's positive effect on SCI has also been reported to be mediated through its ability to reduce lipid peroxidation (Kaptanoglu et al., 2004). Because of this recognized importance of lipid peroxidation and ROS the use of antioxidants have been tested in animal models of SCI. Upregulation of glutathione (an antioxidant) by L-2-oxothiazolidine-4-carboxylate treatment decreased oxidative stress, increased white matter sparing, and improved function after a thoracic SCI (Kamencic et al., 2001). Melatonin, another scavenger of oxyradicals and peroxynitrite, decreased inflammation and preserved tissue with subsequent better functional outcome after a SCI (Genovese et al., 2005). Temperol, a nitroxide antioxidant, also increased preservation of spinal cord tissue and produced positive behavioral results (Hillard et al., 2004). As briefly mentioned earlier (see section 1.3), the neurotrophin BDNF which has been shown to enhance recovery after SCI (Liu et al., 1999; Kim et a l , 2001; Ruitenberg et al., 2003; Schwartz et al., 2003) is an antioxidant (Joosten and Houweling, 2004). The extensive line of research outlined above points to the importance of reducing ROS and subsequent lipid peroxidation. What makes this line of research interesting is that increasing various antioxidants appears to be an attainable goal that should lead to reduced ROS and lipid peroxidation offering neuroprotection and result in improved behavioral recovery.  I.3.B.  The adult mammalian CNS is inhibitory to axonal growth  The adult CNS expresses a number of molecules that are inhibitory to axonal growth. The inhibitory components are associated with the glial scar and with the degenerating myelin.  10  1.3.B.L Glial scar The glial scar which forms after SCI is inhibitory to axonal growth (Rudge and Silver, 1990) and is composed primarily of astroctyes, meningeal cells, fibroblasts, and oligodendrocyte precursors (David and Lacroix, 2003; Klapka and Muller, 2006). After an injury to the CNS, astrocytes undergo activation which is called reactive gliosis, but only a limited amount of cell division occurs (Silver and Miller, 2004). The main response is glia hypertrophy, which is marked by an increase expression of the intermediate-filament glial fibrillary acidic protein (GFAP). While the glial scar is detrimental to axonal growth it does serve an important purpose of repairing the blood-brain barrier and reducing the inflammatory response (Faulkner et al., 2004). Though the glial scar performs important functions, the byproduct is that when axons encounter the scar they form dystrophic endbulbs. The glial scar primarily consists of activated astrocytes and proteoglycans excreted by activated astrocytes (Carulli et al., 2005). Proteoglycans are components of the extracellular matrix (ECM) and inhibitory to axonal growth. Astrocytes produce at least four different classes of proteoglycans: heparan sulphate proteoglycan (HSPG), dermatan sulphate proteoglycan (DSPG), keratan sulphate proteoglycan (KSPG), and chondroitin sulphate proteoglycan (CSPG). The most widely studied of these groups regarding the glial scar are the CSPGs. The CSPGs includes the familiar aggrecan, brevican, neurocan, NG2, phosphacan, and versican. After a SCI activated astrocytes secrete CSPGs, increasing their expression (Jones et al., 2003). In vitro activated astroctyes produce CSPGs and they are inhibitory to axonal growth (McKeon et al., 1991). The glycosaminoglycan (GAG) side chains of CSPGs are the primary inhibitory component because chondroitinase (an enzyme from the bacterium Proteus vulgari) that cleaves the G A G side chains made CSPGs less inhibitory (McKeon et al., 1995). Chondroitinase A B C (ChABC) treatment after a dorsal column lesion facilitated regenerative growth of both ascending sensory axons and descending corticospinal axons as well as some gain of motor function (but not sensory) (Bradbury et al., 2002). Interestingly, the researchers observed an increase of GAP-43 expression in the D R G neurons with ChABC treatment compared to controls, which demonstrates how reducing inhibitory components can increase a marker for the intrinsic growth state of the neurons (see section 1.3). Proteoglycans are not the only inhibitory to axonal growth molecules expressed as a consequence of the glial scar. The secreted protein semaphorin 3a (Sema3a), a member of the 11  semaphorin family, is produced by fibroblasts that migrate to the lesion site after a spinal cord injury (Pasterkamp et al., 1999). The interaction between Ephrin-B2 expressed by activated astrocytes and fibroblasts expressing EphB (the receptor for Ephrin-B2) seems to participate in the formation of the glial scar (Bundesen et al., 2003). EphrinA4 is also expressed by activated astrocytes after a spinal cord lesion and ephrinA4 knockout studies demonstrate reduced glial scaring and increased regeneration (Goldshmit et al., 2004). Ephrins also seem to be expressed by adult CNS myelin, which contributes to reduced axonal growth (see section 1.3.B.ii). What can be concluded is that the glial scar is composed of numerous inhibitory molecules that represent a serious obstacle to axonal growth. However, there is some hope because a number of these inhibitory molecules appear to use the same general pathway, which is additionally shared by many of the inhibitory proteins expressed by adult myelin.  1.3.B./7. Adult mammalian CNS myelin is inhibitory to axonal growth The second major inhibitory groups of proteins in the adult CNS are produced by the myelin forming oligodendrocytes. While myelin was proposed to be inhibitory to axonal growth for many years, Caroni and Schwab were the first to demonstrate myelin's ability to reduce axonal growth in a series of landmark in vitro studies (Caroni and Schwab, 1988a, b). This was further supported by developmental studies correlating the regenerative permissive periods and the onset of myelination (Kalil and Reh, 1982; Bregman et al., 1993; Hasan et al., 1993; Saunders et al., 1998). As one example, in chicks complete spinal cord transections performed up to E12 (no myelin formation) leads to full functional recovery, while if performed at E l 3-15 (myelination onset) there are permanent functional deficits (Hasan et al., 1993). The importance of the inhibitory properties of myelin was directly tested by x-ray irradiation (Savio and Schwab, 1990) or delaying myelination (Keirstead et al., 1992) which extended the permissive period. Then the question became what are the molecules responsible for myelin's inhibitory properties? The CSPG family of axonal growth inhibitory molecules are most widely known for their role in contributing to the inhibition by the glia scar (see section 1.3.B.i), but at least some CSPGs, such as versican V2 and brevican, are also expressed in myelin (Niederost et al., 1999). Sema4D, a growth cone collapsing protein, is also expressed by oligodendrocytes and is upregulated after a spinal cord injury (Moreau-Fauvarque et al., 2003). Two groups in 1994 found that Myelin-Associated Glycoprotein (MAG) is inhibitory to neurite outgrowth (McKerracher et al., 1994; Mukhopadhyay et al., 1994). Another myelin protein,  Oligodendroycte Myelin glycoprotein (OMgp), initially identified in 1990 (Mikol et al., 1990), was added to the growing list of inhibitory proteins in 2002 (Wang et a l , 2002b). However, Nogo, or at least the then unidentified fractionated myelin protein (NI-35/NI-250), was the first inhibitory fraction of myelin that was explored, and became the most heralded (Caroni and Schwab, 1988a, b). Specific myelin inhibitory fractions were isolated by Caroni and Schwab in 1988, which were identified by their molecular weights: NI-35 and NI-250 (Caroni and Schwab, 1988a). In the same year these two researchers generated a monoclonal antibody against NI-250, termed IN1, that blocked the inhibitory properties of both NI-35 and NI-250 in vitro (Caroni and Schwab, 1988b). Schnell and Schwab furthered this research showing IN-1 also enhanced axonal growth in vivo (Schnell and Schwab, 1990), and resulted in functional recovery (Bregman et al., 1995). An important advance was made in 2000 when at least one of IN-1 recognizing proteins was identified as Nogo (Reticulon 4) by three different groups simultaneously (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000). A large body of in vitro and in vivo research strongly supports that Nogo is inhibitory to axonal growth (Buchli and Schwab, 2005). Interestingly, M A G , Omgp, and Nogo all signal through the same receptor complex, Nogo-receptor complex. The Nogo receptor (NgR) was initially identified as the receptor for Nogo (Fournier et al., 2001), but subsequently it was discovered to also be the receptor for M A G (Domeniconi et al., 2002; Liu et al., 2002a), and for Omgp (Wang et al., 2002b) (recently additional NgR homologues have been discovered (Barton et al., 2003; Pignot et al., 2003). NgR is a GPI linked protein lacking a cytoplasmic domain and therefore cannot transduce a signal. The ubiquitous neurotrophin receptor p75 (see section 1.3.C.i) was discovered as the signaling component for NgR, for all three myelin inhibitory proteins (Wang et al., 2002a; Wong et al., 2002; Yamashita et al., 2002). After several puzzling findings, a new component to the pathway, T R O Y , was found that could perform the same function as p75 in this receptor complex (Park et al., 2005; Shao et al., 2005). This finding may explain why mice carrying p75 gene mutations still failed to show axonal regeneration in some cases (Barker, 2004; Mandemakers and Barres, 2005). Another important component of the complex is the LRR and Ig domain containing Nogo receptor interacting protein (Lingo-1) since it was found to be a necessary part of the complex for myelin to inhibit axonal growth (Mi et al., 2004). Nogo, M A G , or Omgp binding to NgR leads to an increase in active RhoA, and subsequently Rho kinase (ROCK) and its activated form targets various substrates leading to growth cone collapse (for review see: (Sandvig et al., 2004)). Recently, Amino-Nogo and 13  Versican V2, was also shown to activate Rho kinase, but through a NgR independent pathway (Schweigreiter et al., 2004). The Nogo receptor complex and/or the Rho/ROCK kinase pathway might provide a unique convergence point of axonal growth inhibitory signaling from myelin and the glial scar (Lee et al., 2003a). Lately, every few months scientist discover another inhibitory protein or receptor that is expressed in the spinal cord and involved in reducing axonal growth. Recent findings include myelin expressed ephrin-B3 that is argued to be as inhibitory as all the other proteins combined (Benson et al., 2005). The adult CST is EphA4 (the receptor for ephrin-B3) positive and sensitive to the ephrin-B3 expressed by the oligodendrocytes. Previous work had suggested that ephrin-A4 signals through the Rho/ROCK pathway suggesting that ephrin-B3 could also mediate its effect through this common pathway (Yiu and He, 2003). Ephrins are also expressed by the glial scar (see section 1.3.B.i), which suggests that ephrins could be playing an important role in reducing axonal growth after a SCI. The repulsive guidance molecule (RGM) is increased after SCI and also signals through the Rho/ROCK pathway (Schwab et al., 2005a). R G M , like ephrin-B3 is reported to be one of the most inhibitory substrate so far identified and expressed after human spinal cord injuries (Schwab et al., 2005b). Somewhat hard to believe, it appears that every new inhibitory molecule is claimed to be the most inhibitory or as inhibitory as all other previous found inhibitory molecules combined. A n antibody against R G M promoted CST growth and improvement in motor behavior (Hata et al., 2006). In addition, there is the recent finding that the epidermal growth factor receptor (EGFR) might also be a mediator of the myelin and CSPGs inhibitors that acts downstream of NgR in a calcium dependent manner (Koprivica et al., 2005). In summary, there are multiple axonal growth inhibitory proteins expressed in the spinal cord. This suggests blocking any one of these inhibitory proteins by itself would be insufficient, as many others remain active and capable of inhibiting axonal growth. There appears to be a convergence point of many of these inhibitory proteins on the Rho/ROCK pathway. However, blocking this pathway has not produced optimum regeneration, or functional recovery. In mice inhibition of Rho by C3 transferase or inhibition of R O C K by Y-27632 produced an improvement in behavior after SCI (Dergham et al., 2002). In contrast, C3 transferase treatment in rats surprisingly inhibited functional recovery and Y-27632 only accelerated the rate of recovery but did not affect the overall functional recovery (Fournier et al., 2003). Another study in rats reported positive behavioral effects of Y-27632 with high dose treatment but at lower doses the compound had negative effects on motor function (Chan et al., 2005). These data suggest further research is needed to fully understand this pivotal pathway and ways to target it 14  for the promotion of regeneration and recovery. We also await new revelations about novel inhibitory molecules and additional signaling components.  I.3.C. The weak intrinsic axonal growth response of CNS neurons  A positive intrinsic cell body growth response is usually defined by its ability to increase regeneration associated genes (RAGs) and/or promote axonal growth. Neurotrophins increase the intrinsic axonal growth response. However, one must also address what substrate or environment the axons are growing on/in. To better understand intrinsic axonal growth response I will give a brief background on neurotrophins.  I.3.C./. Neurotrophins  Neurotrophins are neuronal growth factors essential for neuronal health. They play a role in survival, differentiation, and activity driven plasticity (Lu et al., 2005). In mammals, four main neurotrophins are expressed in the brain: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4). There are two classes of transmembrane receptors for the neurotrophins: p75 neurotrophin receptor (p75  NTR  ), a member  of tumor necrosis factor (TNF) receptor family, and tyrosine receptor family of tropomyosinrelated kinase (trk) receptors, which includes trkA, trkB, and trkC. N G F preferentially binds to trkA, BDNF and NT-4 to trkB, and NT-3 to trkC (and can also function through trkA and trkB). What all the neurotrophins listed have in common is that they can bind to p75. The neurotrophins have a low affinity for the p75 receptor, which was designated as the low affinity receptor. In contrast, neurotrophins have a high affinity for their respective trk receptor. p75 expression enhances the on-rate of neurotrophin binding to trk receptors. However, neurotrophin activation of p75 in the absence of trk receptors leads to cell death, whereas the activation of trk receptors promotes cell survival. O f course, this dichotomy is an oversimplification. The exact combination of receptors and their ratio(s) along with their isoforms, amount, and ratios of neurotrophins eventually determines the fate of the cells. I will briefly illustrate this using BDNF and its receptors as an example, since they play a prominent role in chapter 2 and 4 of this thesis. The majority of neurons in the CNS express trkB (Klein et al., 1989) while the expressions of trkA and trkC are more limited (Holtzman et al., 1992; Tessarollo et al., 1993). BDNF, like other neurotrophins, is produced as a precursor propeptide, which is then 15  proteolytically cleaved to form the mature protein. The mature protein, a 13.5 kDa secretory peptide forms a dimer to exert its biological action (Jungbluth et al., 1994; Kolbeck et al., 1994). When bound to trkB, the BDNF dimer induces receptor dimerization resulting in autophosphorylation of intracellular tyrosine domains (Jing et al., 1992). The phosphorylation induces changes to the docking sites and thereby a host of adaptor and effector proteins binds to the receptor. Multiple intracellular transduction pathways can then be initiated that lead to a diversity of signals and actions including altered gene expression (Kaplan and Miller, 2000). The pathways activated include Ras/MAPK, PI-3-K/Akt pathway and PLCy pathway (Kaplan and Miller, 2000). Ras activates the M E K / M A P K (ERK) pathway, which leads to the activation of the transcription factor, cAMP response element binding protein (CREB), and the anti-apoptotic gene Bcl-2. Additionally, the activation of Ras leads to PI-3-K/Akt pathway signaling which plays a major role in cell survival. Downstream of this pathway is the inactivation of Bcl-xL/Bcl-2-Associated Death Promoter (BAD) and caspase-9, which are proapoptotic. Activity of the PLCy pathway results in production of both diacylglycerol (DAG) and IP3 (inositol-1,4,5-triphosphate). D A G activates PKC and results in further activation of M E K / M A P K (ERK). IP3 causes release of ubiquitous signaling dication C a from intracellular 2 +  stores. The increased C a  2 +  activates Ca -dependent signaling molecules, which include CREB. 2+  It is generally argued that the pro-forms of neurotrophins are damaging to the system and they lead to preferential activation of p75 leading to cell death (Lee et al., 2001; Beattie et al., 2002; Harrington et al., 2004). Specifically, pro-NGF leads to increased cell death in the spinal cord via the p75 receptor (Beattie et al., 2002). However, the detrimental effect of the pro-form might not be true for all neurotrophins. Interestingly, the pro-form of B D N F can also activate trkB (Kolbeck et al., 1994; Lee et a l , 2001; Mowla et al., 2001). More recently, pro-BDNF was found to activate Erkl/2 and induce neurite outgrowth (Fayard et al., 2005). The pro-form of BDNF also binds to p75 but at a reduced strength as compared to mature BDNF. Pro-BDNF might therefore be more effective at eliciting survival and axonal growth as compared to the mature form which was used in the various experiments (including SCI) (Fayard et al., 2005). In addition to the dual forms of BDNF, the trkB receptor exists in more than one isoform (Middlemas et al., 1991; Barbacid, 1994). Beyond the well known full-length tyrosine kinase containing isoform of trkB (trkB , or alternatively called TK+) that separates by SDS-PAGE at fl  140-145 kDA, there exist three ( T l , T2, T-Shc) intracellularly truncated isoforms generated by alternative splicing. They as a group are called trkB , or alternatively called T K - (90-95 kDa), tc  lack the tyrosine kinase domain, and therefore are incapable of signaling via tyrosine 16  phosphorylation, (Klein et al., 1990a; Middlemas et al., 1991). Only one truncated form, T I , is expressed in humans (Shelton et al., 1995). The function of the three different C-terminally truncated receptor isoforms is not fully known, however, the expression data of trkB in rats might offer some hints. The highest expression of trkB rnRNA and protein is found in neurons of the thalamus, 11  hippocampus and cortical layers, with pyramidal neurons in the hippocampus and layer V of the cortex being particularly prominent (Klein et al., 1990b; Beck et al., 1993; Armanini et al., 1995; Fryer et al., 1996). In contrast, trkB is mainly expressed in the ependyma, choroid plexus and tc  non-neuronal cells (Beck et al., 1993; Frisen et al., 1993; Biffo et a l , 1995). However, both the full-length and truncated isoforms can co-localize in the same cells such as adult motoneurons (Armanini et al., 1995). Developmentally trkB is expressed earlier than the truncated isoforms fl  with trkB* reaching adult levels by the time of birth while the peak for the truncated versions 1  occurs two weeks postnatally (Masana et al., 1993; Fryer et al., 1996). The delayed expression of the truncated isoforms of trkB leads to the suggestion that these isoforms might modulate trkB  11  signaling. The ratio of trkB :trkB decreases developmentally due to an increase in truncated n  tc  trkB during the period of cell death in retinal ganglion cells (Allendoerfer et al., 1994). This developmental shift from trkB to trkB occurs in both mouse and rat brains and corresponds to 11  tc  reduced phosphorylation levels of trkB (Escandon et al., 1994; Knusel et al., 1994; Fryer et al., 1996). It has been suggested that the truncated trkB isoforms expressed mainly in non-neuronal cells inhibit BDNF signaling in neurons by taking up BDNF and sequestering it from the fulllength trkB isoform expressed in neurons (Beck et al., 1993; Biffo et al., 1995; Rubio, 1997; Alderson et al., 2000). When neurons are grown on a monolayer of NIH 3T3 cells transfected with the TI truncated form of trkB, the transfected cells bind and internalize the exogenously added BDNF and hence the neurons fail to extend neurites (Fryer et al., 1997). In vivo if nonneuronal cells take up the limited amounts of extracellular BDNF, sequestering this vital source, neurons receive less and the downstream positive effects of B D N F is reduced (cell survival and axonal outgrowth). Alternatively, the truncated isoforms can alter signaling by forming inactive heterodimers with trkB in cells that co-express the two isoforms (Eide et al., 1996; Ninkina et 11  al., 1996; L i et al., 1998; Gonzalez et al., 1999). The formation of trkB -trkB heterodimers fl  tc  results in the absence of BDNF induced signaling and hence a reduction in appropriate cellular outcome, including a reduction in cell survival (Eide et al., 1996; Ninkina et al., 1996; Haapasalo et al., 2001). Mice lacking only the full-length trkB isoform with continous expression of the 17  truncated isoforms displayed an increased loss of neurons in direct comparison with mice with all isoforms of trkB knocked-out (Luikart et al., 2003). This line of work strongly supports the negative effects of the truncated isoforms on cell signaling/survival. Another group explored the effect of overexpression of truncated trkB in a stroke model. In areas of the brain that overexpressed truncated trkB there was a ~ 200 % increase in damage, but not in areas where normal levels of truncated trkB were expressed (Saarelainen et al., 2000). These same animals demonstrated less BDNF signaling in response to kainic acid which resulted in reduced expression of B D N F in the hippocampus (Saarelainen et al., 2001). Additionally, this strain of mice did not respond to antidepressant treatment which is mediated via BDNF-trkB signaling (Saarelainen et al., 2003). The increase of truncated trkB isoforms resulted in a decrease in trkB  fl  signaling in these mice. Recently, it was discovered that truncated trkB isoforms are capable of some form of signaling, though the mechanism is not fully understood. The truncated isoforms are implicated in the outgrowth of dendritic filopodia in conjunction with the p75 receptor (Hartmann et al., 2004). In astrocytes which predominately express the T l isoform, B D N F elicits a release of intracellular stores of calcium from the astrocytes via the truncated trkB receptor (Rose et al., 2003). In vitro the T l isoform was found to bind Rho GDP dissociation inhibitor 1 (GDI1) and regulate astrocyte morphology in a BDNF dependent manner (Ohira et al., 2005). BDNF leads to dissociation of GDI1 from T l and thereby inhibits the conversion of Rho GTPases GDP-bound inactive form to the GTP-bound active form and alter the astrocytes morphology (though the authors did not specifically argue for a reactive astrocyte morphology). It is probable that, like the non-catalytic receptor p75, in order to signal, the truncated isoforms of trkB require additional interacting factors that have yet to be discovered. What is interesting for this present thesis is the alteration of trkB expression after injury. Truncated trkB expression is increased after hippocampal lesion in non-neuronal cells (Beck et al., 1993), after kainite induced seizures (Dugich-Djordjevic et al., 1995; Venero and Ffefti, 1998), and spinal cord lesion (Frisen et a l , 1993; King et al., 2000; Liebl et al., 2001; Widenfalk et al., 2001). Interestingly, Frisen and collaborators reported a strong increase of truncated trkB in glial cells after SCI but did not observe the same effect after a peripheral nerve axotomy (Frisen et al., 1993). After a peripheral nerve axotomy Schwann cells downregulate their expression of truncated trkB (reminder - PNS regeneration is considerably more than CNS regeneration) (Funakoshi et al., 1993). In contrast, full-length trkB expression is reduced or not detected after SCI at the injury site, or on the axons of injured tracts (rubrospinal and 18  corticospinal) (King et al., 2000; Liebl et al., 2001; Lu et al., 2001; Kwon et al., 2002b; Kwon et a l , 2004). The result of these changes in the CNS would be a drastic reduction in the ratio of full-length to truncated trkB receptors. The differential expression of full-length and truncated trkB (and resultant ratio changes) after axonal injury between the PNS and CNS might partially explain the differences in regeneration potential between the two. In summary, while the truncated isoforms is likely to keep offering new surprises in their functions, the majority of studies indicate the increase of trkB after SCI has a strong inhibitory tc  function on normal BDNF-trkB* signaling in neurons. The increased trkB expression in 1  tc  conjunction with the reduced full-length trkB expression results in a substantially decreased trkB :trkB ratio, which may play a role in increased cell death and reduced axonal growth after fl  tc  SCI.  I.3.C.//. Differences in intrinsic cell body response  Injury to a peripheral D R G axons induces a robust programmed gene response including regeneration associated genes (RAGs) and subsequent regeneration (Schreyer and Skene, 1993) (though the regeneration and recovery is not as good as we are led to believe: see section 1.2.A). This is an example of positive intrinsic cell body response to injury. In contrast, injury to the central axons of DRGs in the dorsal root produces far weaker R A G expression and the axons grow at half the rate within the Schwann cell environment of the dorsal root and halt at the dorsal root entry zone (DREZ), which is the area of transition into CNS tissue (Bradbury et al., 2000). One of the most prominent RAGs is growth associate protein 43 (GAP-43), which increases after a PNS axotomy and correlates with regenerating axons (Skene and Virag, 1989). When CNS axons are injured, GAP-43 does not increase in the cell body of the injured neurons if the injury occurs at a significant length away from the cell body, and the increase in GAP-43 is only transient when the injury is close to the cell body (Fernandes et al., 1999). Specifically, rubrospinal neurons upregulate GAP-43 transiently after axotomy at the cervical level (Tetzlaff et al., 1991) but no increased is observed if the injury occurs in the thoracic region. This distance-dependent regulation of GAP-43 expression correlates with the ability of the injured axons to grow into a peripheral nerve graft (PNG), i.e. it fails if the transplants are inserted into the thoracic cord. One of the theories behind the lack of a sufficient cell body growth response to injury by CNS neurons is an inadequate supply of trophic factors. B D N F expression is not increased in the 19  red nucleus after a cervical spinal cord injury (Kobayashi, 1998), and trkB expression is slightly downregulated (- 30 %) by 7 days after injury (Kobayashi et al., 1997), or maintained (Liebl et al., 2001). In response to axotomy, motoneurons (peripheral nerve projections) display a substantial increase in BDNF and trkB, and the elevated expression is maintained for 2-3 weeks (Piehl et al., 1994; Kobayashi et al., 1996). In corticospinal neurons the lack of adequate trophic supply is even more dramatic. Approximately, 50 % of corticospinal neurons die after an axotomy at the internal capsule level, a further loss results when function-blocking antibodies to BDNF are administered (Giehl et a l , 1998). The application of B D N F rescues 100 % of the injured neurons pointing out the need for adequate trophic support (Giehl and Tetzlaff, 1996). Therefore, it appears that after axotomy CNS neurons lack a sufficient supply of neurotrophins and this could contribute to their weak intrinsic axonal growth response (transient and/or limited increase in R A G expression) and hence the failure to regenerate. The lack of a sufficient cell body response and low neurotrophin expression after injury provided the rationale to test if trophic factors could create a more significant increase of RAGs. Cell body treatment of the red nucleus with BDNF (or NT4) induced a stronger and more sustained expression of GAP-43 and Ta-1 tubulin after a cervical lesion and for the first time after thoracic lesion, and prevented rubrospinal atrophy (Kobayashi et al., 1997; Kobayashi, 1998) . In addition, increased numbers of injured axons grew into a cervical PNG, and some grew into a thoracic PNG (Kobayashi 1998). The application of trophic factors to the lesion site (cut axons) has also been extensively tested using a number of delivery techniques including infusion pump (Novikova et al., 2000), gelfoam (Bregman et al., 1998), and genetically altered cell lines (Menei et al., 1998; Liu et al., 1999) . Application of trophic factors to freshly cut axons prevented atrophy and in some cases promoted regeneration. For example, a number of different trophic factors have shown efficacy in both enhanced sprouting/regeneration and more importantly in improving functional recovery: NT3 (Grill et al., 1997; Houweling et al., 1998; Blits et al., 2000; Ruitenberg et al., 2003), BDNF (Liu et al., 1999; Kim et al., 2001; Ruitenberg et al., 2003; Schwartz et al., 2003), CNTF (Ye et al., 2004), GDNF (Tang et al., 2004), or the combination of B D N F and NT3 (Cao et al., 2005b; Mitsui et al., 2005). However, over time the cut axons appear to become non-responsive to trophic treatment. In chronic experiments, 14 to 22 weeks delayed treatment failed to prevent cell atrophy and atrophy induced by a second injury, while if treated 4 to 8 weeks after injury the treatment was effective (Houle and Ye, 1999). Positive functional effects of neurotrophins delivered to the 20  injury site were observed with applications of neurotrophins at 2-4 weeks after injury in combination with embryonic transplant or delivered by transplanted transfected fibroblasts (Coumans et al., 2001; Jin et al., 2002). However, if the treatment of the spinal cord lesion site was delayed until 6 weeks after injury, no positive effects in functional recovery were observed in a number of different tests (Shumsky et al., 2003). More so, cell transplants performed at this later time point resulted in a greater deficit than before transplant. This line of research indicates a time-limited window when the treatment should be applied at the lesion site. In contrast, BDNF infused to the cell body of the red nucleus, rather than the lesion site, even 1 year after a cervical axotomy reversed the atrophy of the rubrospinal neurons and stimulated GAP-43 and Ta-1 tubulin expression (Kwon et al., 2002b). More importantly, this delayed cell body treatment induced a significantly higher number of chronically lesioned axons to grow into a PNG. However, not surprisingly, a lesser number of chronically injured axons grew into the P N G as compared to acutely injured axons (Kobayashi et al., 1997; Kwon et al., 2002b). One plausible explanation for the apparent greater effectiveness of cell body treatment versus lesion site treatment in a chronic setting is the regulation of trophic factor receptors. King and colleagues failed to observe immunoreactivity of full-length trkB receptor in the spinal cord lesion site and reported an upregulation of the truncated form of trkB (no tyrosine kinase signaling) (King et al., 2000). In a contusion model of spinal cord injury full-length trkB expression was also lost at the lesion site 7 days post injury, but the cell bodies of the injured rubrospinal neurons maintained their expression of trkB (Liebl et al., 2001). Lu and collaborators also reported downregulation of full-length trkB at the lesion site but in contrast there was maintained expression at the cell body level after a midthoracic dorsal hemisection (Lu et al., 2001). The protein expression of full-length trkB is reported to be non detectable on injured rubrospinal axons at 2 months after injury even though the cell body of these neurons still expressed the full-length receptor, along with the intact rubrospinal tract on the non-injured side (Kwon et al., 2004). Interestingly, Kwon and company reported the continued expression of fulllength trkB receptor in the rubrospinal neurons even 1 year after axotomy and the growth of chronically injured axons into a PNG with delivery of BDNF to the cell bodies (Kwon et al., 2002b). These series of papers suggest that for chronic injuries the cell body appears to the best location for treatment because of the continued expression of full-length trkB receptor, which contrasts with the loss of this receptor at the lesion site. However, the clinical feasibility of treating the cell body of rubrospinal neurons could be a problem, but the principals learned could 21  be transferred to the more assessable, and in humans more relevant, corticospinal tract. More importantly, the behavioral outcome of cell body treatment of injured descending supraspinal neurons has yet to be determined. Chapter 2 of this thesis examines this question.  22  1.4. Overcoming CNS inhibitory molecules by increasing the intrinsic cell body response This next set of sections focuses on the inter-relations of the main obstacles that are responsible for the lack of C S N regeneration. Specifically, I discuss how increasing the cell body response can overcome the inhibitory nature of the glial scar and myelin.  1.4.A. Conditioning lesion As mentioned earlier, central root axotomy of DRGs results in a limited regenerative response and all growth halts at the dorsal root entry zone (DREZ). Only a very limited number of axons regenerate into a transplanted PNG after axotomy in the dorsal columns of the spinal cord. However, if a peripheral axotomy is performed at the same time as the central axotomy, there is increased growth of the centrally projecting DRGs into a P N G (Richardson et al., 1982; Richardson and Issa, 1984; Richardson and Verge, 1987). The in vitro growth of conditionally lesioned DRGs was also substantially increased (Hu-Tsai et al., 1994; Smith and Skene, 1997). Neumann and coworkers demonstrated that a conditioning lesion of the sciatic nerve prior (one week prior produced the largest effect) to a subsequent dorsal column lesion allowed the central branches to cross the hostile environment of the CNS scar (Neumann and Woolf, 1999). In a more clinically relevant model, performing a peripheral conditioning lesion at the time of a dorsal column injury followed by a second sciatic nerve re-injury one week later increased axonal growth was observed (Neumann et al., 2005). This manipulation of the PNS and CNS allowed some growth of the injured sensory axons across the dorsal column scar and into the rostral CNS environment.  I.4.B. Cell body priming of neurons to increase the intrinsic growth response  In vitro studies indicated that prior exposure of DRGs to neurotrophins allowed their axons to overcome inhibition by myelin (Cai et al., 1999). However, if the neurotrophins were added to the neurons at time of plating on inhibitory substrates no increase of growth was observed. The neurite promoting effect was dependent on the P K A pathway and subsequent increase in cAMP in the treated neurons. Therefore, it appears that neurotrophins (e.g. BDNF) are upstream of the increase of cAMP in these neurons, and neurotrophins are good agents to initiate this growth-promoting pathway. 23  Peripheral axotomy induces an upregulation of cAMP in DRGs and subsequent regeneration, while central axotomy fails to induce an upregulation of cAMP, which correlates with their reduced regenerative ability (see section 1.3.C.ii). Extending this line of research, two separate groups directly elevated cAMP in DRGs using db-cAMP 48 hrs or one week prior to a dorsal column lesion and reported enhanced growth in the CNS as well as in vitro on both M A G and myelin substrate (Neumann et al., 2002; Qiu et al., 2002). Further support for the importance of cAMP is led by the switch from high expression of cAMP in embryonic (P5) neurons to low levels in maturing/adult neurons, which could account for the switch of adult neurons to become responsive to myelin associated inhibitors (Cai et al., 2001). Polyamines were identified as one downstream effector of cAMP mediated by increased arginase I levels, the rate limiting enzyme in polyamine synthesis (Cai et al., 2002). Overexpression of either arginase I or polyamines allowed D R G neurons to overcome inhibition by myelin (Cai et al 2002). The ability of both neurotrophins and cAMP to increase neurite outgrowth over an inhibitory substrate was mediated by activating tyrosine kinase receptors (e.g. trkB) and increasing E R K activity levels (Gao et al., 2003). If tyrosine kinase receptors were blocked, neither cAMP nor neurotrophic treatments produced an increase in the axonal growth response. The same group led by Dr. Mary Filbin showed that the transcription factor CREB was required for cAMP-enhanced neurite growth, both in vitro and in vivo, on myelin substrate (Gao et al., 2004). Multiple factors and pathways are involved in the increased intrinsic growth response activated by cAMP and neurotrophins, and novel ones are being added to the list. Gene array screening revealed several genes that displayed increased expression after both the conditioning lesion paradigm or cAMP treatment, and recently confirmed the involvement of IL6 in neurite outgrowth (Cao et al., 2006). cAMP is not the only purine that can influence neurite outgrowth. In vitro, cGMP can reverse the repulsion by the soluble semaphorin-3 A (see section 1.3.B.i and 1.3.B.ii) into attraction (Song and Poo, 2001). Conceivably, both nucleotides cAMP/cGMP play an important role in the response of growing axons to inhibitory proteins. Therefore, manipulating either of these nucleotides could prove useful for spinal cord sprouting/regeneration. In summary, multiple pathways are stimulated by neurotrophins or conditioning lesions. These include tyrosine kinase receptors and subsequent downstream components such as cAMP and CREB, eliciting an enhanced axonal growth response. Therefore, in-vivo treatment with neurotrophic factors could possibly increase the axonal growth response of CNS neurons.  24  I.4.C. Priming supraspinal descending neurons in vivo with growth factors While the effects of a conditioning lesion and pretreatment priming of neurons has been extensively tested in peripheral D R G neurons, pretreatment priming of descending supraspinal neurons has not been attempted. Therefore, I examined the effect of pretreatment of rubrospinal neurons with the neurotrophin BDNF prior to a cervical spinal cord injury in chapter 2 of this thesis. Additionally, I tested if cell body treatment of rubrospinal neurons at time of spinal cord injury would have any effect on the behavioral outcome since it had been reported to increase axonal growth into the permissive environment of a PNG (Kobayashi et al., 1997).  I.4.D. Side effects of priming neurons with neurotrophins Pretreatment of neurons with neurotrophins in addition to eliciting axonal growth could produce physiological effects possibly confounding our conclusions. Interestingly, many of the neurotrophic factors used in spinal cord injury experiments reduce food intake. B D N F infused into the cerebral ventricles at small doses of 1.5 ug per day caused a reduction in weight (Lapchak and Ffefti, 1992; Pelleymounter et al., 1995). Pelleymounter concluded the effect of B D N F on weight was not due to a toxic effect but rather it reduced appetite and thereby food intake (Pelleymounter et al., 1995). Subsequent work using mice with heterozygous BDNF deletions further demonstrated the important role of BDNF in food consumption as knockouts became obese (Kernie et a l , 2000). The effect of BDNF on eating behavior is partially mediated via the hypothalamus (Xu et al., 2003). Infusion of either BDNF or NT4/5 into the ventricles rescued the B D N F +/- obesity phenotype (Kernie et al., 2000). More recent work suggested that the effect of BDNF on food consumption might be mediated at more than one location in the brain. The dorsal vagal complex in the brain stem alters food intake via B D N F (Bariohay et al., 2005). Further support of BDNF-trkB signaling on food intake comes from mutant mice that express full-length trkB at 25 % of normal levels become hyperphagic and overweight (Xu et al., 2003). The importance of trkB in food consumption is additionally supported by the finding that a spontaneous mutation of the trkB receptor in humans, which elicits reduced BDNF-trkB signaling, is associated with obesity (Yeo et al., 2004). Interestingly, even peripherally administered BDNF has an effect on food intake and weight gain. Five subcutaneous injections per week of B D N F caused ~ 25 % reduction in weight gain (Tonra et al., 1999). Daily subcutaneous injections of B D N F resulted in reduced food intake 25  and weight loss (not just reduction of weight gain) (Ono et al., 2000). Even when the daily injection was stopped, food intake was reduced for at least one week. This research emphasizes the powerful effect of BDNF on eating behavior. BDNF is not the only neurotrophin that causes reduced food intake. N G F infusion into the ventricles also caused a reduction in food intake and thereby affected weight gain (Williams, 1991). Intraparenchymal infusion of NGF can cause weight loss if it diffuses into the CSF (Tuszynski, 2002). NT-3 infused into the substantia nigra caused decreased food intake and weight reduction (Martin-Iverson and Altar, 1996). GDNF was also reported to result in weight loss if infused into the intracerebral ventricle (Giehl et al., 1998). This weight reduction by GDNF is mediated through reduced food intake (Turner et al., 2006). C N T F produced an extreme reduction in weight and it might be acting by increasing neurogenesis in one of the feeding centers of the brain, the hypothalamus (Lambert et al., 2001; Kokoeva et al., 2005). CNTF, or its analog Axokine, is actively being pursued as a treatment for obesity (Preti, 2003). Therefore, altering trophic factors or their receptors seems to universally play a role in controlling appetite and thereby the weight of animals. Reduced food intake could play a role in functional recovery after spinal cord injury. Reduced food intake, better known as dietary restriction (DR), elicits positive effects in several brain diseases/pathologies if implemented prior to insults (see section 1.6.E) including stroke, Parkinson's, Huntington's, and Alzheimer's disease (Duan and Mattson, 1999; Y u and Mattson, 1999; Duan et a l , 2003b; Patel et a l , 2005). DR is neuroprotective if started prior to insult (Bruce-Keller et al., 1999; Duan et al., 2001a; Duan et al., 2001b; Anson et al., 2003). Therefore, the unintentional DR induced by neurotrophin pretreatment could be neuroprotective and/or promoting plasticity.  26  1.5. Rationale of pretreatment studies Obviously, we cannot act on a premonition to start infusing trophic factors into the brain before a person is an unfortunate victim of a spinal cord injury. However, there are at least two scenarios where pretreatment could prove valuable. In the first case, when a patient arrives in emergency with a spinal cord injury, other than neuroprotective treatment, it is unlikely that other invasive treatments to the spinal cord would be considered immediately. After circulation and respiration are stabilized it is more likely the patient will be monitored to examine the extent of injury and functional loss, and if there is evidence for spinal canal obstruction, decompression surgery is performed in many centers (Fehlings and Perrin, 2006). Only then, maybe two or three weeks (or longer) after injury, would an intervention to the spinal cord itself be considered. At this time point the glial scar (see section 1.3.B.i) would have been mostly formed and scar tissue removal might be required which likely results in further injury. In this scenario, pretreatment with trophic factors could be used to prime the neurons with or without the addition of other treatment molecules to e.g. reduce the general inhibition by myelin. Other scenarios that I envision for the possible application of pretreatment of neurons with trophic factors are the non-invasive natural ways of increasing endogenous trophic factor production in the CNS. There are two well-documented methods that will increase various trophic factors, specifically BDNF, in adult mammalian brains: exercise and dietary restriction.  1.5.A. Exercise Voluntary exercise and the subsequent increase in trophic factors in various brain regions have a positive effect on neurogenesis, learning, depression, and recovery from brain insult (Vaynman and Gomez-Pinilla, 2005). Voluntary wheel running in rats consistently increased BDNF (mRNA and protein) expression, in not only the expected motor cortex but also the hippocampus. Other trophic factors such as NGF (Neeper et al., 1996) and FGF-2 (GomezPinilla et al., 1997) are induced to a lesser extent and more transiently. More specifically, as little as seven days of running increased BDNF protein expression in the lumbar spinal cord by approximately 50 % along with increased mRNA levels of trkB (Gomez-Pinilla et al., 2002). NT-3 was also increased by 7 days of running along with a transient increase in trkC (Ying et al., 2003). This suggests that exercise could prime neurons to regenerate more efficiently if an injury 27  occurs. Recently this hypothesis was tested in the peripheral nervous system and exercise prior to a sciatic nerve lesion was found to increase regeneration (Molteni et al., 2004). However, BDNF, trkB, and N G F are downregulated when animals stop exercising, in some cases to levels lower than before they started 'training' (Widenfalk et al., 1999; Radak et al., 2006). Whatever activity level a person is normally engaged in during their daily life (from extensive exercise to carrying groceries), the level of activity will drastically decrease acutely after injury and hence it is likely that BDNF and trkB expression will decrease. Exercise appears to be beneficial when started after SCI. Running increased BDNF expression after SCI as compared to controls, along with p-CREB and GAP-43 (Ying et al., 2005), and treadmill training improved motor function and reduced allodynia (Multon et al., 2003; Hutchinson et al., 2004; Engesser-Cesar et al., 2005). However, partial SCI rats appear capable of performing higher levels of activity after SCI compared to humans. The level of activity needed to increase BDNF or other appropriate proteins might be difficult to reach in humans with a spinal cord injury. Nevertheless, whatever level of exercise (rehabilitation) can be performed by patients, it should be highly encouraged to promote plasticity and hopefully some form of recovery. While the best exercise regimen for humans is still debated, some form of aggressive rehabilitation scheme is deemed beneficial (Dobkin et al., 2006). Additionally, it is standard practice for humans with SCI to get extensive rehabilitation  I.5.B. Dietary restriction  Dietary restriction (DR) without malnutrition has profound effects on multiple systems. It is important to note that excessive dietary restriction is detrimental for an organism's health and the positive effects of DR are dependent on avoiding malnutrition. There are two main forms of dietary restriction: calorie restriction (CR) in which overall calories are reduced from 10 to 40 % and intermittent fasting (IF). CR by 30-40 % appears to be optimal depending on species. Throughout this thesis the percentages regarding CR mean the percentage of calories restricted, e.g. 40 % CR indicates the animals are receiving 60 % as much as the control animals. The main form of IF is every-other-day fasting (EODF) in which organisms eat ad libitum for 24 hours followed by 24 hours of fasting (no calories consumed with the only oral consumption being non-caloric fluid intake - water) (Anson, 2004; Mattson, 2005). E O D F is the form of dietary restriction used in this thesis (chapter 3 and 4). The effects of DR/EODF will be covered in sections 1.6 to 1.8. 28  1.6. Dietary restriction has diverse effects on the biological system 1.6.A. Dietary restriction increases lifespan Francis Peyton Rous, a Nobel Prize winner, published in 1914 that DR reduced the incidence of cancer in rodents (Rous, 1914). Since cancer is a major cause of death in laboratory rodents, the work hinted that DR could extend lifespan if specifically measured. Two researchers from Yale, Osborne and Mendel, followed this up and published two papers in Journal of Biological Chemistry and Science in 1915 and 1917 demonstrating that DR delayed the loss of fertility and increased lifespan when started soon after weaning (Osborne and Mendel, 1915; Osborne et al., 1917). McCay from Cornel University subsequently published a number of papers showing various forms of food restriction extended lifespan in rats (McCay et al., 1935; McCay et al., 1937). Research continued to explore the effects of DR but most of the work was ignored or marginalized by mainstream science. The next major finding occurred when Walford's group published their finding in Science that CR extends lifespan even when started in middle age (12 months of age) mice (Weindruch and Walford, 1982). The life extension effect was not as effective when started at middle age as compared to soon after weaning (which is typically used in CR longevity studies). However, Masoro's group found that CR extended lifespan equally when started at 6 months of age as compared to the traditional paradigm of soon after weaning (Yu et al., 1985). 40 % CR initiated in 19 month old male B6C3F1 mice started to increase mean time to death within 2 months after induction of the diet and these finding were found throughout the rest of the experiment (Dhahbi et al., 2004). Importantly, in older animals the CR diet regime should be introduced gradually (Weindruch and Walford, 1982; Dhahbi et a l , 2004; Rae, 2004) because some groups have failed to observe an effect if adult animals were directly switched to 40 % CR (Forster et al, 2003). What is striking about the effect of dietary restriction is its universality in extending lifespan. DR extended lifespan in yeast (Saccharomyces cerevisiae) (Jiang et al., 2000; Jiang et al., 2002; Lin et al., 2002; Anderson et al., 2003a; Anderson et al., 2003b; Kaeberlein et al., 2004), nematode worm (Caenorhabditis elegans) (Klass, 1977; Lakowski and Hekimi, 1998; Houthoofd et al., 2003), fruit fly (Drosophila) (Chapman and Partridge, 1996), C3B10RF1 mice (Weindruch et al., 1986), Sprague-Dawley rats (Berg and Simms, 1960, 1965), Fischer 344 rats (Yu et al., 1982), Labrador retriever dogs (Kealy et al., 2002; Lawler et al., 2005), and Hereford cows (Pinney et al., 1972). More specifically, a meta-analysis of 14 studies in rats revealed that 29  DR animals on average increased maximum lifespan by 33 % and the average time to double the rate of mortality was increased by 74 % (Finch, 1990). Additionally, another form of DR, everyother-day fasting also extends lifespan in rats (Goodrick et al., 1982, 1983a, b; Beauchene et al., 1986) and mice (Talan and Ingram, 1985; Ingram and Reynolds, 1987). It would be hard to find any other treatments that have proven itself repeatedly and in such diverse set of species.  I.6.B. How dietary restriction works There are a number of theories that attempt to explain the effect of DR on lifespan. They include developmental delay (which is now discounted (Weindruch and Walford, 1982)), reduced metabolic rate (now in question due to reports of CR animals having equal or higher metabolic rate (Masoro et a l , 1982; Nisoli et a l , 2005)), glucocorticoid cascade, decreased fat, reduced reactive oxygen species, cell survival hypothesis, increased protein turnover, decreased glucose and insulin levels, decreased IGF-1 signaling, and the hormesis hypothesis (Sinclair, 2005). DR's effect is likely due to the combination of many of these mechanisms. The hormesis hypothesis states that D R is a mild stressor that elicits a survival response in the organism, which protects it from stressors that are more serious and thereby leads to an extension in lifespan. Interestingly, it is not just the stress from DR that extends lifespan but a number of other stressors that include chemotherapeutic agents, ionizing radiation, antibiotics, heavy metals, pesticides, chloroform, pro-oxidants, and hyper-gravity also increase lifespan (Rattan, 2004). One can think of hormesis like an exercise program, each time we exercise our body receives a stress signal and responds by altering gene responses to cope better with the next bout of exercise. However, too much stress is detrimental. One cannot start an exercise program by running a marathon; the body would not be able to respond to that level of stress and would probably be in pain and unable to move for a week. In addition, if one ran a marathon every day, the probability of an unfavorable overall outcome would likely increase. The same is true for stressors to the body that extends lifespan. The dose response curve to these stressors is an inverted U . For example, high doses of irradiation decreased lifespan, but low doses enhanced lifespan in adult housefly (Allen and Sohal, 1982), mice (Lorenz et al., 1955; Ina and Sakai, 2004, 2005) and human males (Mine et al., 1990). From the above research, one can view CR as a mild stressor that causes adaptations in the organism to fight off aging more efficiently. While using exercise to explain CR's hormesis theory, exercise had only a small effect on lifespan (average longevity increased ~ 9 %) and caused no increase in maximum longevity in rats 30  (Holloszy et al., 1985; Holloszy, 1993, 1998) in contrast to CR (increase average longevity (based on meta-analysis of 14 studies) by ~ 33 % and maximum longevity) (Finch, 1990). Numerous molecular pathways change in response to DR. The involvement of these pathways was supported in single-pathway knockout animals that demonstrated an increase in longevity, however to a lesser degree than the animals on D R (Barger et al., 2003). Even though DR only partially blocks/reduces particular pathways while the knockout experiments completely eliminate each of these pathways, the increase in longevity in these knockout experiments is still always less than the simple DR experiments. These results argue for three points: i) targeting only one pathway is not the best way to treat aging (and probably most other biological diseases/injury), ii) DR's effects on longevity (and probably most other effects) involve multiple pathways, and/or iii) completely blocking these pathways might not elicit the desired effect. Despite a considerable body of research, we do not know exactly how such a simple paradigm like DR can extend lifespan and exert multiple other positive effects. A definitive single explanation of the DR's effect is lacking because D R affects many systems and biological pathways simultaneously.  1.6.C What are the positive effects of dietary restriction on diseases and leading causes of death? The top ten leading causes of death in humans are listed in Table 1.1 (Kochanek et al., 2004). Included in this list are the effects of DR on these leading causes of death, and the relevant citations. As you can see from the table, CR/EODF has positive effects on 9 of the top 10 leading causes of death in animal models. Hence, CR/EODF could affect 74.3 % of the 78.7 % of the leading causes of death (the top 10 accounts for 78.7 %). The only cause of death in the top 10 not affected by CR is accidents. In the light of this, the life extending effect of CR/EODF does not come as a surprise. CR/EODF delays the onset of these death-causing diseases significantly and in some cases almost eradicates them. The reason I am presenting this data is to demonstrate the global/inclusive effects of CR/EODF. DR not only affects a few subsystems of the organism, instead it treats the whole body. The multiple-system effect of DR is important for the understanding of this thesis. I would also like to note that many of the leading causes of death are inter-related. Heart disease and stroke are directly linked, with atherosclerosis playing an important role in both conditions. Improving 31  the health of the circulatory system will reduce both heart disease, and the incidence and severity of stroke. The circulatory system is negatively affected by the development of diabetes, as is kidney disease. This emphasizes possible common underlying biological mechanisms and the link between organ systems, i.e. the injury/disease in one system can have cascading effects on other systems. Heart disease is the # 1 cause of death in humans. CR/EODF has repeatedly been shown to have positive effects on heart function. CR diminished the accumulation of oxidative damage to the heart (Pamplona et al., 2002), which is a consistent theme in the CR field for all tissues studied. I will examine the importance of CR/EODF effect on the heart and circulatory system (including stroke, # 3) and its relevance to SCI patients in chapter 5 (see section 5.5.B). Lifetime DR dramatically reduced the incidence of cancer (#2), and it might play a large role in the increased life span in rats on DR. Additionally, while most studies started animals on DR soon after weaning, reducing calories also reduced cancer incidence when started in middle age mice (12 month age) (Pugh et al., 1999). Another method of DR, EODF, also reduced tumorigenicity in rodents (Kritchevsky, 1990). Interestingly, not only severe D R reduces cancer even a mere one day of fasting a week reduced the incidences of cancer in p53 +/- transgenic mice (Berrigan et al., 2002). The one day a week of fasting resulted in about one half the effect of 40 % CR. CR implemented after a radiation dose, known to induce cancer in myeloid leukemia prone mice, was effective in reducing cancer death (Yoshida et al., 2006). I might venture forward the hypothesis that many of the chemotherapies used for cancer treatment might be working at least partially by the dramatic reduction in food intake caused by the toxic treatments. After having this idea I found that I am not alone in raising this possibility (Mukherjee et al., 2004). Most people can probably understand/accept that CR/EODF reduces the probability of developing diabetes (# 6). However, it might be less intuitive that there is also experimental support to suggest that CR/EODF might protect an organism from chronic lower respiratory disease (# 4) (Dong et al., 1998; Vasudevan et al., 2006), developing influenza (# 7) (Effros et al., 1991), and against septicemia (# 10) (Perkins et al., 1998). I will discuss effect of DR on septicemia in chapter 5 at greater length (see section 5.5.B). C R / E O D F has a very positive effect on reducing kidney disease (# 9) and I will discuss DR's effect on stroke (# 3) and Alzheimer's disease (# 8) in section I.6.E. CR/EODF's effects go beyond just extending lifespan; it can have positive effects on a diverse range of biological functions that would increase the quality of life. For example, age-related cataract formation is reduced with CR. At 22 months of age, 90 % of 32  control rats had grade 5 cataracts while only 18 % of the CR animals had this same grade of cataracts (Mura et al., 1993). These results were confirmed by two other papers demonstrating reduced cataract formation in CR animals (Taylor et al., 1989; Mura et al., 1993; Taylor et al., 1995). CR also reduces or fully prevents age related loss of hearing in mice (Sweet et al., 1988; Someya et al., 2006). In summary, I hope the readers have acquired an impression of the powerful and diverse effects of CR/EODF from Table 1. Usually at this point people quickly start to build up arguments against CR/EODF to defend their 'reasoning' of why they would not, or others should not, implement either regimen. I will counter against one of the first points usually raised against CR/EODF for the purpose of demonstration.  I.6.D. Dietary restriction/Every-other-day fasting will not make you weak and frail One of the first arguments raised against any form of CR/EODF is that the person will waste away and become weak and frail. The truth might be exactly the opposite. On a 30 % C R diet mammals will lose approximately 30 % in weight over time. However, most of this loss would be body fat and going by the current average percentage of fat in humans, and obesity rate, this would be a positive outcome. On an EODF diet though, there would be little weight loss. Mice on EODF eat close to twice the amount on days they receive food and end up only weighing ~ 5 % less than control animals (Goodrick et al., 1990; Anson et al., 2003). Short-term human studies indicate similar results with the people on EODF either losing only a small amount of weight (mostly fat) or none at all (Halberg et al., 2005; Heilbronn et al., 2005b; Heilbronn et al., 2005a). Beyond weight maintenance, how does CR/EODF affect muscle function? As organisms age there is a general decline in muscle mass and strength. CR reduced the age-related increase of TNF-a levels (inflammatory marker) in the muscle and serum in 26 month old rats and prevented muscle loss in both soleus (slow-twitch) and superficial vastus lateralis (fast-twitch) (Phillips and Leeuwenburgh, 2005). Other groups found similar results with reduced loss of muscle fibers and size in CR aging rats in a number of muscle groups (vastus lateralis, rectus femoris) (McKiernan et al., 2004). Alternating fasting for 3 days a week attenuated the agerelated loss of motoneuron in rats (Kanda, 2002). The apoptotic loss of muscle cells in aging animals is accompanied by increased caspase activation (e.g. 350 % increase in procaspase-12) (Dirks and Leeuwenburgh, 2004). CR resulted in a reduction in procaspase-12 levels and a 33  reduced apoptotic index in aging muscles (Dirks and Leeuwenburgh, 2004). Apoptosis repressor with a caspase recruitment domain (ARC), an apoptotic inhibitor, which inhibits cytochrome c release, normally declines with age but increases in animals on CR. Therefore, A R C could be responsible for the reduced apoptosis of muscle cells in CR animals. Even a mild form of CR (20 %) resulted in reduction of mitochondrial damage and morphological changes in aging rat muscles (Usuki et al., 2004). While CR might reduce muscle apoptosis and decrease the reduction of muscle mass, does it actually help muscle function? Rats on 40 % CR started at weaning displayed higher muscle force at 25 and 32 months of age, both in soleus (slow-twitch) and extensor digitorum longus (fast-twitch) compared to the control group at both time points (Mayhew et al., 1998). Control animals showed the typical age-related decline (from adulthood - 25 months, to old rats - 32 months) of ~ 30 % reduction in muscle force with the CR animals performing significantly better. Interestingly, even in the adult animals (not old) the CR group had higher muscle force than age matched controls, suggesting that not only did CR prevent the age related decline but it also improved muscle function at the adult age before the main deterioration period in muscle strength occurred. With aging there is an increase in extracellular space in the muscles and hence the organism has a reduced muscle mass/body mass ratio. CR (40 %) attenuated the age-related increase of extracellular space and resulted in a greater muscle mass/body mass ratio along with increased strength-to-body mass ratio in fast-twitch muscle (but not slow-twitch muscles) (Payne et al., 2003). Interestingly, for fast-twitch muscles the CR animals actually had higher muscle force than young animals, which is truly remarkable. What about CR's effect on the aerobic ability of skeletal muscle? Researchers found a ~ 45 % reduction in VO2 max as rats age from 10 months to 35 months while 40 % CR animals showed absolutely no decline in muscle aerobic ability (Hepple et al., 2005). This research group also found no reduction in muscle force/kg of the 35-month-old CR groups (aged CR animals performed as well as the 10 month old control rats) while there was a drastic reduction in aged control animals. While most of the above-mentioned studies used in vitro preparations to test muscle force etc., researchers have also explored the in vivo effect on general motor function in CR/EODF animals. For example, the maximum coordinated running speed on a rotarod test was increased in CR animals (40 % CR started at 4 months of age) at 6, 16, and 22 months of age along with better avoidance learning (Dubey et al., 1996). The increased motor performance occurred in conjunction with reduced oxidative damage in the brain. CR also had a positive effect on  34  numerous other motor tasks that include time to climb out of an obstacle, ability to resist slipping at increasing angles, and duration of hanging from a bar (Ishihara et al., 2005). In summary, in contrast to CR/EODF making you frail this diet appears to have very pronounced effects on the reduction of the age-related loss of muscle fibers but more importantly on the prevention of the decline of muscle aerobic capacity and muscle specific force. There is even evidence that CR can increase muscle specific force in old animals to levels greater than young animals. This line of research is further supported by an increase in motor performance of animals on CR.  I.6.E. Effect of dietary restriction on the central nervous system  Since this thesis is interested in the effects of DR on the central nervous system, I will go into further details briefly. CR/EODF if followed for several months has positive effects on a number of CNS diseases/insults. The only known CNS disease that does not benefit from CR/EODF is amyotrophic lateral sclerosis (ALS), in which CR was actually detrimental (Pedersen and Mattson, 1999; Hamadeh et al., 2005). However, the range of positive effects CR/EODF has on other CNS diseases/injury is remarkable. Stroke is one of the leading causes of death in humans (table 1.1). Due to the numerous effects that DR has on improving, or reducing, the aging induced negative effects on the vascular system, the probability of stroke would be reduced (see table 1.1, heart disease and stroke). More specifically in stroke prone rats, CR (40 %) started in adulthood reduced blood pressure, dramatically decreased the percentage of animals that suffered strokes, and increased lifespan (Stevens et al., 1998). Interestingly, a reduction in the function/health of the circulatory system has been linked to numerous CNS disorders that include Alzheimer's disease and age-induced dementia (Kalaria and Ballard, 1999; Kalaria et a l , 2001; Lewis et al., 2006). Beyond the reduction of stroke incidences, prophylactic DR protects against stroke induced brain damage. The cerebral ischemia induced by stroke results in degeneration and death of neurons. Three months of EODF treatment prior to a middle cerebral artery occlusion-reperfusion (MCAO-R) dramatically reduced the infarct volume and increased both heat-shock protein 70 (HSP-70) and glucose-regulated protein 78 (GRP-78) (Yu and Mattson, 1999). The stress proteins, HSP-70 and GRP-78, are neuroprotective (Lowenstein et al., 1991; Y u et al., 1999). More importantly, EODF resulted in improved behavioral recovery.  35  The devastating effects of Alzheimer's disease (another leading cause of death: table 1.1) is a growing problem in our society and DR has produced favorable results. Zhu and coworkers demonstrated in presenilin-1 K O mice (one model of Alzheimer's disease) that 3 months of EODF prior to kainate injections into the hippocampus protected neurons from death (Zhu et al., 1999). A measure of oxidative stress and lipid peroxidation (4-hydroxynonenal) were decreased in EODF animals. The authors concluded that EODF counteracted the death enhancing effect of presenilin-1 mutation and suggested that it may be beneficial to humans with familial forms of Alzheimer's disease. Six years later this work was followed up by another group that found as little as 6-14 weeks of CR (40 %) reduced the amount of AP-plaques by 40-55 % in two different strains of Alzheimer's disease transgenic mice (Patel et al., 2005). CR (30 %) prevented the generation of AP peptides through increased a-secretase activity (ADAM-10) (Wang et al., 2005). Elucidating another possible mechanism of CR's effect, CR at 32 % was found to reduce the age-related increase of p75 expression and Ap. p75 increases the p cleavage of amyloidprecursor protein (resulting in AP) via the increase of the second messenger ceramide (Costantini et al., 2005). CR increased trkA expression and reduced the age-related increase in p75, ceramide, BACE1 and Ap expression. Recently, another group found that CR decreased Ap production in the brain by increasing sirtuin 1 (SIRT1: see section 1.7.A) and decreasing ROCK1 protein levels (see section 1.6.2) (Qin et a l , 2006). In summary, five different groups have observed positive effects of CR/EODF for Alzheimer's disease, and recent works have elucidated some of the possible mechanism behind this effect. Another destructive neurodegenerative illness is Parkinson's disease. One animal model for this disease is the use of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) to damage dopaminergic neurons. EODF followed for several months prior to insult caused a significant preservation of dopamine neurons in the substantia nigra compared to control treated animals (Duan and Mattson, 1999). The control animals had a deficit on the rotarod test while EODF animals were significantly better and returned to baseline pre-injury levels. This work was extended into a primate model of Parkinson's disease. Rhesus monkey on six months of 30 % CR prior to a MPTP insult had higher levels of dopamine in the striatal region and increased glial derived neurotrophic factor (GDNF) expression after the injury, along with increased distance and speed of movement (Maswood et al., 2004). There was a trend toward increased survival of dopamine neurons in the striatum and increased manual dexterity, but neither reached statistical significance.  36  EODF also has positive effects in Huntington's disease. 3-nitropropionic acid (3NP) administration (daily i.p. doses for three days) which produces a selective loss of striatal neurons and motor deficits was used as a model for this disease in 1999 (Bruce-Keller et al., 1999). Animals on the EODF regime for 4 months prior to the start of 3NP injections showed a dramatic reduction in lesion volume. While the control animals showed a progressive deterioration in motor performance on the rotarod test, the EODF animals performed at normal uninjured levels. In transgenic mice expressing mutant human Huntingtin protein, EODF (started at 8 weeks of age) delayed the onset of the disease, increased overall survival, and improved performance on a rotarod test (Duan et al., 2003b). Additionally, E O D F increased levels of BDNF and HSP-70 in the striatum and cortex, which were decreased in the transgenic control animals. Brain atrophy and caspase-1 activation were also decreased in the EODF animals in both the cortex and striatum. In summary, CR/EODF if followed for several months has very positive effects on both biochemical and behavioral outcomes on four major brain diseases/insults that affect humans. These robust results suggest CR/EODF could have a beneficial effect on a number of other CNS insults including neurodegeneration and injury. The effect of CR/EODF on SCI has not been tested previously and is one focus of my thesis (chapter 3 and 4).  37  1.7. Dietary restiction may improve the outcome of spinal cord injuries via multiple mechanisms As I have outlined previously (see section 1.3) the three main reasons why functional recovery is limited after SCI is due to secondary damage, glial scar/myelin inhibitory proteins, and the weak intrinsic axonal growth response. Hence, a combinatorial approach would most likely elicit an optimal response. The diverse effects of dietary restriction suggest that DR could have positive effects on all three of the obstacles. A microarray study examining the effect of EODF on a number of different regions of the CNS (cerebral cortex, hippocampus, striatum and cerebellum and spinal cord) interestingly found that the greatest number of changes occurred in the spinal cord (twice as many as the other CNS tissues studied) (Xu et al., 2005b). I will briefly outline how DR could possibly have positive effects on the three main obstacles to recovery after SCI.  1.7.A.  Dietary restriction may reduce secondary damage and replace lost cells There are a number of reasons why dietary restriction would likely reduce secondary  damage to the injured spinal cord. As outlined in earlier in the introduction (see section 1.3.A) one of the primary triggers of the cascade of secondary damage is ischemia induced by the initial physical trauma. Therefore, it is interesting that EODF animals, if started on the diet regimen 3 months before a focal brain ischemia (MCAO-R), showed more than a 50 % reduction in infarct volume along with improvements in neurological scores (Yu and Mattson, 1999) (see section 1.6.E). Therefore, it appears that EODF could interfere with the ischemia-induced cascade that normally results in loss of neurons and tissue. The possibility of reducing the detrimental outcome of one of the main initiator of the secondary cascade would offer a great upstream opportunity to reduce subsequent damage and thereby promote functional improvement. EODF was noted to increase the levels of B-hydroxybutrate while no increase was found in control or 40 % CR animals (Anson et al., 2003).fi-hydroxybutrateis a fat derived fuel that is used by the brain when carbohydrate supplies are low, including in the fasted state (Seyfried et al., 2003; Seyfried and Mukherjee, 2005). Interestingly,fi-hydroxybutrateis known to protect neurons in animal models of Parkinson's and Alzheimer's diseases (Kashiwaya et al., 2000; Veech et al., 2001). Hence, the preferential increase of B-hydroxybutrate in EODF animals might  38  partially explain the increased neuronal survival after kainic acid injury when compared to either control or 40 % CR animals (Anson et al., 2003). A number of studies have shown beneficial effects using glucocorticoid as a treatment for SCI (see section 1.3.B.ii). Corticosteroids such as methylprednisolone act via the glucocorticoid receptor (though the actual mechanism of its positive SCI effects might be mediated via reduced lipid peroxidation - see section 1.3.B.ii). Interestingly, CR causes an increase in glucocorticoid hormone levels (Sabatino et al., 1991). CR-induced increase in corticosteroids could mediate a neuroprotective effect similar to MP treatment. One hypothesis is that the increased corticosteroid production in CR animals represents the adaptive stress response mediated by CR. However, long-term elevated corticosteroids levels are actually detrimental, causing neurodegeneration (Patel et al., 2002). It is argued that increased corticosteroid production in CR animals is offset by the increase in other neuroprotective mechanisms (Berner and Stern, 2004). In addition, CR decreased mRNA levels of glucocorticoid receptors in the dentate gyrus and CAI region of the hippocampus, and the corresponding protein levels in the cortex (Lee et al., 2000b). Hence, animals would be less responsive to the negative effects of chronic increased glucocorticoid levels. There is considerable controversy in the spinal cord field on the actual mechanism of action and clinical effectiveness of methylprednisolone (Hall and Springer, 2004). The interesting component of DR's  possible use in SCI is that it does not rely on just affecting  one pathway/mechanism. Therefore, it is possible that the small positive effects of separate pathways could accumulate into meaningful positive outcome with D R treatment. Dietary restriction has been repeatedly shown to decrease excessive inflammatory responses (Chung et al., 2002). The ability to dampen the inflammatory response after SCI could be beneficial. One of the molecular triggers of the secondary cascade after trauma to the spinal cord is the transient increase of the nuclear factor K B ( N F K B ) and tumor necrosis factor alpha (TNF-a) (Tyor et al., 2002; Brambilla et al., 2005). N F K B plays a major role in the inflammatory response by regulating the transcription of a number of pro-inflammatory molecules (TNF-a, IL6 and COX-2) (Chung et al., 2002). Inhibiting N F K B in astrocytes reduced inflammation and improved recovery after spinal cord injury (Brambilla et al., 2005). CR reduced the age related increase in N F K B (Kim  et al., 2002). More specifically, CR also decreased N F K B in the cortex  while at the same time increased the N F K B inhibitor, I F K B (Lee et al., 2000a). In addition, C R decreased the production of TNF-a , IL-ip, and IL-6 (Chung et al., 2001). One way to reduce the effect of the increased TNF-a level is reduction of its receptor, TNFR. A 25 % reduction in food intake caused a reduction in the membrane form of TNFR (mTNFR) and the soluble form of 39  TNFR (sTNFR) (Raina et al., 1999). A n intervention that reduced TNF-a after SCI has shown positive effects on reducing inflammation and tissue injury, resulting in improved functional outcome (Genovese et al., 2006). The ability of CR to reduce N F K B and TNF-a could prevent the progressive nature of secondary damage to the spinal cord after injury and thereby lead to better functional recovery. In addition to the inflammatory secondary cascade after SCI, further damage is induced by the strong upregulation of free radicals and oxidation. Sullivan demonstrated that the spinal cord produced more free radicals in the uninjured state than cortical cells (Sullivan et al., 2004); and reactive oxygen species (ROS) are further increased after SCI (Luo et al., 2002a; Luo et al., 2002b; X u et al., 2005a). Oxidative stress plays a major role in SCI secondary damage (Hulsebosch, 2002; Bao and Liu, 2004). Various experiments have shown the efficacy of CR in reducing oxidative damage (Yu, 1993) including in regions of the brain (Dubey et al., 1996). Dubrey et al., found as little as 6 weeks of CR caused a reduction in oxidation in whole brain homogenate as compared to control fed animals. Interestingly, protein oxidative damage (as measured by carbonylation) can be reduced by as little as 3-6 weeks of CR, which matched the effect of long-term CR (Forster et al., 2000). The mechanism by which CR reduces oxidation is believed to be due to reduction of ROS (Sohal et al., 1994). CR appears to fundamentally change most cells in the body (tested tissue: brain, heart, liver, brown adipose tissue) to become more efficient by increasing mitochondrial biogenesis, decreasing oxygen consumption, maintaining or increasing ATP content, and drastically reducing ROS production (Nisoli et al., 2005; Lopez-Lluch et al., 2006). Some studies suggest increased oxidative metabolism in CR animals (when adjusted for weight) (Masoro et al., 1982; Nisoli et al., 2005) while maintaining close to normal oxygen consumption. Six-month-old rats on 40 % CR demonstrated only a 25 % reduction in oxygen consumption, and 26-month-old CR rats showed only a 2 % reduction in oxygen consumption (Selman et al., 2005). These CR animals produced normal or increased levels of ATP while having reduced ROS and consuming near normal oxygen levels. This might be one of the few cases of a 'free lunch'. ROS were decreased in EODF animals as compared to the control group after either an increase in oxidative stress (amyloid P-peptide and Fe ), or metabolic insult (the mitochondrial 2+  toxin 3-nitropropionic acid) (Guo et al, 2000). Guo and colleagues suggested that the observed greater survival of synapses after injury in EODF animals was due to the decreased ROS and increased levels of heat shock proteins. Increased numbers of functioning synapses in the perimeter of a spinal cord injury site could preserve more functional ability. 40  In addition to the increased efficiency qf mitochondria that allows the production of normal levels of ATP but reduced ROS, there is also an increase in the antioxidant scavenger system which may play a role in the reduced ROS production after injury. E O D F animals showed an increase in the antioxidant scavenger system (Cu-Zn SOD, catalase, glutathione (GSH)) along with reduced lipid peroxidation and increased neuronal survival after kainic acid insult as compared to control animals (Sharma, 2005). Many neuroprotective agents used in spinal cord injuries act by reducing lipid peroxidation (Hall and Springer, 2004) (see section 1.3.A.iii). A n increase of free radical scavenging after experimental spinal cord injury can improve functional outcome (Kamencic et a l , 2001; Hillard et al., 2004; Guizar-Sahagun et al., 2005). Therefore, it is conceivable that CR/EODF may reduce secondary damage and improve functional recovery after spinal cord injury due to its ability to reduce ROS via mitochondrial efficiency, and increase antioxidants, which results in reduction of lipid peroxidation. Beyond the mitigation of the secondary cascade and the production of ROS, other molecules can significantly affect secondary damage after neuronal insults. Induced upregulation of epithelial nitric oxide synthase (eNOS) in stroke models reduced lesion size and improved functional recovery (Endres et al., 1998; Jung et al., 2004; Asahi et al., 2005). In a traumatic brain injury model, L-arginine was used to increase eNOS, which resulted in reduced lesion size in wild type animals but not in eNOS -/- (Hlatky et al., 2003). A recent paper showed that EODF increased eNOS in the brain along with the induction of increased mitochondrial biogenesis (Nisoli et al., 2005). The EODF-induced increase of eNOS could produce a number of beneficial effects including increased blood flow to re-supply the injured ischemic area and decreased lesion size (neuroprotective) - all leading to a better functional outcome. Nisoli and coworkers reported along with the increased eNOS expression in the brains of EODF rats, there was also an increase in a number of other proteins which included sirtuin 1 (SIRT1: closely related to yeast Sir2 (silent information regulator 2) protein) (Nisoli et al., 2005). Sirtuin 1 is a NAD -dependent histone deacetylase that affects a number of molecules including: +  p53, Ku70, FoxOl, N F K B , PPARy, p300, and PGC-la. This group confirmed results from Cohen's group that CR increased SIRT1 expression in a number of tissues including the brain (Cohen et a l , 2004); and recently an additional group also reported increased SIRT1 in the brain with 30 % CR followed for 6 months (Qin et al., 2006). Serum from DR rats increased the production of SIRT1 in human cells and protected the cells from apoptosis. SIRT1 has been reported to reduce neurodegeneration, to spare axons, and is actively being pursued in treating axonopathy and neurodegeneration (Araki et al., 2004). It is possible that CR/EODF could 41  reduce secondary damage to unlesioned axons in the spinal cord due to an increase of SIRT1. In the model studied in this thesis the corticospinal tract was left intact but both the collaterals and the tract could be damaged by the expanding lesion cavity of the nearby dorsolateral funiculus due to secondary damage. Therefore, we see a number of upstream effects of CR/EODF that could lead to reduced secondary damage to the spinal cord. What about some of the downstream molecules in the cell death pathway? Duan and coworkers reported decreased cleaved (active) caspase 1 levels in EODF animals in both the cortex and striatum in a model of Huntington's disease (Duan et al., 2003b). The frontal cortex of aging CR animals displayed a reduction of caspase-2 activity along with an increase of apoptosis repressor with a caspase recruitment domain (ARC) (a known inhibitor of caspase-2, of which cleavage (activation) is normally stimulated by TNF-a), and an inhibition of cytochrome c release (Shelke and Leeuwenburgh, 2003). X-linked inhibitor of apoptosis (XIAP), an inhibitor of the cell death pathway, was increased in the frontal cortex of aged CR rats along with decreased cleaved (activated) caspase-3 (Hiona and Leeuwenburgh, 2005). This evidence suggests that CR/EODF can positively affect a number of molecules involved in the apoptosis pathway and thus promote survival. While I have outlined some of the possible pathways and molecules affected by CR/EODF to reduce secondary damage, this does not necessarily affect cell survival and the morphology of damaged nervous system tissue. EODF has been proven repeatedly to be neuroprotective by promoting survival of neurons after insult. The most widely studied paradigm of neuroprotection using EODF is kanic acid induced damage. E O D F if started prior to insult (8 weeks or more) consistently increased neuronal survival and functional outcome (Bruce-Keller et al., 1999; Zhu et a l , 1999; Duan et a l , 2001b; Anson et al., 2003; Sharma and Kaur, 2005). The robustness of the effects of EODF on neuroprotection has also been reported in other models. EODF greatly reduced the loss of striatal neurons after application of 3NP, and E O D F animals demonstrated improved function on the rotary rod test (Bruce-Keller et al., 1999). Animals on EODF for 3 months were more resistant to substantia nigra damage of their dopamine neurons induced by MPTP and these animals also performed better on the rotarod behavioral test (Duan and Mattson, 1999). Specific measurements of cortical infarct size after a stroke were reduced in animals on the EODF regimen for 3 months prior to insult (Yu and Mattson, 1999). Additionally, striatal lesion size after either 3NP or malonate administration was reduced in EODF rats (BruceKeller et al., 1999). These data lead to my hypothesis that EODF could also reduce lesion size and loss of neurons after a SCI, which could lead to an increase in functional recovery. 42  Although EODF reduces the neuronal loss after various neuronal insults, there is still some degree of residual neuronal death. The adult mammalian brain does contain neural precursor cells (NPC) that can differentiate into neurons and glia (Gould et al., 1999b; Gould et al., 1999a). These endogenous neural progenitors are activated after SCI (McTigue et al., 2001; Yamamoto et al., 2001) but they are obviously not sufficient to mediate recovery to the levels we desire. Three months of EODF increased overall survival of neurons (and hence total number) produced from neurogenesis in the hippocampus which coincided with increased BDNF expression and higher trkB^trkB ratio (Lee et al., 2002b). The direct role of B D N F in the 10  EODF-induced survival of newly generated neurons was shown in B D N F +/- transgenic mice; however the overall production was not affected (Lee et al., 2002a). Similarly, while not increasing the amount of neurogenesis, CR (40 % gradually introduced) promoted the survival of newly generated neurons hence resulting in greater number of neurons (Bondolfi et al., 2004). The increased number of new neurons in the hippocampus in E O D F animals was N M D A receptor independent (Kitamura et al., 2006) while running-induced neurogenesis was N M D A dependent (Kitamura et al., 2003). These results suggest the combination of exercise and EODF could compound the total number of new neurons. Neurogenesis could not only repopulate the area of lost neurons but also replace lost oligodendroctyes after SCI. Remyelination by endogenous or implanted neural precursors could promote an increase in functional recovery (Enzmann et al., 2006). A 34 % CR diet followed for only 15 days before induction of experimental allergic encephalomyelitis (EAE), which is used as a model for multiple sclerosis, fully protected the rats from the behavioral deficit normally observed after this treatment (Esquifino et al., 2004). This suggests that CR could either produce more oligodendrocytes to remyelinate the damaged spinal cord and/or possibly reduce the initial damage to myelin. In summary, I hope I have convinced the readers that E O D F / C R can be neuroprotective and beneficial via multiple mechanisms: increased anti-oxidants, reduced ROS, reduced lipid peroxidation, increased B-hydroxybutrate, decreased inflammatory proteins, increased eNOS, increased SIRT1, increased inhibitors of apoptosis, decreased initiation of apoptosis (e.g. caspases), increased trophic factors and their receptors. These mechanisms lead to reduced lesion sizes, increased survival of damaged neurons, and greater number of newly generated neuronal cells. These combined mechanisms could be beneficial after a SCI.  43  I.7.B. Dietary restriction may reduce the inhibitory to axonal growth properties of the adult C N S myelin/glial scar While it is important to decrease secondary damage, it is equally important to reduce the inhibitory nature of the glial scar and myelin. The glial scar is usually considered to be predominantly made up of activated astrocytes which upregulate their expression of glial fibrillary acidic protein (GFAP) (see section 1.3.B.i). Aging results in increased astrocyte activation as demonstrated by increased GFAP mRNA expression and immunostaining in older rats (Nichols et al., 1993; Linnemann and Skarsfelt, 1994; Berciano et al., 1995). CR reduced the age-related increase in GFAP in the hippocampus (Major et al., 1997; Morgan et al., 1997). Morgan and colleagues suggested that the age-related increase of GFAP expression is due to oxidative stress. CR animals displayed decreased GFAP expression in the hippocampus, basal ganglia, and in the white matter tract of the corpus collusum (Morgan et al., 1999). Interestingly, short-term CR (6 weeks) reduced amyloid-P induced activation of astrocytes (as measured by GFAP immunostaining) (Patel et al., 2005). Reducing astrocyte activation after injury by DR could inhibit the production of the glial scar and thereby allow a greater plasticity response by the injured spinal cord. The second main inhibitory obstacle to sprouting/regeneration is the inhibitory nature of adult myelin (see section 1.3.B.ii). Adult myelin expresses a number of proteins that are inhibitory to axonal growth including Nogo, Mag, Omgp, Versican, and Sema4D. Semaphorins are also expressed in the glial scar. Interestingly, the repulsive nature of semaphorins can be reversed to attraction if neurons were treated to increase their cGMP content (Song and Poo, 2001). It has recently been documented that EODF increases cGMP in the brain (Nisoli et al., 2005). Therefore, EODF could reverse the repulsive nature of semaphorins expressed by the glial scar and adult myelin. Most, if not all, myelin-associated inhibitory proteins appear to signal through the NgR/p75/lingo receptor complex resulting in increased Rho/ROCK activation and subsequent growth cone collapse. Inhibiting the NgR/Rho-A pathway can lead to increased plasticity/regeneration along with improved functional outcome after SCI (Dergham et al., 2002; Fournier et a l , 2003; Chan et al., 2005; Mueller et al., 2005). The expression of the low affinity neurotrophin receptor p75 increases after spinal cord injury (Beattie et al., 2002; Dubreuil et al., 2003). Age also results in increased p75 expression in motor and sensory neurons, along with the downregulation of trk receptors (Bergman et al., 1999; Johnson et al., 1999). Similarly, with 44  increasing age both the cortex and hippocampus showed an upregulation of p75 and a decrease in trkA expression (Costantini et al., 2005). Interestingly, CR (32 % reduction) reduced this agerelated increase in p75 expression and halted the trkA downregulation (trkA in older CR mice was actually higher than control young mice) (Costantini et al., 2005). Therefore, it is possible that CR could reduce the SCI-induced upregulation of p75 and thereby inhibit the downstream Rho/ROCK activation. This could lead to both increased axonal growth and reduced cell death (Dubreuil et a l , 2003). The expression of NgR, the other main receptor mediating inhibition by myelin-associated proteins, is downregulated following wheel running (Josephson et al., 2003). Since CR and exercise appear to produce many of the same effects, it is conceivable that CR might also downregulate NgR expression. One commonality between exercise and CR is their reduction of cholesterol synthesis. Reduction of cholesterol production leads to reduced activation of the NgR/p75/lingo/Rho-A pathway in vitro (Yu et al., 2004), pointing to the importance of the lipid rafts for Rho-A signaling. Wamsley and coworkers demonstrated that NgR is endogenously cleaved to produce a soluble fragment thereby reducing Rho-A activation (Walmsley et al., 2004). Further, cholesterol depletion increased the cleavage of NgR, which is not mediated by A D A M metallopeptidase domain 10 (ADAMI0). Interestingly, cholesterol depletion increased A D A M 10 (kuzbanian) and A D A M 17 (tumor necrosis factor-a-converting enzyme (TACE)) expressions and thereby increased shedding of the IL-6 receptor (Matthews et al., 2003). Therefore, the cholesterol depletion mediated increase in A D A M 1 0 , ADAM17 could lead to increased NgR shedding and enhanced plasticity/regeneration. A combinatorial treatment of three different neurotrophins (BDNF, NT-3, and FGF2) increased T A C E expression along with increased shedding of both the p75 and NgR receptor and a 30 % downregulation of Rho-A activation (Logan et al., 2006). This combinatorial treatment and subsequent downstream changes of p75/NgR/Rho-A led to increased cell survival and promoted axon regeneration. If H M G - C o A reductase is inhibited (via the cholesterol lowering drug lovastatin) or if methyl-Pcylcodexrin treatment is used to lower cholesterol levels there is a resulting increase of amyloid precursor protein cleavage which is thought to occur due to increased a-secretase activity of ADAM10 (Kojro et al., 2001). CR reduces cholesterol levels and, as suggested from the research outlined above, at least A D A M 10 has been shown to be upregulated (Wang et al., 2005). Therefore, CR/EODF mediated increase of A D A M 10 and/or T A C E ( A D A M 17) expressions could result in reduced p75/NgR/Rho-A signaling and thereby increase sprouting/regeneration. Recent work adds further support to the idea that CR could alter this inhibitory pathway as C R (30 %) caused a decrease in ROCK1 levels, which is downstream of RhoA (Qin et al., 2006). 45  I mentioned in the section covering CR's effect on neuroprotection that EODF increases catalase (Sharma and Kaur, 2005). Interestingly, catalase is known to inhibit EGFR (Finch et al., 2006), which was recently reported to inhibit axonal growth (Koprivica et al., 2005) (see section 1.3.B.ii). Therefore, the CR-induced increase in catalase could inhibit E F G R and thereby promote greater axonal growth. In summary CR/EODF could affect multiple parts of the redundant pathways induced by axonal growth inhibitory proteins associated with the glial scar and CNS myelin. Instead of targeting one particular molecule of these pathways, CR/EODF could act in a combinatorial fashion to overcome the inhibitory nature of the injured adult CNS.  I.7.C. Dietary restriction may stimulate the intrinsic response ofinjured neurons after SCI  The lack of adequate trophic support reduces the intrinsic growth response of neurons after injury (see section 1.3.C). A number of different trophic factors have been shown effective in both enhancing sprouting/regeneration and more importantly in improving functional recovery: NT3 (Grill et al., 1997; Houweling et al., 1998; Blits et a l , 2000; Ruitenberg et al., 2003), BDNF (Liu et al., 1999; Kim et al., 2001; Ruitenberg et al., 2003; Schwartz et a l , 2003), CNTF (Ye et al., 2004), GDNF (Tang et al., 2004), or the combination of B D N F and NT3 (Cao et al., 2005b; Mitsui et al., 2005). Therefore, any increase in neurotrophins singly, or in combination, could significantly improve functional recovery after SCI. The first hint that DR could affect neurotrophin levels came from a microarray experiment that reported a 1.8-fold increase of B D N F mRNA in the neocortex of C R (26 %) animals as compared to control animals (Lee et al., 2000a). Mattson's group was the first to demonstrate that EODF increased BDNF protein levels in the brain (Lee et al., 2000c; Duan et al., 2001a; Duan et al., 2001b; Duan et a l , 2003b; Duan et a l , 2003a). Specifically, they demonstrated that BDNF protein levels were increased in the hippocampus, striatum and cortex of EODF mice, but saw no changes in N G F levels (animals on EODF for 3 or more months). EODF caused an increase of BDNF mRNA in the CA1 and CA3 region of the hippocampus but not in the cortex, indicating a differential regulation of mRNA and protein levels (Lee et al., 2002b). Using immunohistochemistry Lee and coworkers found that B D N F was increased in the cortex of wild type mice on EODF, and EODF restored the decreased production of BDNF in heterozygous BDNF +/- mice (Lee et al., 2002a). Further, using ELISAs, Mattson's group demonstrated that BDNF protein levels were increased in the hippocampus, striatum, and cortex 46  in both wild type mice and BDNF +/- mice compared to their respective controls (Duan et al., 2003a). The reduced B D N F protein expression in striatal and cortex tissue in an animal model of Huntington's disease was partially rescued with EODF (Duan et al., 2003b). The transgenic animals on EODF fell off the rotarod fewer times and were able to remain on the rotarod longer than control animals. The receptor for BDNF, trkB, exists in several isoforms: the full-length version is a functional tyrosine kinase receptor whereas the truncated forms may negatively regulate signaling by sequestering BDNF (if expressed in glial cells) or acting in a dominant negative manner if co-expressed with the full-length form in neurons (see section 1.3.C.ii). Increasing BDNF expression can only be beneficial if the ratio of full-length trkB to the non-signaling truncated isoform is increased, or at least maintained. Three months of E O D F increased the ratio of the full-length to truncated isoform of trkB in the hippocampus by approximately 25 % (but not the cortex) (Lee et al., 2002b). Therefore, it appears that EODF can either increase BDNF and/or its receptor signaling propensity which could promote plasticity. BDNF is not the only trophic factor that is altered by EODF. NT3 was increased in the dentate gyrus in CR animals (Lee et al., 2002b). Additionally, GDNF was increased in the MPTP injured striatum of CR primates (Maswood et a l , 2004). Therefore, it is possible that DR in the form of E O D F could increase various trophic factors throughout the CNS including the spinal cord. Combined with the extensive literature demonstrating positive effects of neurotrophins on improving functional outcome after SCI (see section 1.3.C.ii), it is possible that EODF could produce similar behavioral effects through the induction of neurotrophins. Interestingly, CR and EODF might produce differential regulation of trophic factors. Twenty weeks of 40 % CR reduced serum levels of IGF-1, while the same time period of EODF resulted in an increase of IGF-1 (Anson et al., 2003). In vitro data suggests that IGF-1 increases neurite outgrowth in vestibulospinal, spinal projecting neurons from the raphe nuclei, and DRGs (Kimpinski and Mearow, 2001; Salie and Steeves, 2005). A recent paper found the subcutaneous administered IGF-1 increased the functional outcome after a thoracic contusion injury (Koopmans et al., 2006). The increase of IGF-1 in EODF treated animals offer a potential to increase functional recovery after SCI. The above research also suggests that EODF would be a better DR regimen for SCI compared to CR. Molecules less well known for their role in neurite outgrowth might possibly increase CNS plasticity and neuroprotection in EODF animals. EODF increased levels of IFN-gamma and its receptor in the hippocampus; and in vitro work suggests that pretreatment of neurons with 47  IFN-gamma is neuroprotective (Lee et al., 2006). Interestingly, IFN-gamnia promoted neuronal differentiation and neurite outgrowth (Wong et al., 2004; Song et al., 2005). In summary, EODF alters a number neurotrophins and additional proteins that could increase the intrinsic axonal growth response of injured axons.  48  1.8. Further considerations on how dietary restriction may be beneficial after SCI 1.8.A. Short-term dietary restriction can produce molecular changes Most research focusing on DR has used long-term dietary restriction (3 months or more) when examining functional outcome or gene expression. In this thesis I examined short-term DR: one month prior to injury and continuing for one month in the post-injury time period (chapter 3), or initiated after the injury and maintained for one month post-injury (chapter 4). Therefore, the question could be raised whether short-term DR changes gene expression. There is experimental evidence demonstrating that short-term dietary restriction can promptly elicit changes at the mRNA and protein level. Six weeks of 40 % CR in mice reduced the amount of GFAP immunostaining in a mouse Alzheimer's disease model along with reduced amyloid-beta levels (Patel et al., 2005). Three weeks of 26 % CR increased both muscle and liver mitochondrial oxidative capacity (Barazzoni et al., 2005). Dhahbi, using microarrays, demonstrated that 2 weeks of 40 % CR in adult mice (19 months old) produced 61 gene expression changes in the liver as compared to controls, even though the body weight did not differ between the groups (Dhahbi et al., 2004). The 61 gene expression changes in the short-term CR group was 50 % of the total changes observed with long-term CR. Interestingly, by 2 months after the start of the CR diet there was a reduced mortality rate even though CR was not induced until 'middle age'. In the heart, 8 weeks of CR in male B6C3F1 mice produced 19 % of the changes seen with long-term CR (Dhahbi et al., 2006). A number of gene changes found in mRNA microarrays were confirmed with immunoblots. Twenty-one days of EODF increased extracellular striatal glutamate levels after MPTP induced injury in male C57BL/6J mice (Holmer et al., 2005). Fifteen days of 34 % CR protected Lewis rats from developing experimental allergic encephalomyelitis, which is used as a rodent model of multiple sclerosis (Esquifmo et al., 2004). In humans, alternating fasting with 20 hours of fasting followed by a full day of eating, for a total period of 15 days resulted in increased whole body glucose uptake induced by insulin indicating an increased sensitivity to insulin (Halberg et al., 2005). Additionally, there was increased circulating adiponectin, which is correlated with insulin sensitivity. In adult male Fischer rats, seven days of 26 % CR reduced blood levels of insulin and leptin while increasing gherlin; but more interestingly, it increased the protein expression of eNOS in the aorta and produced physiological changes in vascular function (Zanetti et al., 2004). As little as 5 days of 40 % CR increased the protein levels of phosphorylated-AMPK (AMP49  activated protein kinase) in the hippocampus of female Sabra mice (Dagon et al., 2005). In humans, 8 days of CR decreased total cholesterol, triglycerides, and a parameter of oxidative stress (plasma levels of malondialdhyde) while increasing |3-hydroxybutyrate (neuroprotective) and superoxide dismutase (antioxidant) (Skrha et al., 2005). The above examples suggest that both mRNA and protein levels can be altered in mice, rats, and humans over a relatively short time period in various tissues including the CNS.  I.8.B. Calorie restriction or every-other-day fasting?  One reason to test the effect of EODF instead of CR is that mice and humans do not necessarily have to lose weight on the EODF diet regimen (see section 1.8.C). More importantly, research by Mattson's group suggest EODF is more neuroprotective than 40 % CR (Anson et al., 2003). In a direct comparison of 40 % CR and EODF followed for 24 weeks, EODF produced greater survival of hippocampal neurons after kainic acid insult. The possible mechanism for the differences between the two DR regimes could be the greater increase in B-hydroxybutrate in the EODF group compared to the CR group. Additionally, EODF surprisingly caused an increase in serum insulin-like growth factor 1 (IGF-1), while all other CR papers (including the control group in the above-cited paper) show decreased IGF-1 expression in animals on a reduced diet. IGF-1 was recently implicated in neurite outgrowth and increased functional improvement after SCI (Koopmans et al., 2006) (see section 1.7.C). Further to this, an increased BDNF expression in the CNS was only seen in animals on the EODF regimen (Lee et al., 2000c; Duan et al., 2001a; Duan et al., 2001b; Duan et al., 2003b; Duan et al., 2003a) but not a 40 % CR regimen (Hiona and Leeuwenburgh, 2005; Newton et al., 2005). Since E O D F appears to be more neuroprotective, has a greater effect on increasing P-hydroxybutrate, IGF-1 and BDNF, I chose to test the effect of EODF on SCI instead of 40 % CR.  I.8.C. Human dietary restriction/every-other-day fasting studies  Another concern raised by the doubters of the effect of CR is that it has not been tested in humans: that it will not work in humans. One natural study in which the residents of Okinawa, Japan, who consume fewer calories than the rest of Japan (or most other countries) but are not malnourished, have a longer lifespan and lower incidences of vascular diseases and cancer (Kagawa, 1978). The positive effects of reduced calorie consumption in Okinawa residents are 50  even more remarkable when one considers that Japan's population has the longest life expectancy in the world (Mathers et al., 2001). Research studying the effects of the Dutch famine during world war II suggests reduced incidences of cancer (van Noord and Kaaks, 1991). The problem with interpreting this work is that malnutrition is a factor under these wartime conditions. Another hint of CR/EODF affecting humans can be gathered from a Spanish study that occurred in the 1950's (Arias-Vallejo 1957 as discussed in (Anson et al., 2005)). This study examined people over the age of 75 who resided in a retirement home and were divided into two groups. One group received normal food consumption (2,300 kcal/day) while the second group received 2,300 calories one day followed by only 1,000 calories the second day (and the diet sequence was repeated). These senior citizens were followed for three years: the control group had 44 illnesses and 13 deaths while the restricted diet group only had 25 illnesses and 6 deaths (not significant although). No firm conclusions can be drawn from this experiment but fortunately, in recent years there is finally interest in performing human studies with various forms of DR. Fontana and colleagues studied 18 people who voluntarily implemented long-term (6 year average) CR as a lifestyle choice and compared various parameters with matched control group (Fontana et al., 2004). The CR group had similar blood profiles to the control group prior to their start of CR. However, CR dramatically altered their blood profiles: total cholesterol, lowdensity lipoprotein cholesterol, ratio of total cholesterol to high-density lipoprotein cholesterol, triglycerides, fasting glucose, fasting insulin, C-reactive protein, platelet-derived growth factor A B , and systolic and diastolic blood pressure were all markedly lower, whereas high-density lipoprotein cholesterol was higher. Additionally, carotid artery intima-media thickness was 40 % less iii the CR group. All of these are indicative of a healthier heart and circulatory system. Interestingly, the vast majority of the effects of CR appeared within 1 year (and possibly sooner but no earlier time point was reported). A follow-up study with 25 subjects and matched controls found similar blood profiles changes as above (Meyer et al., 2006). In addition, CR reduced TNF-a, and TGF-pi levels and attenuated the normal age related impairments in diastolic function (as measured by Doppler echocardiography) to a degree that the middle aged CR subjects were similar to young subjects (Meyer et al., 2006). There are a number of ongoing human experiments (which include EODF groups) from this same group published in abstract form, and the field looks forward to viewing the published results. Currently only the effect of short-term EODF on humans have been tested and fully reported. Non-obese subjects (8 men (average age = 34, body fat 22 %) and 8 women (average 51  age = 30, body fat 25 %)) underwent 22 days of alternating fasting days (Heilbronn et al., 2005a). Absolute and relative resting metabolic rate did not change while fat oxidation (not the same as lipid peroxidation) doubled and carbohydrate oxidation decreased by more than 50 % on fasting days at the end of the test period. Fasting insulin levels were lower on day 22 (fasting day) along with a 7-fold increase in P-hydroxybutyrate while free fatty acid concentrations doubled. Overall, the subjects only lost a small amount of weight (0.6 kg of fat free mass and 0.8 kg of fat mass). In a second study, individuals with slightly lower starting body fat levels (20.1 %) underwent 15 days of alternating 20-hour fasts and showed no weight loss, even while maintaining their normal activity levels (Halberg et al., 2005). This suggests that humans (similar to mice) are able to consume sufficient calories on feeding days to compensate for the lack of calories on fasting days while maintaining normal activity. The researchers found similar increases in p-hydroxybutyrate and free fatty acids on fasting days, and also reported an increase in whole body insulin sensitivity. Therefore, CR and EODF appear to have positive effects in humans (not surprising since they have positive effects on a wide variety of species) and EODF is possibly more easily tolerated in humans than 30-40 % CR since weight loss is not a necessary consequence.  52  1.9. Basis of experimental paradigm 1.9.A. What do SCI patients want? As scientists in the SCI field, we should be cognitive of what are the desires and needs of the people who have suffered a SCI to improve the quality of their lives. Anderson specifically asked different subpopulations of people living with SCI what they wanted basic researchers to concentrate on in terms of needs they think are most important for their quality of life (Anderson, 2004). Arm-hand function was clearly the most important need for quadriplegics (~ 49 %), followed by sexual function (-13 %) and trunk stability (-12 %). Walking movement was 5  th  on the list at around 8 %. For paraplegics sexual function topped the list at - 27%, followed by bladder/bowel/autonomic dysreflexia (-18 %). Walking movement was only fourth in their ranking. It is interesting that the clear majority of animal studies examine thoracic lesions and assess hind limb motor recovery (Webb and Muir, 2005) when walking is not a top priority for SCI patients. Therefore, if we listen to the patients' wants and needs for a higher quality of life we should study the return of upper limb function along with sexual function.  I.9.B. What SCI model to study?  While spinal cord researchers have typically used thoracic lesion models there might be good reasons to pour some of our resources into cervical injury models. As reported above, quadriplegics ranked regaining hand function far higher than walking (Anderson, 2004). In examining the level and severity of injuries most researchers report more than 50 % of injuries are at the cervical level (Ackery et al., 2004; Jackson et al., 2004) and incomplete cervical injuries represent the individual category with the highest incidence (Dobkin and Havton, 2004). Additionally, recovery from this type of injury might only require a limited amount of neuroprotection, or some form of sprouting/regeneration, to produce meaningful functional improvements. To gain function after a thoracic lesion one might expect growth covering 3-4 segments is required; however, after a cervical lesion, growth or sprouting over only one or two segments can bring important functional improvements. The return of upper limb use would dramatically improve the quality of life of people with cervical spinal cord injury. The loss of arm function entails a high degree of dependence on others. This loss of independence is an important psychological factor in the well being of SCI patients (DeSanto-Madeya, 2006) and 53  the main factor in the high health care cost for cervically injured individuals (Dobkin and Havton, 2004; Anderson et al., 2005). Animal models of spinal cord injury can for simplicity sake be divided into two types: compression-contusion injuries and partial/full transections. Compression and contusion SCI models in rats better represent the histopathology of human SCI and therefore are better clinical models (Metz et al., 2000). The transection models allow analysis of the loss of specific tracts/columns and assessment of axonal regeneration (Kwon et al., 2002a). Therefore, both types of lesion models have their own strengths and weaknesses. A crush injury, as used in this thesis, could be argued, combines some aspects of both main SCI models. Similar to a partial laceration type injury, the crush injury model used completely severs the rubrospinal tract but the injury model.also produces substantial secondary damage. The ratio of males to females who experience a spinal cord injury is 4:1 (Jackson et al., 2004). So I find it interesting that the majority of rat animal studies use female as their experimental test subjects (though there are additional valid reasons for choosing females such a lower rate of urogenital tract infections). Human spinal cord injuries result in large cavity formations due to the extensive loss of initially spared tissue during the wake of secondary damage (see section 1.3.A - 1.3.A.ii). This same effect is seen in rats but not in most mice strains (Kuhn and Wrathall, 1998; Sroga et al., 2003). Therefore, rats might be a better model for human spinal cord injuries than mice. In conclusion, for the several above listed reasons I decided to examine functional recovery after a partial cervical crush/lesion model in male rats.  I.9.C. Lesion Model  The main interest of this thesis is the recovery from a spinal cord injury: specifically, a left C4 dorsolateral funiculus crush of the spinal cord in male Sprague-Dawley rats (see figure 1.1). This lesion model axotomizes the descending rubrospinal tract (RST) (arising from the contralateral red nucleus), the ascending dorsal spinocerebellar tract (arising from the ipsilateral Clarke's nucleus), Lissauer's tract containing axons from the lateral division of dorsal roots, and the lateral corticospinal tract (CST) (while sparing both the dorsal and ventral CST). Some of the gray matter and most of the ventral funiculi and medial aspects of dorsal funiculi are spared on the lesion side. This lesion model (or variants) has been widely used to examine regeneration/sprouting and/or functional recovery (Liu et al., 1999; Liu et al., 2002b; Murray et  54  al., 2002; Ruitenberg et al., 2003; Schwartz et al., 2003; Webb and Muir, 2003; Tobias et al., 2005) . The main descending surpraspinal tract injured in the lateral funiculus using this injury model is the rubrospinal tract (RST). The RST is involved in both skilled motor functions (Whishaw et al., 1998), and general motor actions that include locomotion (Muir and Whishaw, 2000). Therefore, a number of behavioral testing paradigms have been used to assess the return of function after a RST lesion. These tests include horizontal rope crossing (Murray et al., 2002; Ruitenberg et a l , 2003; Tobias et al., 2005; Hendriks et al., 2006), horizontal ladder crossing (Soblosky et al., 2001; Webb and Muir, 2003), ground reaction forces during walking/running (Muir and Whishaw, 2000; Webb and Muir, 2003), catwalk (including base of support) (Hendriks et al., 2006), and Schallert rearing task (Liu et al., 1999; Soblosky et al., 2001; Schwartz et al., 2003; Webb and Muir, 2003; Neuhuber et al., 2005; Tobias et al., 2005). Bilateral lesions of the rat rubrospinal tracts lead to long term functional deficits that are more severe than a dorsal column lesion taking out both sides of the dorsal corticospinal tracts (while sparing the lateral and ventral CSTs) (Hendriks et al., 2006). This suggests that at least in rats the RST is more important than the CST (the opposite might be true for humans). The ipsilateral dorsal corticospinal tract (the most important CST for forelimb function) is left intact in the lesion model used in this thesis. When the rubrospinal system is eliminated there is no spontaneous sprouting of the CST (Jeffery and Fitzgerald, 2001; Hendriks et al., 2006) . After a rubrospinal system injury the CST could be induced to sprout with NT3 treatment, however, Jeffery and Fitzgerald did not examine if this sprouting had any functional consequences (Jeffery and Fitzgerald, 2001). Another group studying both bilateral dorsolateral funiculus lesions (taking out the rubrospinal tract on both sides), or dorsal column lesions (taking out the dorsal CST) also found no spontaneous sprouting of the CST when the rubrospinal tracts were lesioned (Hendriks et al., 2006). When the dorsal CST was lesioned researchers observed sprouting of the unlesioned ventral CST caudal to the lesion site (Aoki et al., 1986; Weidner et al., 2001), which can result in behavioral gains, or sprouting of collaterals rostral of the lesion site of the lesioned CST (Fouad et al., 2001). Therefore, while the CST might not sprout spontaneously when the RST is lesioned, it is possible that the CST could compensate behaviorally if triggered appropriately. Extensive research points out that the RST and CST are parallel systems that have overlapping connections and functions (Lawrence and Kuypers, 1968a, b; Whishaw et al., 1990; Schrimsher and Reier, 1993; McKenna and Whishaw, 1999). In  55  the rat spinal cord there is an overlap of target areas in laminae IV, V , VI and the dorsal area of VII for these two tracts (Brown, 1974; Antal et al., 1992; Raineteau et al., 2002). Interestingly, the RST can sprout and compensate behaviorally for the loss of the CST if treated with an antibody against the myelin inhibitory protein Nogo, but not without this intervention (Raineteau et al., 2001; Raineteau et al., 2002). In summary, it appears in the untreated situation after a spinal cord injury the RST and CST do not sprout in compensation of the loss of the other, but with the appropriate intervention each tract can sprout and compensate behaviorally for the other.  56  1.10. Chapter overviews and hypotheses Cell body treatment of rubrospinal neurons with B D N F can promote growth into the permissive environment of a peripheral nerve graft. However, we do not know if this treatment has the same growth promoting properties in the inhibitory environment of the CNS, or if it has positive effect on functional recovery. Priming neurons in vitro and pretreatment of peripheral DRGs with neurotrophins or cAMP elicit a greater axonal growth response than when treated at time of injury. However, pretreatment has not been tested in vivo on descending supraspinal neurons. In chapter 2 of this thesis I hypothesized  that acute treatment of rubrospinal  neurons with BDNF promotes sprouting/regeneration (la) and increases motor recovery (lb) after a cervical dorsolateral funiculus injury; and that pretreatment of rubrospinal neurons with BDNF one week prior to injury further enhances regeneration (2a) and motor recovery (2b). As a positive control, I examined cell size of rubrospinal neurons to verify that a cell body response was elicited with BDNF treatment. To test if BDNF treatment enhanced axonal growth I examined anterogradely labeled rubrospinal axons to see if they grew across the lesion site and/or sprouted in the grey matter at the cervical level. The forelimb usage during vertical exploration was assessed to measure return of the ipsilateral forelimb function. The increase in behavioral recovery in the BDNF pretreated rats combined with their reduced weight gain (chapter 2) prompted me to review the extensive research regarding dietary restriction and its positive effects on various neuronal injury models. From these readings, in chapter 3  I hypothesized that prophylactic dietary restriction in the form of every-other-day  fasting started one month prior to a cervical dorsolateral funiculus injury is neuroprotective (3a) and promotes functional recovery (3b). I used lesion size and the number of NeuN-positive neurons as measurements of the neuroprotective effects of EODF; and I examined GFAP expression to see if there was a reduction in the glial scar. The Schallert cylinder was used to assess forelimb recovery during vertical exploration. While the results of prophylactic dietary restriction are interesting scientific findings, it is unlikely to be useful to the average human population because a very small percentage would decide to practice such a food regime even though it has been shown to extend lifespan in numerous different species. The potential diverse and multiple effects of DR on a host of obstacles that normally inhibit functional recovery (see section 1.5.4-1.6.6) prompted me to explore a more clinical model in chapter 4:1  hypothesized that EODF implemented after a  cervical dorsolateral funiculus spinal cord injury is neuroprotective (4a), promotes 57  plasticity of spared spinal cord tracts (4b), increases neurotrophin/receptor expressions (4b),  and leads to an increase in motor recovery (4d). I measured the density of NeuN-  positive neurons and lesion size to examine the neuroprotective effect of EODF. I anterogradely labeled the intact corticospinal tract to test if EODF promoted plasticity of intact spinal cord tracts. Western blots and ELISAs were used to examine protein expression of B D N F and trkB, which had been previously reported in the literature to be altered with long-term prophylactic EODF regime. Finally, I used three different behavioral tests to more thoroughly examine the recovery of forelimb motor function.  58  Table 1.1. Effect of CR/EODF on the top 10 leading causes of death. Cause of death  %of total death  Experimental effect of CR/EODF  Heart disease  28.5  Cancer  22.8  Stroke  6.7  Chronic lower respiratory disease Accidents Diabetes  5.1  CR reduced aging of the heart (Maeda et al., 1985) CR reduced oxidative damage to heart (Pamplona et al., 2002) EODF reduced cardiovascular risk factors (Wan et a l , 2003) EODF and CR reduced age related fibrosclerosis (Castello et al., 2005) CR in humans reduced risk of atherosclerosis (Fontana et al., 2004; Meyer et al., 2006) EODF reduced myocardial infarction size (Ahmet et al., 2005) DR reduced incidences of cancer (Rous, 1914) CR reduced incidences of cancer in mice even if started at middle age (Pughetal., 1999) EODF reduced tumorigenicity (Kritchevsky, 1990) One day a week of fasting reduces cancer (Hursting et al., 2004) CR started in adulthood reduced death by stroke in stroke prone rat model (Stevens et al., 1998) CR/EODF reduced cardiovascular risk factors (see heart disease) EODF reduced infarct lesion size, improved recovery (Yu and Mattson, 1999) CR animals had an increased resistance to gram-positive bacteria in the lung (Dong etal., 1998) CR reduced pathogenesis marker of asthma (Vasudevan et al., 2006)  4.4 3.0  Influenza and pneumonia Alzheimer's disease  2.7  Kidney disease  1.7  Septicemia  1.4  2.4  Not tested EODF reduced the incidences of diabetes (Pedersen et al., 1999) Two days a week of fasting reduced the incidences of diabetes (Pedersen et al., 1999) CR prevented development of diabetes (Colombo et al., 2006) CR promoted a better immune response to influenza (Effros et al., 1991) EODF protected neurons in presenilin-1 K O mice (Zhu et al., 1999) CR reduced the amount of AB-plaques/peptide (Costantini et a l , 2005; Patel et al., 2005; Wang et al., 2005; Qin et al., 2006) CR reduced death by kidney disease (Stern et al., 2001) CR reduced incidence and severity of kidney disease (Everitt et al., 1982; Masoro et al., 1989; Gumprecht et al., 1993; Keenan et al., 1995) CR reduced death from septicemia after skin lesions (Perkins et al., 1998)  59  Figure 1.1. 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Introduction Adult mammalian CNS axons typically fail to regenerate after injury and functional recovery is minimal. One of the main obstacles limiting axonal growth is the weak intrinsic response of the axotomized CNS neurons (for review see (Plunet et al., 2002; Filbin, 2003)) leaving axons highly susceptible to the inhibitors associated with myelin (Caroni and Schwab, 1988a, b; Buchli and Schwab, 2005) and with the glial scar that forms after a spinal cord injury (for review see (Silver and Miller, 2004)). The intrinsic growth response of neurons can be enhanced by a conditioning lesion (Richardson and Issa, 1984), by pretreatment with cAMP (Neumann et al., 2002; Qiu et al., 2002; Lu et al., 2004), by inflammation in the vicinity of the neuronal cell bodies (Lu and Richardson, 1991; Leon et al., 2000; Fischer et al., 2001; Yin et al., 2003; Lorber et al., 2005; Yin et al., 2006), by over-expression of more than one regeneration-associated gene (Bomze et al., 2001), or by cell-body treatment with trophic factors (Kobayashi et al., 1997; Kwon et al., 2002). Specifically, we observed that cell-body treatment of rubrospinal neurons with BDNF promoted increase growth into the permissive environment of a peripheral nerve graft (PNG) (Kobayashi et al., 1997; Kwon et al., 2002). However, we have not examined if this same treatment is effective in promoting axonal growth into the less permissive CNS environment, or can improve functional recovery. The timing of the inventions to enhance the intrinsic growth response is critical. Increasing the cell-body response by a conditioning lesion of the sciatic nerve performed 7-14 days prior to a dorsal column lesion of the spinal cord stimulated the growth of the central branches of the D R G axons into a PNG inserted into the dorsal columns (Richardson and Issa, 1984) or into the hostile CNS lesion site (Neumann and Woolf, 1999). In vitro, prior exposure (priming) of DRGs to neurotrophins (BDNF or GDNF) overcame neurite growth inhibition by myelin in part via increasing cAMP (Cai et al., 1999). No effect was observed if neurotrophins were added simultaneously with the exposure to myelin. In vivo, pretreatment of DRGs with cAMP 48 hrs or one week prior to a dorsal column lesion stimulated the axotomized central axons to grow into the lesion site (Neumann et al., 2002; Qiu et al., 2002). Whether these principles learned in the D R G model can be translated to the supraspinal CNS neurons projecting into the spinal cord has not yet been addressed.  90  Here we tested two experimental questions: (1) does acute cell-body treatment of rubrospinal neurons with BDNF at time of injury, which can promote growth into a PNG, increase axonal growth in the CNS and promote recovery after a spinal cord injury; (2) will priming of rubrospinal neuronal cell bodies with BDNF one week prior to injury allow the injured axons to overcome the inhibitory nature of the adult CNS spinal cord and improve functional recovery? We failed to observe regeneration into, or across, the lesion site with any of the two paradigms. Both the BDNF-acute and BDNF pretreatment groups displayed less atrophy of the rubrospinal neurons and reduced dieback of the rubrospinal tract. Both B D N F groups also showed ~ twice the density of rubropsinal axon branches/sprouts rostral to the lesion site, although this parameter did not reach significance. Despite these effects in both BDNF groups, only the B D N F pretreatment improved the usage of the ipsilateral forelimb.  91  2.2. Material and Methods  Animal procedures. Eighteen male Sprague-Dawley rats weighing 260-300 g (UBC/Charles River Breeding Laboratories) were used in this study. All procedures performed on the rats were in accordance with the guidelines of the Canadian Council for Animal Care and approved by UBC's animal care committee. According with these guidelines, one animal in the BDNF-acute group was removed from the study due to autotomy of its right rear foot in week 2 of the experiment. Injury model. For all surgeries rats were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, Ontario, Canada) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, Ontario, Canada). Animals were placed in a stereotaxic frame, and a hemi-laminectomy was performed at the fourth cervical vertebra. After the dura was opened, a fine needle (30 gauge) was inserted vertically at the juncture between the dorsal gray matter and the dorsal column white matter on the left hand side of the spinal cord. Fine forceps with tips 0.2 mm wide by 2 mm long (customized Dumont #5, Fine Science Tools, Vancouver) were inserted into the needle hole to a depth of 2.0 mm and the lateral spinal cord crushed with the forceps held closed for 20 seconds, severing the rubrospinal tract but leaving the dorsal corticospinal tract intact. The surgeon was blinded to the treatment groups. We have used this lesion extensively (>120 times) and never found evidence of spared rubrospinal tract axons ((Ramer et a l , 2004; Richter et al., 2005); plus in 2 ongoing projects). A priori criterion for exclusion of animals in this injury model was histological confirmation of corticospinal tract damage. No animals in this study were eliminated based on this criterion. BDNF administration.  One week prior to cervical spinal cord injury, a canula attached to  an osmotic mini pump, (Alzet no. 2002, 0.5 ul/hr for 2 weeks; Alzet, Palo Alto, CA) was implanted lateral to the red nucleus (6.0 mm posterior to bregma, 1.7 mm to the right of midline, 6.5 mm below the dura) for rats in both the BDNF pretreatment (n = 6) and the control pretreatment (n = 6) groups. The BDNF-acute group (n = 5) received its canula and pump at the time of the C4 injury (Fig. 2.1 A). The osmotic mini pumps delivered 12 ug of BDNF (gift from Regeneron Pharmaceuticals) per day (0.5 ul/hr at a concentration of 1.0 ug/ul) in a vehicle solution of 20 m M sterile PBS, 100 units of Penicillin/Streptomycin and 0.5 % rat serum albumin. The control pretreatment pump contained only the vehicle solution. One week prior to pump installation and every subsequent week the weights of the animals were recorded.  92  Anterograde tracing. The rubrospinal tract was anterogradely traced after the fourth week of behavioral testing. While anaesthetized as above, all animals received 0.6 ul 25 % biotin dextran amine (BDA; Molecular Probes, OR) through a fine glass pipette inserted stereotaxtically medial to the rubrospinal neurons at 6.3 mm posterior to bregma, 0.7 mm to the right of midline, and 7.0 mm below the dura. Forelimb usage during vertical exploration in cylinder rearing task. Animals were placed in a 30 cm by 30 cm plexiglass cylinder for 5 minutes (initially), and videotaped with a digital camcorder for later scoring of forelimb usage during vertical exploration with their forelimbs against the cylinder. For each week of testing, all animals were required to perform a minimum of 15 initial rearing touches of the cylinder wall and a minimum of 20 limb usage scores for the completion of the task. If animals did not complete the task within the five minutes they were given a minimum of 1 hour rest before being re-exposed to the cylinder, and this was repeated until the task was completed each week. Animals were tested two weeks prior to cervical spinal cord injury (and prior to pump installation), and once a week post injury at 7, 14, 21 and 28 days. An observer blinded to the groups replayed the videotapes and recorded the limbs used for contact when vertically exploring the cylinder. Both forelimbs would have to be removed from the ground and at least one forelimb would have to contact the vertical surface of the cylinder to be counted as a scoring rear. The total score consisted of all touches of the wall while rearing as traditionally measured (Schallert et al., 2000). A l l scores were converted into percentage usage of left limb, right limb and both limbs. Dieback and Gray matter sprouting. At 5 weeks post injury, rats were anesthetized with a lethal dose of Ketamine/Rompun mixture and perfused transcardially with lx PBS, followed by a solution of 4 % paraformaldehyde in phosphate buffer. CI to C7 cervical spinal cord segments were removed, postfixed in 4 % paraformaldehyde overnight, and cryoprotected in 22 % sucrose for 24 hours. Post-fixed, cryoprotected spinal cords were frozen and sectioned at 20 um horizontally (C3-C7), or transversely (C1-C2) on a cryostat. Sections were washed in 0.01 M PBS and then incubated overnight at 4 °C with Cy3-conjugated streptavidin (1:400; Jackson Immunological Research, PA) to label the B D A traced rubrospinal axons. The distance between the most caudal end of the axons and the rostral end of the lesion was used as a measure for dieback. The 5 B D A labeled rubrospinal axons closest to the rostral border of the lesion site from 3 different sections containing the rubrospinal tract were measured and averaged for each animal. To quantify rubrospinal axonal profiles in the gray matter, six longitudinal sections from each animal spaced 80 um apart from each other were used. The gray matter region rostral to the 93  rostral border of the lesion site was cropped out using Photoshop CS2 (Adobe, USA) for later image analysis with Sigma Scan Pro 5 (SPSS, Chicago, IL). A n omni-directional edge detecting filter (Laplace) equated axons to equal thickness (this eliminated the apparent differences in axonal size in the groups to make fair comparisons) and reduced non-specific background noise (Scott et a l , 2005). Applying a threshold to the filtered images provided an on or off measurement of immunopositive axons. The final normalized density measurement is a measure of proportional area of the gray matter occupied by immunopositve rubrospinal axons from 02000 urn rostral to the most rostral border of the lesion and divided by the number of traced RST at the CI level for each animal to normalize for labeling efficiency. Cell size of rubrospinal neurons. Forty 20 um coronal sections were collected from the midbrain. Collection started when 4-10 rubrospinal neurons were visible in the caudal end of the red nucleus. Sections 5, 10 and 15 were stained with NeuralTrace 500/525 green fluorescent Nissl stain (1:200; Molecular Probes, Eugene, OR). Magnocellular rubrospinal neurons in both the injured and contralateral sides were circled in Sigma Scan Pro 5 to obtain a cross-sectional area for each neuron. Cell size of neurons for each section was expressed as % contralateral and averaged for each animal and subsequently for the three groups. Statistics. SPSS 13.0 (SPSS, Chicago, IL) was used to run all statistics. A One-way A N O V A was used to compare dieback and cell size of rubrospinal neurons amongst the groups. A Two-way repeated measure A N O V A was implemented for gray matter spouting (treatment and distance), weight, and % use of Left+Both forelimbs (treatment and time). When significance was reached in the A N O V A s a Scheffe post-hoc comparison was implemented due to the unequal n's in the groups. Standard error of the mean (+/- S.E.M) are reported and displayed in the graphs.  94  2.3. Results BDNF treated rats gained less weight Prior to being enrolled in this study the young adult male rats gained approximately 5060 grams (g) per week (Fig. 2.1B) (vehicle pretreatment 59.16 g +/-1.70, n = 6; BDNF pretreatment 57.17 g +/- 2.00, n = 6; BDNF-acute 52.40 g +/- 1.66, n = 5). In the post pump installation time period there were differences in weight gain per week between the groups (p < .01, Two-way repeated measure A N O V A ) . BDNF-acute (p < 0.01, Scheffe) and BDNF pretreatment rats (p < 0.05) gained less weight than the vehicle pretreatment group. In the week after pump installation (but before spinal cord injury) the vehicle control rats gained 62.67 g +/4.29, while the B D N F pretreatment group gained only 22.67 g +/- 4.39 (p < 0.001, Scheffe). These results are consistent with the literature showing appetite suppression by BDNF (Pelleymounter et al., 1995). A l l animals gained less weight one week after injury (vehicle pretreatment 18.17 g +/- 3.11, BDNF pretreatment 1.67 g +/- 3.79, BDNF-acute - 21.40 g +/4.26) as compared to previous control weeks, which is in part attributed to the general response to spinal cord injury. However, the animals receiving BDNF (acute and pretreatment) gained significantly less weight during this period as compared to the control group (p < 0.05). BDNFacute animals gained less weight than BDNF pretreatment animals (p < 0.01). During the second week after the spinal injury there was no difference in weight gain between the groups. However, in the third week after injury the BDNF-acute group (even though the pump delivers BDNF for only two weeks) gained less weight than either the control or B D N F pretreatment groups (p < 0.001). The BDNF-acute group resumed normal weight gain two weeks after the pump had ran out of B D N F (4 weeks post-injury).  BDNF pretreatment improved forelimb usage Forelimb usage during exploratory rearing is a natural behavior in rats and does not require training (Schallert et al., 2000). Hence, the animals were tested once before the cervical injury (and prior to pump installation) to establish their baseline performance and weekly for one month after the injury. During a vertical exploration before injury, the percent usage of the 'left' or 'right' forelimb is approximately 25 % each and the % score of 'both' is in the vicinity of 50 %. The animals were injured on the left side of the cervical spinal cord and therefore favored the use of the right (contralateral) forelimb after injury. Rats after injury typically rear up placing  95  their right forelimb on the cylinder wall for weight support and at times include their left limb for further exploration. The involvement of the left forelimb is a measure of recovery. One convention is to record the summed percentage usage of the ipsilateral (left) alone plus both ('Left+Both') forelimbs as a measurement of usage of the ipsilateral limb (Schwartz et al., 2003; Shumsky et a l , 2003; Tobias et al., 2005). Prior to operation, baseline forelimb usage of 'Left+Both' did not differ among the groups (Fig. 2.2) and ranged from 73 % to 78 % (vehicle pretreatment 73.12 % +/- 4.03, n - 6; BDNF-acute 77.01 % +/- 3.01, n = 5; B D N F pretreatment 78.00 % +/- 3.06, n = 6). One week after the C4 dorsolateral funiculus crush there was a considerable drop in the 'Left+Both' forelimb usage to values below 35 %. Specifically, the vehicle pretreatment group scored a mean of 33.16 % +/- 7.49, the BDNF-acute group showed mean scores of 22.12 % +/- 13.25, and the BDNF pretreatment group of 23.84 % +/- 7.14. These one week post injury scores indicate that the BDNF-acute and B D N F pretreatment groups were at least as severely injured as the control group. A two-way repeated measure A N O V A revealed differences among the groups in the post injury period (Fig. 2.2) (p < 0.01). Importantly, at 21 and 28 days post injury the BDNF pretreatment group performed significantly better compared to both the vehicle pretreatment and the BDNF-acute groups (p < 0.05, Scheffe) (vehicle pretreatment 31.44 % +/- 7.85, 26.75 % +/- 3.76; BDNF-acute 27.84 % +/- 4.23, 28.27 % +/8.50, B D N F pretreatment 59.44 % +/- 6.45, 53.38 % +/- 6.43, at the two times respectively). Neither the vehicle pretreatment group nor the BDNF-acute group displayed any sign of recovery on this measurement over the experimental period. The lack of recovery in the control animals is in agreement with other researchers using similar lesion models (Liu et al., 1999; Schwartz et al., 2003; Tobias et al., 2005).  BDNF cell body treatment (acute or pretreatment) rescued red nucleus neuron size after cervical spinal cord injury Typically after cervical rubrospinal tract lesion rubrospinal neurons undergo atrophy which is readily apparent in comparison to the intact contralateral neurons (Kobayashi et al., 1997; Kwon et al., 2002). All groups displayed a decrease in rubrospinal neuron cell size 5 weeks after axotomy as compared to the uninjured contralateral side (Fig. 2.3A-F). However, the axotomized cell bodies of both BDNF-treated groups (Fig. 2.3C,E) appeared less atrophic than those of the vehicle pretreatment group (Fig. 2.3A). Quantification of cell size expressed as a percentage of the uninjured contralateral red nucleus indicates significant differences between groups (p < 0.01, one-way A N O V A ) (Fig. 2.3G). The cell sizes of both BDNF-acute (82.30 % 96  +/- 2.55, n = 5) and B D N F pretreatment (75.41 % +/- 4.58, n = 6) groups were significantly greater compared to the vehicle pretreatment group (61.33 % +/- 2.23, n = 6) (p < 0.05, Scheffe).  BDNF cell body treatment (acute or pretreatment) did not promote regeneration but did prevent rubrospinal axon retraction Crush injury of the dorsolateral funiculus of the rat spinal cord typically leads to the formation of a cavity, occasionally sparing a thin residual strip of tissue under the pia matter, but not sparing rubrospinal tract axons. Anterograde tracing with B D A did not reveal any rubrospinal axons in this residual strip of spared subpial tissue, in any treatment group. Neither did any group show any anterogradely traced rubrospinal axons grown across, nor around, the lesion site (Fig. 2.4A-C). Hence, we did not observe any evidence for rubrospinal tract regeneration. Typically, rubrospinal axons retract by approximately 500 pm following cervical spinal cord injury (Houle and Jin, 2001). Both BDNF-acute and B D N F pretreatment groups displayed less dieback than the control group (Fig. 2.4A-C). While the vehicle pretreated group (n = 6) retracted by 664.51 pm +/- 78.06, the BDNF-acute (n = 5) and BDNF pretreatment (n = 6) groups showed mean retraction distances of 222.53 pm +/- 25.77 and 202.34 pm +/- 40.03 respectively, which are significantly less than those of the vehicle pretreated group (p < 0.001, one-way A N O V A for group differences followed by Scheffe test, p < 0.001) (Fig. 2.4D). There was no difference between the BDNF-acute and the BDNF pretreatment groups.  BDNF treatment did not increase rubrospinal axonal arborizations/sprouts in the gray matter rostral to the lesion site We also entertained the possibility that the cell body pretreatment of the rubrospinal neurons might have enhanced their sprouting proximal to their site of injury (Fig. 2.5A-C). Such sprouting could facilitate rubrospinal signaling caudal to the lesion via propriospinal neurons. While both BDNF-acute (n = 5) and BDNF pretreatment (n = 6) groups displayed ~ 2 times higher density of axonal arborizations into the gray matter rostral to the lesion site than the control group (n = 6), there were no significant group differences (Fig. 2.5D) (p > 0.05, Twoway repeated measure A N O V A ) .  97  2.4. Discussion  Here we report that neither the BDNF-acute treatment nor 7 day B D N F pretreatment of rubrospinal neurons promoted regeneration after a cervical spinal cord injury. Both acute treatment as well as pretreatment with BDNF showed a partial rescue of rubrospinal neuron cell size and reduced dieback of the rubrospinal tract indicating a cell body response was triggered, but the ~ 2-fold increase in rubrospinal axon branches projecting into the gray matter of the spinal cord cranial to the lesion site did not reach significance. The BDNF-acute treatment also failed to improve functional recovery. Surprisingly, despite the lack of an increase in rubrospinal axonal growth/plasticity the BDNF pretreatment group showed a strong effect on functional recovery. While we have previously reported positive effects of acute cell body treatment of rubrospinal neurons in promoting growth into a PNG (Kobayashi et al., 1997; Kwon et al., 2002), in this experiment we failed to observe an increase in axonal growth in the CNS after a spinal cord injury. The well known evidence of a PNG being more permissive to axonal growth as compared to the inhibitory environment of the CNS could explain these results. This would suggest that B D N F rubrospinal cell body treatment while promoting growth in the permissive PNG environment is not sufficient to enhance growth into the inhibitory environment of the CNS. Pretreatment of neurons in vitro with neurotrophins (including BDNF) prior to exposure to myelin allowed them to overcome myelin's inhibitory properties, but no increase of growth was observed if neurotrophins were applied at the same time as exposure to myelin (Cai et al., 1999). Additionally, either a priming lesion, or pretreatment of DRGs with cAMP stimulated growth of the central axons into the CNS which normally fails (Neumann and Woolf, 1999; Neumann et al., 2002; Qiu et al., 2002). In our case however, pretreatment of rubrospinal (descending supraspinal) neurons with the neurotrophin B D N F failed to induce regeneration. Additionally, the 2-fold increase of axonal branches/sprouts rostral to the lesion site did not reach significance and was not different from that of the BDNF-acute group. This lack of axonal regeneration is unlikely due to technical difficulties with the BDNF delivery from the osmotic minipumps as both BDNF-treated groups showed attenuated rubrospinal neuron atrophy and axonal dieback. The effect of BDNF-acute treatment on cell size seen in this experiment 35 days post injury (BDNF-acute 82.30 % +/- 2.55, control = 61.33 % +/2.23) is very similar to the results reported by Kobayashi examining rubrospinal neurons 28 days 98  post injury (BDNF = 83 %, control = 56 %) (Kobayashi et al., 1997). Additionally, both BDNFtreated groups displayed reduced weight gain compared to control indicating effective B D N F delivery as previously reported (Lapchak and Hefti, 1992; Pelleymounter et al., 1995). Hence our data suggests that neurotrophin pretreatment of this group of CNS supraspinal neurons (rubrospinal) does not produce the same axonal growth promoting effect as similar pretreatment of peripheral D R G neurons (Neumann et al., 2002; Qiu et a l , 2002). The difference in the results is likely due to the higher intrinsic growth capacity of PNS neurons compared to CNS neurons (Fernandes, 2000). The axonal branching/sprouting data failed to reach significance, which might be due to several compounding factors. There is an inherent variability in the efficacy of anterograde tracing between animals and hence a normalization of axonal branching in the cervical gray matter was performed. We counted the number of traced axons in cross sections of the CI level of the spinal cord in order to generate a measure of the efficacy of tracing. However, these crosssections displayed higher (but not significant) numbers of axons in both B D N F treatment groups (acute and pretreatment). We conclude that the trophic factor treatment per se might have enhanced the capacity of the rubrospinal neurons to take up and transport the tracer and/or to form collateral sprouts at all levels of the axon. Such effect is likely, since the rubrospinal neurons in control rats underwent massive atrophy as well as axonal shrinkage and retraction after axonal injury, while the BDNF-treated ones did not (Kobayashi et al., 1997). Hence, the tracing of the BDNF-treated rubrospinal axons may also reflect this trophic effect indicative of a normalized or enhanced functional state of these neurons with maintained/spouted axonal arborization, including at the CI level used for normalization. Such normalization would likely diminish or even mask the BDNF-induced effects, and hence is very conservative. The extent of functional recovery in the BDNF pretreatment group was remarkable, given that this system shows no spontaneous recovery over the 4 weeks of observation. Forelimb usage is a complex parameter and we can only speculate on mechanisms for the improved functional recovery. There was no evidence for rubrospinal axon regeneration and a prevention of rubrospinal retraction was seen in the BDNF pretreatment as well as the BDNF-acute groups. Yet, the BDNF-acute group failed to recover forelimb usage while the B D N F pretreatment group demonstrated substantial improvement. The infused B D N F could have affected multiple additional populations. Diffusion of BDNF is limited and the bulk of the infused BDNF stays close to the site of injection (Kobayashi et al., 1997). However, it is conceivable that some infused B D N F reached the nearby 99  corticospinal and brainstem-spinal axons. In addition, B D N F infusion into the red nucleus increases BDNF mRNA in the rubropinal neurons (Kobayashi, 1998), which is likely translated, transported down the axon, and secreted into the cervical spinal cord. Increased BDNF levels could exert neuroprotective as well as plasticity promoting effects on other trkB receptor expressing populations. These might include the corticospinal, brainstem-spinal, and serotonergic axonal tracts. A large body of literature demonstrated the role of these axonal populations in functional recovery after spinal cord injuries (Deumens et al., 2005). With pretreatment, B D N F levels in the spinal cord could have built up prior to injury. In vitro, in order to overcome myelin inhibition only pretreatment with B D N F was effective in promoting neurite outgrowth (Cai et al., 1999). The possible increase in B D N F levels in the spinal cord might have diminished the axonal susceptibility to the inhibitory effects of myelinassociated proteins and thus enhanced sprouting/plasticity. Moreover, any possible neuroprotective effects of BDNF are highly time sensitive and an accumulation of BDNF (and activated BDNF signaling) prior to injury might have been critical in preventing secondary damage and/or promoting plasticity, giving the pretreated rats a behavioral advantage. The BDNF-treated animals showed attenuated weight gain that did not return to normal until 1-2 weeks after the BDNF pump had run out. These results are consistent with the effects reported for B D N F infusions into the ventricles (Lapchak and Hefti, 1992; Pelleymounter et al., 1995). Pelleymounter concluded the B D N F effect on weight was not due to a toxic effect but rather it reduced appetite and thereby food intake. Subsequent work suggests the BDNF food reduction effect is mediated via the hypothalamus (Xu et al., 2003) and/or the dorsal vagal complex in the midbrain (Bariohay et al., 2005). These results indicate that BDNF applied in the vicinity of the red nucleus led to the un-planned reduction of caloric intake and hence attenuating weight gain in our BDNF experimental groups. Reduced food intake, better known as dietary restriction (DR), if implemented prior to insult has been shown to positively affect several models of brain disorders including stroke, Parkinson's, Huntington's, and Alzheimer's diseases, (Duan and Mattson, 1999; Y u and Mattson, 1999; Duan et al., 2003; Patel et al., 2005). DR increases neurotrophins and is neuroprotective if started prior to an insult (Brace-Keller et al., 1999; Duan et al., 2001; Anson et al., 2003). The BDNF pretreatment group was subjected to D R before injury. Therefore, the unintentional DR induced by our BDNF pretreatment could have been neuroprotective and/or promoted plasticity. Hence, the enhanced behavioral outcome in the B D N F pretreatment group might have been in part due to DR. 100  BDNF pretreatment of rubospinal neurons promoted recovery in forelimb usage compared to either control pretreatment or BDNF-acute treatment. However, the increased functional recovery is unlikely mediated by the rubrospinal tract. Both B D N F treatment groups displayed less atrophy of the rubrospinal neurons and less dieback, but no regeneration or increase of sprouting of rubrospinal axons occurred in these two groups. Our results suggest that BDNF-acute cell body treatment of rubrospinal neurons does not promote axonal growth in the CNS. Additionally, in vivo neurotrophin pretreatment of supraspinal CNS neurons might not produce the same growth inducing properties that priming elicits in peripheral neurons.  101  Figure 2.1. Experimental timeline and weekly weight gain of animals (A) The BDNF pretreatment group (n = 6) started receiving B D N F 7 days prior to spinal cord injury and a vehicle control group (n = 6) was run in parallel. The infusion lasted for 14 days using an osmotic minipump with an attached cannula that was implanted in the vicinity of the red nucleus. A third group, the BDNF-acute group (n = 5) received a 14 day B D N F infusion starting at the time of injury. Post injury behavior was tested every 7 days for 4 weeks after injury. At 28 days after injury the anterograde tracer B D A was injected into the vicinity of the red nucleus in all groups. (B) Both BDNF pretreatment and BDNF-acute animals gained less weight than the control group (p < 0.01, Two-way repeated measure A N O V A ) . (* , B D N F pretreatment group gained less weight than control pretreatment and BDNF-acute groups (prior to pump installation) (p < 0.001, Scheffe); **, BDNF pretreatment group gained less weight than control pretreatment group (p < 0.05) but more than BDNF-acute group (p < 0.01); ***, BDNF-acute group gained less weight than control pretreatment and BDNF pretreatment groups (p < 0.01). On the graph, -2 weeks pre-injury for the control pretreatment and BDNF pretreatment groups and -1 week preinjury for the BDNF-acute group represents weight gained in a one-week period prior to treatment (normal). At -1 week pre-injury for the pretreatment groups (control and BDNF pretreatment) and 1 week post-injury for the BDNF-acute group on the graph represents the weight gained during the first week of treatment (via mid brain cannula attached to osmotic mini-pump delivery vehicle or BDNF - shaded area of the data lines). The pump delivered the treatment for 2 weeks, which means the pretreatment groups no longer receive treatment after 1 week post-injury and the BDNF-acute group treatment ended at 2 weeks post-injury. On the graph, 2 weeks after injury for the pretreatment groups (control and B D N F pretreatment) and 3 weeks after injury for the BDNF-acute group represent the weight gained during the first week in which the animals were no longer receiving treatment via the installed pump. There is no "0" week because the graph depicts weight gained over a 1-week period, so there is no weight given for a particular day (e.g day of surgery). Hence, there is only weight gain per week for the weeks prior to injury (-2, -1) or post-injury (1, 2, 3, 4).  102  <  Behaviour  Lesion Day-7  0  I  L  I ~ V  7  ^  14  .  I  L  1  1  \  1  1  BDNF-acute  Kill  28  35  I  1  1  1  1  BDNF pretreatment 1  BDA tracing  I  Control pretreatment  1  21  >  1  1  Figure 2.2. BDNF pretreatment increased ipsilateral forelimb usage in the Schallert cylinder task Baseline preoperative forelimb usage scores did not differ between the groups for the '% use of Left+Both'. The BDNF pretreatment group ( n = 6) had significantly higher '% use of Left+Both' compared to both control pretreatment (n = 6) and BDNF-acute (n = 5) groups in the post injury period (p < 0.01, Two-way repeated measure A N O V A ) with 21 and 28 days post injury being significant in pair-wise comparisons (p < 0.05, Scheffe). Neither the control pretreatment nor the BDNF-acute group showed any recovery of function over time.  104  Days after injury  Figure 2.3. BDNF-acute and B D N F pretreatment rescued red nucleus cell size NeuralTrace staining of the red nucleus 5 weeks after cervical spinal cord injury in the three groups. (A,B) In the control group the injured red nucleus (A) displayed typical neuronal atrophy compared to the uninjured contralateral (B). (C,D) BDNF-acute group also have reduced neuron cell size on the axotomized side (C) but to a lesser extent than the control group. (E,F) BDNF pretreatment neuron cell size is in between the control and BDNF-acute treatment groups. Scale bar = 50 pm. (G) Quantification of red nucleus neuron size 5 weeks after injury reveals overall significant difference between groups (expressed as % of contralateral; p < 0.01, one-way A N O V A ) . The rubrospinal neurons of both BDNF-acute treatment (p < 0.01, Scheffe, n = 5) and BDNF pretreatment (p < 0.05, Scheffe, n = 6) had less atrophy as compared to the control pretreatment group (n = 6).  106  107  Figure 2.4. BDNF-acute and BDNF pretreatment reduced rubrospinal dieback BDA-labeled rubrospinal axons descending in the lateral funiculus are depicted in the three pictures and the lesion sites are seen at the right margin of the figures. While the control pretreated group displayed typical dieback from the lesion site (A), both BDNF-treated groups maintained many axons close to edge of the lesion (B: BDNF-acute, C: B D N F pretreatment). No regeneration of the rubrospinal tract was observed in any group. Scale bar = 200 pm. (D) Quantification of rubrospinal dieback revealed differences among the groups (p < 0.001, oneway A N O V A ) . Both BDNF pretreatment (n = 6) and BDNF-acute (n = 5) groups displayed significantly reduced dieback of their labeled axons when compared to control pretreatment (n = 6) group (p < 0.001, Scheffe).  108  109  Figure 2.5. BDNF-acute or BDNF pretreatment failed to promote rubrospinal tract sprouting in the gray matter rostral to the lesion site The control pretreated group exhibited only a consistent limited number of labeled rubrospinal axons in the gray matter rostral to the lesion site (A). In contrast both B D N F treatment groups (B: BDNF-acute, C: BDNF pretreatment) showed highly variable amount of axonal profiles. Scale bar = 200 pm. (D) Quantification of normalized rubrospinal axonal profiles in the gray matter rostral to the lesion site. Despite a two fold increase in the mean density of axon profiles in the gray matter rostral to the lesion site in both BDNF treatment groups, there were no significant differences among the groups (p > 0.05, Two-way repeated measure A N O V A , control group n = 6, BDNF-acute n = 5, BDNF pretreatment n = 6).  110  •  ,V-  S.\«  •""'f""'--'.-f.  ;  v .........y •  '•  •  ':  ;  v  * y -'  A as.:. - -v  CD C " Q . CO CO CD  O  i  =  n 2 P CL  0.04  —•— Control pretreatment - O - BDNF-acute — T — BDNF pretreatment  o 20.03 W CO  73  .4-J  CD CD £ CD  0.02 0.01  CD  0.00  Distance in u m rostral t o lesion site  ill  2.5. 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Fischer D, Heiduschka P, Thanos S (2001) Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats. Exp Neurol 172:257-272. Houle JD, Jin Y (2001) Chronically injured supraspinal neurons exhibit only modest axonal dieback in response to a cervical hemisection lesion. Exp Neurol 169:208-217. 112  Kobayashi N (1998) Neurotrophins and the neuronal response to axotomy. PhD Thesis, University of British Columbia. Kobayashi NR, Fan DP, Giehl K M , Bedard A M , Wiegand SJ, Tetzlaff W (1997) B D N F and NT4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talphal -tubulin mRNA expression, and promote axonal regeneration. J Neurosci 17:9583-9595. Kwon B K , Liu J, Messerer C, Kobayashi NR, McGraw J, Oschipok L, Tetzlaff W (2002) Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury. Proc Natl Acad Sci U S A 99:3246-3251. Lapchak PA, Hefti F (1992) BDNF and N G F treatment in lesioned rats: effects on cholinergic function and weight gain. Neuroreport 3:405-408. Leon S, Yin Y , Nguyen J, Irwin N , Benowitz LI (2000) Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci 20:4615-4626. Liu Y , Kim D, Himes BT, Chow SY, Schallert T, Murray M , Tessler A , Fischer I (1999) Transplants of fibroblasts genetically modified to express B D N F promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J Neurosci 19:4370-4387. Lorber B, Berry M , Logan A (2005) Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors. Eur J Neurosci 21:2029-2034. Lu P, Yang H, Jones L L , Filbin M T , Tuszynski M H (2004) Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury. J Neurosci 24:6402-6409. Lu X , Richardson P M (1991) Inflammation near the nerve cell body enhances axonal regeneration. J Neurosci 11:972-978. Neumann S, Woolf CJ (1999) Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23:83-91. Neumann S, Bradke F, Tessier-Lavigne M , Basbaum A l (2002) Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron 34:885-893. Patel N V , Gordon M N , Connor K E , Good RA, Engelman RW, Mason J, Morgan D G , Morgan T E , Finch C E (2005) Caloric restriction attenuates Abeta-deposition in Alzheimer transgenic models. Neurobiol Aging 26:995-1000. Pelleymounter M A , Cullen MJ, Wellman C L (1995) Characteristics of BDNF-induced weight loss. Exp Neurol 131:229-238. Plunet W, Kwon BK, Tetzlaff W (2002) Promoting axonal regeneration in the central nervous system by enhancing the cell body response to axotomy. J Neurosci Res 68:1-6. Qiu J, Cai D, Dai H, McAtee M , Hoffman PN, Bregman BS, Filbin M T (2002) Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34:895-903. Ramer L M , A u E, Richter M W , Liu J, Tetzlaff W, Roskams A J (2004) Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord inj ury. J Comp Neurol 473:1 -15. Richardson P M , Issa V M (1984) Peripheral injury enhances central regeneration of primary sensory neurones. Nature 309:791-793. Richter M W , Fletcher PA, Liu J, Tetzlaff W, Roskams A J (2005) Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord. J Neurosci 25:10700-10711. Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000) CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39:777-787. 113  Schwartz ED, Shumsky JS, Wehrli S, Tessler A, Murray M , Hackney D B (2003) Ex vivo M R determined apparent diffusion coefficients correlate with motor recovery mediated by intraspinal transplants of fibroblasts genetically modified to express BDNF. Exp Neurol 182:49-63. Scott A L , Borisoff JF, Ramer MS (2005) Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. Eur J Neurosci 21:81-92. Shumsky JS, Tobias C A , Tumolo M , Long WD, Giszter SF, Murray M (2003) Delayed transplantation of fibroblasts genetically modified to secrete B D N F and NT-3 into a spinal cord injury site is associated with limited recovery of function. Exp Neurol 184:114-130. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146-156. Tobias C A , Han SS, Shumsky JS, Kim D, Tumolo M , Dhoot NO, Wheatley M A , Fischer I, Tessler A , Murray M (2005) Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression. J Neurotrauma 22:138-156. Xu B, Goulding E H , Zang K, Cepoi D, Cone RD, Jones KR, Tecott L H , Reichardt L F (2003) Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin4 receptor. Nat Neurosci 6:736-742. Yin Y , Cui Q, Li Y , Irwin N , Fischer D, Harvey AR, Benowitz LI (2003) Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci 23:2284-2293. Yin Y , Henzl M T , Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R, Benowitz LI (2006) Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat Neurosci 9:843-852. Y u ZF, Mattson MP (1999) Dietary restriction and 2-deoxyglucose administration reduce focal ischemic brain damage and improve behavioral outcome: evidence for a preconditioning mechanism. J Neurosci Res 57:830-839.  114  Chapter 3 Dietary restriction is neuroprotective and promotes functional recovery from cervical spinal cord injury  A version of this chapter has been submitted for publication. Ward T. Plunet, Clarrie K. Lam, Jae H. T. Lee, Jie Liu, Wolfram Tetzlaff. Dietary restriction neuroprotective and promotes functional recovery from cervical spinal cord injury  3.1. Introduction  Regeneration and functional recovery after spinal cord injury are restricted in adult mammals. Since most clinical injuries are anatomically incomplete, one avenue of experimental treatments focus on the prevention of secondary degeneration in the spinal cord and the enhancement of endogenous neural plasticity of the spared spinal cord (Bareyre et al., 2004; Park et al., 2004). Other repair strategies aim at axonal regeneration by addressing various obstacles such as the weak intrinsic growth response of the injured neurons (Plunet et al., 2002; Filbin, 2003) and axonal growth inhibitors associated with myelin and reactive glial scar (Silver and Miller, 2004; Buchli and Schwab, 2005). In the light of these multiple obstacles, preclinical assessments often employ combinatorial experimental strategies to leverage the effects of individual approaches (Ramer et al., 2005). However, combinatorial approaches are more difficult to translate into clinical settings due to safety concerns and regulatory hurdles. A n ideal regimen would be safe, inexpensive and simple, while effectively treating the multiple biological aspects simultaneously to improve functional recovery after spinal cord injury. Dietary restriction (DR) could offer a safe combinatorial approach. When implemented prior to disease/insult DR provides neuroprotection and promotes plasticity (Mattson et al., 2003). Prophylactic DR has biochemical and functional benefits in a wide range of animal injury/disease models including stroke (Yu and Mattson, 1999), Parkinson's disease (Maswood et a l , 2004 ), Huntington's disease (Bruce-Keller et al., 1999), and Alzheimer's disease (Zhu et al., 1999; Patel et al., 2005). However, its efficacy has not been tested in spinal cord injury. The multiple effects of DR include the increased expression of trophic factors, (e.g. BDNF) (Mattson, 2005), and antioxidative and anti-apoptotic proteins (Yu and Mattson, 1999; Shelke and Leeuwenburgh, 2003; Hiona and Leeuwenburgh, 2005; Sharma and Kaur, 2005). Moreover, DR attenuates the age- or disease- related astrocytic reactivity measured by glial fibrillary acidic protein (GFAP) immunostaining (Morgan et al., 1997; Patel et al., 2005). Collectively, these changes may provide neuroprotection (Bruce-Keller et al., 1999), reduce glial scaring, increase the supply of trophic factors, and thereby enhance plasticity/regeneration. The two most common forms of DR are 30-40 % daily reduction of caloric intake (calorie restriction - CR) and every-other-day fasting (EODF). Neuroprotection is more effective with EODF compared to 40 % CR (Anson et al., 2003). Therefore, we hypothesized that EODF  116  increases neuronal survival, reduces lesion size and glial scarring, hence contributing to enhanced functional recovery after spinal cord injury.  117  3.2. Materials and Methods  Animals and Surgery. Male Sprague-Dawley rats (n = 10) 260-300 g (UBC/Charles River Breeding Laboratories) were used in accordance with the guidelines of the Canadian Council for Animal Care with procedures approved by the University Animal Care Committee. The control group (n = 5) received food and water ad libitum. For the every-other-day fasting (EODF) group (n = 5) food was removed from the animals' cage at 8:00 a.m. (ad libitum supply of water) every second day for a 24 hour period beginning 4 weeks prior to the C4 spinal cord injury and continued throughout the experiment. Both groups received the same food source consisting of 59.8 % carbohydrates, 28.0 % protein, and 12.1 % fat (LabDiet 5001; Purina Mills Brentwood, MO). The surgery was performed on a fasting day for the EODF group. The surgeon was blinded to the pretreatment of the animals and kept naive about this project. The animals were anesthetized with ketamine hydrochloride (72 mg/kg;i.p.; BimedaM T C , Cambridge, Ontario) and xylazine hydrochloride (9 mg/kg; i.p.; Bayer Inc., Etobicoke, Ontario, Canada) and a hemi-laminectomy was performed at the fourth cervical vertebra. A fine needle (30 gauge) was inserted vertically between the dorsal gray matter and the dorsal column white matter on the left side of the spinal cord to create a needle hole. Fine forceps with parallel blades 0.2 mm wide and 2 mm long (customized Dumont #5, Fine Science Tools, Vancouver) were inserted into the needle hole to a depth of 2.0 mm. The lateral spinal cord was crushed with the forceps held closed for 20 seconds. This lesion model crushes the rubrospinal tract (RST) and lateral cortical spinal tract (CST) while leaving the ipsilateral dorsal cortical spinal tract (CST) and ventral CST intact. Behavior. Forelimb usage during vertical exploration was tested in a transparent cylindrical container (30 cm diameter, 30 cm height, Nalgene labware, N Y ) and videotaped to score the frequency of forelimb usage as the rats reared up to touch the side of the container. Animals were given five minutes on their initial trial for each testing week. Subsequent exposure to the cylinder occurred after at least a one hour rest period between trials until the animals reached 20 wall touching rears in total. Secondary exposures to the cylinder were all less than five minutes long. The person scoring the behavior was blinded to the treatment groups and kept naive about the premise of this research project. All touches of the wall during vertical exploration - with the left, right or both forelimbs, were counted and expressed as percentage of total touches (Schallert et al., 2000). With the lesion on the left side, percent usage of the left limb and both limbs (% use of Left + Both) reflects how often the ipsilateral limb was employed 118  (Schwartz et al., 2003; Tobias et al., 2005). A baseline pre-operation score was obtained from each rat prior to injury. Post injury behavioral testing was performed on feeding days (at least 8 hours after the E O D F group was fed) every 8 days (7, 15, 23, 31 days post injury). A n a priori criterion for removal of animals was inadvertent dorsal CST damage, which in this spinal cord model is designed to be left intact. One animal belonging to the E O D F group was eliminated from this experiment due to this criterion. Histology. To anterogradely trace the rubrospinal axons, on day 33, all animals, while anaesthetized as above, received 0.6 ul of 25 % biotin dextran amine (BDA; Molecular Probes, OR) through a fine grass pipette inserted stereotaxtically medial to the rubrospinal neurons at 6.3 mm posterior to bregma, 0.7 mm to the right of midline, and 7.0 mm below the dura as described previously (Kwon et al., 2004). One week later, following anaesthesia overdose animals were perfused with 4 % paraformaldyhde. The spinal cords were post-fixed, cryoprotected in 22 % sucrose overnight, frozen, and cut at 20 um in the longitudal-horizontal plane. From each animal, a series of 3 horizontal sections spaced 240 pm apart, with the most dorsal one running just ventral to the main dorsal CST, were stained with anti-NeuN (1:100, Chemicon International Inc., CA). NeuN images captured under same exposure settings were then equally thresholded. An area of 2 mm in length centered at the lesion site was used for counting the number of undamaged neurons in the gray matter. NeuN immunostaining is widely used as a marker for neuronal viability. However, we recently reported that facial motoneurons down-regulate NeuN expression without dying as a response to axotomy (McPhail et al., 2004). Hence, we are reluctant to designate the NeuN-negative population as dead and rather refer to them as damaged. Sections spaced 120 pm apart spanning the dorsal-ventral distance of the spinal cord were stained with anti-GFAP (1:400, Sigma-Aldrich, MO). Images of the three largest lesion areas from each animal were captured and thresholded identically. A person blinded to the treatment groups traced the outlines of the lesion areas marked by GFAP immunoreactivity using Sigma Scan Pro 5 (SPSS, Chicago, IL) to obtain the average lesion area in each animal. The average density of GFAP imunoreactivity in the white matter was quantified in a standard window 2 mm in length centered at the lesion site with Sigma Scan Pro (the same 3 sections used for lesion size measurements). Statistics. SigmaStat 3.0 (SPSS, Chicago, IL) was used to run all statistics. A Student's ttest was used to compare the number of NeuN-positive neurons, lesion size, and GFAP immunostaining differences between the groups. A two-way repeated-measures A N O V A (time and behavior) was used on the behavioral tasks followed by post hoc analysis when appropriate. 119  The Pearson correlation test was used to investigate possible relationships between behavioral outcome and morphological measurements. Standard errors of the mean (+/- S.E.M) were reported and displayed in the graphs.  120  3.3. Results Animals continued to gain weight on the every-other-day fasting diet regime After the initial pre-operative test of forelimb usage during vertical exploration (cylinder test), animals were divided into two groups with similar weights and forelimb usage scores. At the onset of the EODF regimen one month prior to spinal cord injury, the rats in the EODF group weighed on average 297.0 g +/- 9.60 (n = 4) and the control rats 298.4 g +/- 6.76 (n = 5). The EODF group did not lose weight on the restricted food regimen throughout the experiment, and continued to gain weight, albeit at a lower rate than the ad libitium fed group (Fig. 3.1).  EODF animals displayed a greater number of undamaged neurons After a spinal cord injury a cascade of molecular events occurs resulting in secondary damage to the initially undamaged areas of the spinal cord adjacent to the lesion. Ischemia induced increase of reactive oxygen species (ROS), and subsequent lipid peroxidation, among a host of other molecular pathways, result in necrosis and apoptosis in the penumbra of the injured spinal cord (Park et al., 2004). We used a neuronal marker NeuN to quantify the number of undamaged neurons surrounding the lesion site at 6 weeks after injury. The count of NeuNpositive neurons revealed that the EODF group had a greater number of undamaged neurons than the control group (p < 0.05, control = 715.2 +/- 80.6: n = 5, EODF = 1042.7 +/- 49.6: n = 4) (Fig. 3.2). Hence, EODF increased the number of undamaged neuron by -30 %.  EODF reduced lesion size and astrocyte activation Typically, an obvious cavity forms at the site of injury after a crush of the dorsolateral funiculus at the C4 level of the spinal cord. At 6 weeks after injury, the site of injury were outlined in longitudinal sections of the spinal cord by GFAP immuno-fluorescence (Fig. 3.3 A, B). Quantification of the cavity size (Fig. 3.3C) revealed that the rats on the EODF regimen had smaller lesion cavities (~45 %) than the rats in the control group (p < 0.05, Student's t-test, control = 1.03 pm +/- 0.15: n = 5, EODF = 0.56 pm +/- 0.08: n = 4). 2  2  Activated astrocytes are the principal contributor to the formation of glial scar after spinal cord injury, which inhibit axonal plasticity (Rhodes and Fawcett, 2004). Glial fibrillary acidic protein (GFAP) immunostaining visualizes activated astrocytes around the lesion site. Quantification of the density of GFAP immunostaining in the areas depicted in figures 3.3 A and B revealed that the EODF group displayed significantly less GFAP immunoreactivity compared 121  to the control group (control = .1432 +/- 0.0089, EODF = .1161 +/- 0.0035, p < 0.05) (Fig. 3.3D). We did not observe any anterogradely labeled rubrospinal axons (see Methods) regenerating past, or into, the lesion site in any of the animals (data not shown).  EODF improved forelimb recovery The % use of Left + Both forelimbs did not differ between the two groups prior to the injury (control = 74.77 % +/- 3.82, EODF = 73.15 % +/- 3.83) (Fig. 3.4A). A two-way repeatedmeasures A N O V A revealed differences between the groups in the post-injury time points (p = 0.011, control n = 5, EODF n = 4). At 7 days after the crush of the left dorsolateral funiculus at C4, there was a dramatic decline in the usage of the ipsilateral forelimb (control = 24.47 % +/5.71, EODF = 33.54 % +/- 6.39) (Fig. 3.4A). The % use of Left + Both in both groups were not different from each other, indicating the severity of the injuries was equivalent. At 15, 23 and 31 days post injury the EODF group displayed significantly higher % use of Left + Both compared to the control group (p < 0.05, control = 32.31 % +/- 5.15, 21.90 % +/- 4.38, 30.44 % +/- 4.23, EODF = 56.80 % +/- 7.33, 58.81 % +/- 3.36, 52.26 % +/- 5.26) (Fig. 3.4A). The E O D F treated animals average a ~2 fold increase in the use of ipsilateral forelimb use in weeks 2-4 as compared to control animals. In agreement with other studies and several control series from our lab (unpublished observations) with this lesion model, control rats do not recover their ipsilateral limb usages over the 4-week post-operative testing period (Liu et al., 1999; Schwartz et al., 2003; Tobias et al., 2005). The number of rears performed during the initial five minutes exposure to the cylinder was not significantly different between the groups over the 4-week testing period (p > 0.05) (Fig. 3.4B), which is consistent with previous research using a more extensive and exhaustive behavioral monitoring of Sprague-Dawley rats on the EODF diet regimens (Wan et al., 2003). This indicates that increased activity level was unlikely a factor in improving functional recovery after the spinal cord injury.  Behavioral outcome after cervical spinal cord injury correlates with morphological measurements of secondary damage Since we observed less GFAP density surrounding the lesion along with a reduction in lesion size and an increase in NeuN-positive neurons in the EODF treated group we wanted to see if the measured morphological measurements correlated with behavioral outcome. The percent use of Left + Both of the ipsilateral forelimb negatively correlated with lesion size (r = 122  0.668, p = 0.0494) and positively with the number of NeuN-positive neurons (r = 0.848, p = 0.0039), but not with GFAP density (r = 0.541, p = 0.1322).  123  3.4. Discussion  Here we report that every-other-day fasting in adult rats started one month prior to spinal cord injury is neuroprotective, reduces glial scaring, and enhances functional recovery. Only the EODF animals displayed significant functional recovery of ipsilateral forelimb usage. At six weeks post injury, the EODF group had an increase number of NeuN-positive neurons and a reduced density of GFAP immunostaining in the tissue in the vicinity of the lesion. The EODF rats also had a marked reduction in cavitation of the lesion site. Collectively these data show for the first time that prophylactic EODF is beneficial for spinal cord injuries. Axonal plasticity could be enhanced by the reduction in GFAP seen globally around the lesion area, reflecting a reduced activation of astroctyes, which is consistent with previous work with calorie restriction (Patel et al., 2005). Reactive astrocytes are the source of inhibitory molecules such as chondroitin sulfate proteoglycans (Rhodes and Fawcett, 2004). However, in this experiment GFAP density did not correlate with behavioral outcome after cervical spinal cord injury. While no regeneration of the rubrospinal tract was observed in our experiment at the site of injury, such downregulation of inhibitory molecules might have contributed to the plasticity within the uninjured areas of the spinal cord. We are confident that the behavioral recovery of forelimb usage reflects a robust effect of EODF that is corroborated by the reduced lesion sizes in the order of 45 % and increased the number of NeuN-positive neurons. A significant correlation was observed for both lesion size and NeuN-positive neurons in the pneumbra of the lesion with the percent use of the ipsilateral forelimb. Reduction in lesion size correlates well with functional recovery in a number of spinal cord injury models (Basso et al., 1995). In a lesion similar to the one we used, others observed a negative correlation between lesion size and behavioral recovery (Ruitenberg et al., 2003; Schwartz et al., 2003) but failed to find a correlation between the enhanced rubrospinal sprouting and recovery (Ruitenberg et al., 2003). The reduced lesion size seen in our EODF group might have allowed non-rubrospinal descending projections (e.g. corticospinal tract projections) to reach the lower cervical spinal cord and thus enhance function. Our finding of reduced lesion size in the EODF group complements similar reports of diminished lesion size in EODF animals after administration of either malonate or 3NP (Bruce-Keller et al., 1999) and also after focal brain ischemia (Yu and Mattson, 1999). The mechanisms by which EODF protects the injured spinal cord may include a wide spectrum of factors and pathways. Trauma to the spinal cord entails primary mechanical tissue 124  damage, ischemia with energy failure, and early inflammation, which triggers an increase in the production of reactive oxygen species (ROS) and free radicals (Park et al., 2004). These contribute to secondary damage such as lipid peroxidation, and necrotic and apoptotic cell death, in the penumbra of the primary insult and along the initially spared white matter tracts. DR effectively reduces oxidative damage in the brain (Dubey et al., 1996) which is likely due to a reduction in ROS (Sohal et al., 1994; Guo et al., 2000). EODF also increases antioxidants (CuZn SOD, catalase, GSH), reduces lipid peroxidation, and promotes neuronal survival after kainic acid insult (Sharma and Kaur, 2005). Increases in free radical scavenging and/or reduction of ROS are beneficial after spinal cord injury and can improve functional outcome (Kamencic et al., 2001; Sullivan et al., 2004). DR affects multiple components of the cell death pathways thereby promoting cell survival. DR increased anti-apoptotic proteins such as XIAP, apoptosis repressor with a caspase recruitment domain (ARC), and inhibit caspase-3 and caspase-2 activation and subsequent apoptosis (Shelke and Leeuwenburgh, 2003; Hiona and Leeuwenburgh, 2005). Our findings of decreased lesion size and increased number of undamaged neurons in the tissue surrounding the lesion site in the EODF animals are consistent with the findings of ROS reduction and cell death pathway alterations in dietary restricted animals. EODF increases trophic factor expression, namely B D N F in the cortex, hippocampus and striatum, and NT3 in the dentate gyrus (Lee et al., 2000; Duan et al., 2001; Lee et al., 2002). Therefore, it is possible that EODF increases various trophic factors throughout the CNS including the spinal cord. Trophic factors promote neuroprotection, plasticity, and functional recovery after spinal cord injury (Deumens et al., 2005). The enhanced recovery observed in the food-restricted group raises implications for spinal cord and other neurotrauma behavioral paradigms that use some form of food restriction to train/test the animals (including prior to injury). The general use of food restriction in the training and subsequent post injury testing in all experimental groups could be masking results of various treatments. In conclusion, EODF initiated one month prior to spinal cord injury and continued throughout the post-injury period led to a marked recovery of ipsilateral forelimb usage, decreased astrocyte activation, increased neuronal integrity, and reduced lesion size. The wide spectrum of targets reached by this low-risk, drug-free regimen makes this treatment attractive for clinical translation. Humans on EODF (unlike 30-40% calorie restriction) maintain their bodyweight (Halberg et al., 2005). They appear to be able to consume twice the calories on food eating days even while engaging in normal activity levels. The practical use of pre-treatment 125  paradigms may be for elective repair interventions such as cell transplantations into the injured cord, which are likely performed in the subacute to chronic phase. E O D F may enhance neural plasticity and promote significant neuroprotection from the collateral tissue damage associated with repair strategies.  126  Figure 3.1. Rats on EODF gained weight, but at a slower rate than control Weights of the EODF (n = 4) and control rats (n = 5) were similar one month prior to spinal cord injury. The E O D F group started their fasting regime 28 days prior to injury. The EODF animals continued to gain weight on this diet regime throughout the experiment, but at a slower rate than the control group.  127  Figure 3.2. EODF increased the number of NeuN-positive neurons in the penumbra of the lesion The number of undamaged neurons marked by NeuN-positive immunostaining over a 2 mm distance in the gray matter in the vicinity of the lesion was higher in the E O D F group (n = 4) compared to the control group (n = 5) (*, p < 0.05, Student's t-test).  129  Figure 3.3. E O D F reduced lesion size and G F A P immunoreactivity (A, B) Representative pictures of lesion cavities demarcated by GFAP immunostaining; (A) control, (B) EODF. (scale bar = 200 um). (C) Quantification of average lesion size revealed EODF animals (n = 4) had smaller lesion cavities than the control animals (n = 5) (p < 0.05, Student's t-test). (D) Density of GFAP immunostaining in the intact spinal cord tissue depicted in A and B. There was significantly less GFAP density in the E O D F group (*, p < 0.05, Student's t-test).  131  132  Figure 3.4. EODF improved ipsilateral forelimb usage after a cervical spinal cord injury (A) The % use of Left + Both did not differ between the groups prior to injury. The EODF group (n = 4) displayed a significantly higher % use of Left + Both in the post injury testing period compared to control group (n = 5) (p = 0.011, two-way repeated-measures A N O V A ) . The EODF group achieved significantly higher % use of Left + Both at 15,23, and 31 days post injury (*, p < 0.05). (B) The number of rears performed in the rearing cylinder within the first five-minute segment. There was no significant difference between the two groups in the number of rears during the post-injury testing period indicating similar activity levels (p > 0.05, two-way repeated measures A N O V A ) .  133  A  3.5. 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Sullivan PG, Springer JE, Hall ED, Scheff SW (2004) Mitochondrial uncoupling as a therapeutic target following neuronal injury. J Bioenerg Biomembr 36:353-356. 136  Tobias C A , Han SS, Shumsky JS, Kim D, Tumolo M , Dhoot NO, Wheatley M A , Fischer I, Tessler A , Murray M (2005) Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression. J Neurotrauma 22:138-156. Wan R, Camandola S, Mattson MP (2003) Intermittent fasting and dietary supplementation with 2-deoxy-D-glucose improve functional and metabolic cardiovascular risk factors in rats. Faseb J 17:1133-1134. Yu ZF, Mattson MP (1999) Dietary restriction and 2-deoxyglucose administration reduce focal ischemic brain damage and improve behavioral outcome: evidence for a preconditioning mechanism. J Neurosci Res 57:830-839. Zhu H, Guo Q, Mattson MP (1999) Dietary restriction protects hippocampal neurons against the death-promoting action of a presenilin-1 mutation. Brain Res 842:224-229.  137  Chapter 4 Dietary restriction implemented after cervical spinal cord injury is neuroprotective, promotes plasticity, and improves functional outcome  A version of this chapter has been submitted for publication. Ward T. Plunet, Clarrie K. Lam, Jae H. T. Lee, Jie Liu, Wolfram Tetzlaff. Dietary restriction started subsequent to cervical spinal cord injury is neuroprotective, promotes plasticity, and improves functional outcome.  138  4.1. Introduction  Spinal cord injury in adult mammals results in a loss of motor, sensory, and autonomic function with limited recovery. There are three main obstacles to the return of functional recovery: secondary damage (Park et al., 2004), inhibitory environment of the adult central nervous system (CNS) (Silver and Miller, 2004; Buchli and Schwab, 2005), and the weak intrinsic cell body axonal response supporting axonal growth (Filbin, 2003). A n optimal treatment would be combinatorial, addressing all three of these obstacles in a safe manner. The ability of dietary restriction to increase longevity, even if started in adulthood, (Weindruch and Walford, 1982) prompted interest in its potential in CNS diseases/injuries. Dietary restriction if followed for several months has biochemical and functional benefits on Parkinson's disease (Maswood et al., 2004 ), Huntington's disease (Bruce-Keller et al., 1999), and Alzheimer's disease (Zhu et al., 1999; Patel et al., 2005). Prophylactic dietary restriction also has positive neuroprotective and behavioral effects on several neural insult models (Bruce-Keller et al., 1999; Y u and Mattson, 1999; Anson et al., 2003; Maswood et al., 2004; Mattson, 2005; Plunet et al., 2006). The two main forms of dietary restriction are 20-50 % daily reduction of caloric intake (calorie restriction - CR) and every-other-day fasting (EODF). Neuroprotection is more effective with EODF compared to 40 % CR (Anson et al., 2003). As expected, the effects of dietary restriction on a wide variety of systems are accompanied by multiple cellular responses (Cohen et al., 2004; Guarente and Picard, 2005; Mattson, 2005; Nisoli et al., 2005). Dietary restriction increases antioxidative and anti-apoptotic proteins, while decreasing activation of apoptotic proteins (Yu and Mattson, 1999; Shelke and Leeuwenburgh, 2003; Hiona and Leeuwenburgh, 2005; Sharma and Kaur, 2005). Additionally, EODF, if followed for several months, increases BDNF and the ratio of its receptors (full-length to truncated trkB) (Mattson, 2005). Experimental increase of B D N F at the injury site is neuroprotective, increases plasticity/regeneration, and improves functional recovery (Liu et al., 1999; Ruitenberg et al., 2003; Schwartz et al., 2003). We previously observed decreased lesion size and glial scaring, along with improved behavioral recovery if EODF was followed for one month prior to spinal cord injury (Chapter 3) (Plunet et al., 2006). However, it is unknown if a clinically translatable implementation of dietary restriction started at time of spinal cord injury or other neuronal insults would produce the same positive effects. Here we report for the first time that EODF implemented after cervical spinal cord injury can overcome multiple obstacles and promote functional improvement. 139  4.2. Materials and Methods  Male Sprague-Dawley rats (n = 46) (Charles River Breeding Laboratories) were used. A l l animal procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and approved by the Animal Care Committee of the University of British Columbia. For the behavioral and histological aspects of this study 16 rats underwent pre-testing for the three behavioral tasks (see below). The animals were divided into two groups with similar starting weights and forelimb preference. Both animal groups had ad libitum supply of water and the control group (n = 9) had ad libitum access to a standard diet consisting of 59.8 % carbohydrates, 28.0 % protein, and 12.1 % fat (LabDiet 5001; Purina Mills Brentwood, MO). The every-other-day fasting (EODF) group (n = 7) was deprived of this diet at 8:00 a.m. on every second day for a 24 hour period. Food was returned at 8:00 a.m. on feeding days. The EODF group started this every-other-day fasting regime after the cervical spinal cord injury; however, on the day of surgery the first day of food deprivation started between 8 a.m. and 12 p.m., since it took about 4 hours to process the 16 rats in a randomized manner. Animals were weighed on every 8 day at 8:00 a.m. corresponding to the time when both groups had received th  food over the last 24 hours. The surgeon was blinded to the experimental paradigm and naive about this research project. The animals were anesthetized with ketamine hydrochloride (72 mg/kg; i.p.; BimedaM T C , Cambridge, Ontario) and xylazine hydrochloride (9 mg/kg; i.p.; Bayer Inc., Etobicoke, Ontario, Canada) and a hemi-laminectomy was performed at the fourth cervical vertebra. A fine needle (30 gauge) was inserted vertically between the dorsal horn (gray) and the dorsal column (white) on the left side of the spinal cord. Fine straight forceps with 0.2 mm wide and 2 mm long tips (customized Dumont #5, Fine Science Tools, Vancouver) were inserted into the needle hole to a depth of 2.0 mm and closed for twenty seconds, crushing the lateral spinal cord. This lesion model crushes the rubrospinal tract (RST) and lateral corticospinal tract (CST) leaving the ipsilateral dorsal and ventral CSTs intact. Ah a priori criterion for removal of animals was inadvertent dorsal CST damage, which in our spinal cord model is meant to be left intact. One animal belonging to the control group was eliminated from this experiment due to this criterion. Two animals in the control group and one from the EODF group died subsequent to CST tracing and therefore only 6 animals from each group were used for histological analysis. 140  Behavioral testing. The person scoring the behavioral tapes was blinded to the treatment groups and kept entirely naive about the premise of this research project. Baseline taping of the three behavioral tests was performed one week prior to injury. Post-injury video-taping of forelimb use in the rearing cylinder commenced on day 7 (when all animals were fed), and every 8 day thereafter until day 31. The horizontal ladder testing began on day 9 (when all animals th  were fed) and every 8 day thereafter until day 33. On day 33, the rats were tested on a Plexiglas th  footprint run-walk and filmed from below. The evening hours (4-8 pm) of the feeding days (8-12 hours after being fed) were chosen for testing to eliminate the possibility of increased activity/motivation induced by fasting. Ladder. Rats were trained to walk across a 100 cm horizontal ladder (width = 11.5 cm, 17.5 cm height) with irregular rung spacing to a home cage (Metz and Whishaw, 2002). A 42 cm section containing 15 rungs (1 mm diameter) with irregular spacing was used for scoring. Each test rung was a minimum of 1.75 cm apart and a maximum of 4.0 cm. Each animal crossed 4-5 times, and the number of ipsilateral forelimb footsteps and footslips were recorded. A foot-slip error was designated as when the animals completely missed the rungs and the forelimb slipped noticeably below the rung's level. Each week the pattern of rungs were changed so the animal could not learn the sequence of rungs (Metz and Whishaw, 2002). Data were generated from frame-by-frame video replay, and expressed as the percent error (foot-slips). We reported percent error due to EODF animals taking fewer steps to cross the ladder. Forelimb usage during vertical exploration in a cylinder. Animals were tested in a cylindrical container (30 cm diameter, 30 cm height, Nalgene labware, N Y , USA), and videotaped with a digital camcorder for later scoring of forelimb usage as they reared up against the side of the container (Schallert et al., 2000). Initial trials lasted five minutes. Subsequent tests lasted less than five minutes, occurred after at least a one-hour rest period, and repeated until the animals achieved 20 wall-touching rears. The observer replayed the videotapes and recorded the limbs used for initial contact when vertically exploring the cylinder. Successful performance involved removal of both forelimbs from the floor and the placement of at least one forelimb against the vertical surface of the cylinder. Footprint analysis. Animals crossed a transparent plexiglas runway (10.2 cm wide, 91.5 cm long) with darkened 20 cm-high sides while being videoed from below. A predetermined middle section (40 cm) was chosen for footprint analysis to ensure that the animals have attained a consistent gait pattern when entering the scoring area. Five runs for each animal were recorded. 141  Only those crosses in which the animal continuously moved through the scoring area were deemed valid. A minimum of 3 crossings for each animal were scored. Video was replayed on a computer frame-by-frame and each footprint was marked. Stride width was measured between the forepaws to obtain a base of support. Stride length was also measured for each limb and the average standard deviation of stride length for all limbs was determined for each animal as a measure of coordination (Li et al., 2005). Histology. To anterogradely trace the CST axons, on day 35 animals received 1.6 ul (in total) of 10 % Fluoro-emerald (Molecular Probes, OR) (in dH.20), injected into the forelimb area of the right motor cortex using a Hamilton syringe (4 injection sites, each receiving 0.4 ul) (1.8 mm rostral, 1.5 mm and 2.5 mm lateral from Bregma, and 2.8 mm rostral, 1.5 mm and 2.5 mm lateral from Bregma, depth 1.5 mm). Fourteen days after tracer injection (seven weeks after SCI), animals were administered a lethal overdose of anesthetic and perfused through the heart with phosphate buffered saline, followed by 4 % buffered paraformaldehyde. Cervical spinal cords (from C2-C7) were removed, postfixed in 4 % paraformaldehyde overnight, and subsequently cryoprotected in 22 % sucrose for 24 hours. C2 and C6/C7 spinal cord segments were cut transversely and the C4-C5 spinal cord segment was cut in the horizontal plane. Sections were cut at 20 um thickness using a Microm cryostat. After immunostaining with mouse anti-GFAP (1:400; Sigma-Aldrich, MO), twelve horizontal sections from each animal (spaced 240 um apart and extending approximately 2,880 um dorsoventrally) spanning the entire depth of the lesion were digitally captured for the quantification of lesion areas. A person blinded to the treatment groups traced the outlines of the GFAP-bordered lesion cavities using Sigma Scan Pro 5 (SPSS, Chicago, IL) to obtain the area of the lesion in each section. A histogram of these areas at different levels of depth of the lesion was generated and the level with the largest lesion area was used to align the histograms from each rat. From each animal, a series of 3 horizontal sections spaced 240 um apart, with the most dorsal one running just ventral to the main dorsal CST, were stained with mouse anti-NeuN (1:100; Chemicon). NeuN images captured under same exposure settings were then equally thresholded. The density of undamaged (NeuN-positive) neurons next to the lesion edge was measured using two 300 x 300 jam windows. NeuN immunostaining is widely used as a marker for neuronal viability. However, we recently reported that facial motoneurons down-regulate NeuN expression without dying as a response to axotomy (McPhail et al., 2004). Hence, we are reluctant to designate the NeuNnegative population as dead and rather refer to them as damaged..  142  Corticospinal tract sprouting. The C2, C6/C7 transverse sections, along with C4-C5 horizontal sections of the spinal cord were washed in 0.01 M PBS, blocked in 10 % normal donkey serum for 30 minutes, and incubated overnight with goat anti-fluorescein (1:200; Molecular Probes, OR) followed by donkey anti-goat Alexa-488 (1:200; Molecular Probes, OR) to enhance the Fluoro-emerald-traced corticospinal axons. A l l images of the CST were captured with the same camera settings. A person blinded to the treatment groups hand-traced the CST axons using Photoshop CS2 (Adobe, USA). CST axons were measured in 2 standard measurement windows taken from the 3 different horizontal sections each 240 um apart (total of 6 windows for each rat). These sections run through Rexed layers V-VII of the gray matter (including the branches leaving the CST) with one window extending in the gray matter from the edge of the lesion 1 mm rostrally and the other in the gray matter 2 mm caudally. In cross sections (C2, C6/C7), CST axons were measured in three sections spaced at least 200 um apart using standard windows covering the intermediate and ventral layers (Rexed layers V-X) in the gray matter (typical CST innervation areas). The total length of CST axons for each animal was normalized to the CST axons measured at C2 to account for differences in tracing efficacy for the forelimb projection of the CST. Averages were calculated for each animal and subsequently used to compile group averages. Western blots and ELISAS. SDS-PAGE were run with 20 ug of protein extracted at 5 days post injury from the lesion site (n = 6 for both groups), and at 3 weeks post injury from the spinal cord caudal to the lesion (C6/C7) (n = 9 for both groups). Rabbit anti-trkB (1:200; sc-8316 (H-181), Santa Cruz, CA) was used for the western blots to detect both the full-length (140-145 kDa) and truncated isoforms of trkB (90-95 kDa) (Lee et al., 2002; Kingsbury et al., 2003; Deogracias et al., 2004; Palma et al., 2005). A goat anti-rabbit HRP antibody (1:20,000; Jackson ImmunoReseach, PA) was used as secondary and the blots were visualized with a chemiluminescence-based detection kit according to the manufacture's protocol (ECL kit; Amersham Corp., Arlington Heights, IL,USA). We measured the concentrations of BDNF protein in the spinal cord and cortex 3 weeks post injury (n = 9 for both groups) using a commercial BDNF ELISA kit (ChemiKine ™ BDNF Sandwich ELISA Kit, cat. no. CYT306, Chemicon International Inc., Ternecula, C A , USA; BDNF kit sensitivity 7.8 pg/ml, range of detection 7.8-500 pg/ml). Aliquots of 30 ug extracted protein were loaded in triplicate and ELISAs performed as outlined in the manufacturer's instructions. Statistics. SPSS 13.0 (SPSS, Chicago, IL) was used. A Student's t-test was applied to compare protein expression levels in the Western blots, ELISA, CST gray matter axonal profiles, 143  lesion area, NeuN-positive neuron density, and footprint analysis (two-tailed p-values are reported). A two-way repeated measures A N O V A , and if significane was reached followed by a Student's t-test post-hoc to determine whether lesion area (lesion area and distance) and percent errors (foot-slips) on the horizontal ladder (treatment and time) differed between groups. Because of the non-normal data distribution, a non-parametric test (Mann-Whitney U) was used to determine whether forelimb use in the cylinder test differed between groups. The Pearson correlation test was used to investigate possible relationships between behavioral outcome and morphological measurements. Standard errors of the mean (+/- S.E.M) are reported.  144  4.3. Results  Since most human spinal cord injuries are anatomically incomplete and occur in the cervical spinal cord (Ackery et al., 2004), we used a partial spinal cord injury at the cervical (C4) cord in rats as an experimental model. We crushed the dorsolateral funiculus, thus severing the rubrospinal tract (RST) while sparing the dorsal and ventral corticospinal tracts (CST). In rodents, both pathways play a major role in distal limb movements (Hendriks et al., 2006).  Animals continued to gain weight on the every-other-day fasting diet regime. To examine the effects of therapeutic EODF on spinal cord injuries, one group of rats (EODF) did not receive food from the time of surgery until 24 hours later. This was followed by alternating feeding/fasting periods of 24 hours throughout the rest of the experiment, i.e. the rats had access to food the day following surgery, and every second day thereafter. Starting weights of the rats did not differ between groups (Supplementary Fig. la; E O D F = 416.57 g +/- 5.25, control = 425 g +/- 7.58). Aside from a small postoperative weight loss (~ 3 %), all rats continued to gain weight throughout the experiment with the ad libitum-kd controls reaching 471 g +/- 7.94 at 32 days post injury. The EODF group gained weight at a slower rate (433 g +/8.57 at 32 days post injury); however, the two groups differed by only ~ 8 % at the end of the experiment. Activity levels were similar among the groups which is consistent with previous research of rats on EODF (Wan et al., 2003) (fig. S4.1B).  Therapeutic EODF improved forelimb recovery on 3 different behavioral tests. We asked whether EODF resulted in behavioral recovery following spinal cord injury. Deficits in fine motor control and inter-limb coordination are reflected by increased errors in crossing a horizontal ladder (Metz and Whishaw, 2002). One week after injury, there was an equal increase in percent ipsilateral forelimb footslips in both groups, indicative of lesion consistency (Fig. 4.1 A). A two-way repeated-measures A N O V A revealed that EODF animals made fewer % errors in the post injury period (p < 0.05), and by 17 days the errorfrequencyin the EODF group had reached the pre-injury level (full recovery), which was maintained throughout the experiment (Fig. 4.1 A). Extended behavorial testing of another control group using the same injury model demonstrated the same % ipislateral forelimb errors at 3 months post-injury as 4 weeks post-injury, showing no sign of recovery (unpublished observations). Another measure of forelimb function is the forelimb placement against the wall of a transparent 145  cylinder in which the animal are spontaneously rearing. One week after injury the ipsilateral limb was rarely used in either group for initial contact (p = 0.948, Fig. 4.IB). Thereafter, while ipsilateral limb use declined in control rats (a consistent finding in our control rats, fig. S4.2), it gradually increased in EODF rats, such that by 31 days post injury they had reached 12.01 % +/4.22, which was 6 times more frequent than the control group (1.88 % +/- 0.91) (p = 0.017) (Fig. 4.IB). The total % use of ipislateral forelimb for all wall contacts was also significant at 31 days (p = 0.014; control = 0.83 % +/- 0.42, EODF = 7.08 % +/- 3.34). We also analyzed the base of support, which is typically broader after injury. The base of support was narrower in the EODF group than in the control group at 33 days post injury (p = 0.027; Fig. 4.1C), and similar to baseline measurements. Spinal cord injury also affects gait coordination measured by increased variation in stride length in all limbs (Li et al., 2005). E O D F animals had less variation than control animals (p = 0.032, Fig. Id), and was equivalent to preoperative values at 33 days following injury.  EODF decreased lesion size and increased the number of undamaged neurons. We next examined anatomical correlates of EODF-mediated functional recovery. Since prophylactic EODF reduces lesion sizes after experimental stroke, spinal cord injury, and neurotoxic injuries (Bruce-Keller et al., 1999; Y u and Mattson, 1999; Maswood et al., 2004; Plunet et al., 2006), we asked whether similar effects could be brought about by therapeutic EODF following spinal cord injury (Fig. 4.2A,B). At seven weeks post injury the lesion size was significantly reduced in the EODF group (Fig. 4.2C) throughout the dorsal-to-ventral extent of the lesion (p = 0.004, two-way repeated-measure A N O V A ) . The ventral extent of the lesions was equivalent between the two groups indicating a consistent lesion depth (Fig 4.2C). The total lesion area in the EODF group was less than 50 % of the controls (control = 5.08 um +/- 0.72, EODF = 2.26 um +/- 0.30). The density of NeuN- positive neurons in the intact tissue surrounding the lesion was higher in the EODF group (p = 0.012, Fig 2D). These results demonstrate the neuroprotective effects of EODF subsequent to spinal cord injury.  Therapeutic EODF increased intact corticospinal tract sprouting proximal and distal of lesion site. Improved functional recovery might also be brought about by EODF-enhanced axonal plasticity in the spinal cord (Weidner et al., 2001). We failed to observe rubrospinal tract regeneration/sprouting in our prophylactic EODF experiment (Plunet et al., 2006). Therefore, we 146  anterogradely labeled the corticospinal (CS) tract projecting to the lesioned side of the spinal cord to determine whether EODF would prompt CS axons to compensate for injured rubrospinal axons. The total length of labeled CS axonal profiles measured in the C2 gray matter was equal between groups (fig. S4.3), indicating consistent labeling. However, within the rostral millimeter (p = 0.0023) and caudal 2 mm (p = 0.0076) from the lesion boundary, E O D F rats had significantly greater (~ three-fold) total length of labeled axons in the gray matter than that of ad libitum-fed rats (Fig. 4.3A, B, G, H). At C6/C7, 5-8 mm caudal to lesion site, the total length of CS axons in the gray matter was also increased in the EODF animals (Figs. 4.3C-F, I; p= 0.0028).  Lesion size and CST sprouting correlates with behavioral outcome. To test if the difference in morphological measurements (lesion size and CST sprouting) related with the behavioral outcome we statistically tested for significant correlations (table 4.1). Smaller lesions correlated with fewer forelimb errors crossing the ladder (r = 0.744; p = 0.0055) and improved use of the ipsilateral forelimb (r = 0.579; p = 0.0486), but not with forelimb base of support (r = 0.321; p = 0.3097) or stride length variation (r = 0.523; p = 0.0810). Corticospinal tract sprouting correlated with % forelimb errors crossing the ladder (r = 0.669; p = 0.0174) and forelimb base of support (r = 0.628; p = 0.0289), but not with % use of left limb (r = 0.357; p = 0.2539) or stride length variation (r = 0.290; p = 0.3611). It appears that both lesion size and corticospinal tract sprouting independently (% use of left forelimb, forelimb base of support) and in combination (% forelimb errors on ladder) correlate with behavioral outcome after cervical spinal cord injury.  Therapeutic EODF increased the ratio of full-length to truncated form of trkB in the spinal cord. We then asked whether EODF-mediated improvements in behavior, lesion size and CS axonal plasticity were associated with increased trophic support in the injured spinal cord. EODF increases the expression of a number of neurotrophic factors and their receptors, including brainderived neurotrophic factor (BDNF) (Mattson, 2005). BDNF signals through the full-length trkB receptor (trkB ) (Kaplan and Miller, 2000) while truncated trkB (trkB ) sequesters BDNF (Biffo 11  tc  et al., 1995) leading to reduced signaling (Offenhauser et al., 2002). Although we did not detect elevated spinal or cortical BDNF three weeks following injury in the EODF group (Fig. S4.4), we observed changes in trkB expression. After spinal cord injury, the BDNF-sequestering 147  truncated-trkB expression is increased in the lesion penumbra and may interfere with BDNFtrkB signaling (King et al., 2000). However, the rats treated with E O D F had a marked reduction in trkB around the injury by 5 days post-injury compared to control rats (p = 0.008; Fig. tc  4.4A,C). With similar levels of trkB expression (Fig. 4.4a,b), the trkB :trkB ratio was 6 times fl  fl  tc  higher in EODF animals (p = 0.049; Fig. 4.4D). Three weeks following injury, at the uninjured C6/C7 level (caudal to the lesion site) where trkB expression was low in both groups (Fig. tc  4.4E,G), trkB expression was higher in the EODF group (p = 0.012; Fig. 4.4E,F), also fl  increasing the trkB :trkB ratio (~ 2.5 fold) (p = 0.023; Fig. 4.4H). The higher trkB :trkB ratio fl  tc  fl  tc  in the EODF animals both at and caudal to the lesion site would be expected to increase BDNFtrkB* signaling (Offenhauser et al., 2002). 1  148  4.4. Discussion  The neuroprotective and functional benefits of dietary restriction followed for several months prior to injury are well established (Bruce-Keller et al., 1999; Y u and Mattson, 1999; Anson et al., 2003; Maswood et al., 2004; Mattson, 2005; Plunet et al., 2006). The present results demonstrate for the first time that therapeutic EODF implemented after cervical spinal cord injury reduces lesion size, increases plasticity of CS axons, and improves functional recovery. We demonstrate that lesion size and CST sprouting correlated with behavioral outcome after cervical spinal cord injury. This supports the idea that the EODF-induced improvement in recovery was mediated by both neuroprotection and increased CST branching. Correlation scores suggest that EODF mediates its improvement in percent use of left forelimb and possibly stride length variation by its ability to reduce secondary damage. While the improvement in forelimb base of support might be mediated by the EODF-induced increase in CST sprouting. Both reduced secondary damage and CST sprouting appear to contribute to the effect of E O D F on improving percent forelimb errors while crossing a ladder. The reduction in lesion size correlates well with functional recovery in a number of spinal cord injury models (Basso et al., 1995): in a C4 injury model similar to the one used here, there was a strong negative correlation between lesion size and behavioral outcome (Ruitenberg et al., 2003; Schwartz et al., 2003). The initial mechanical injury to the spinal cord causes tissue damage, energy failure, ischemia, and early inflammation, leading to an increase in reactive oxygen species (ROS) (Park et al., 2004). All of these factors contribute to the secondary cascade that involves lipid peroxidation and apoptotic cell death in the penumbra of the primary insult and along the initially spared white matter tracts. The observed reduction in lesion size could be mediated through several pathway that are affected by dietary restriction. Oxidative damage in the brain is reduced with dietary restriction (Dubey et al., 1996) which is likely due to a reduction in ROS (Sohal et al., 1994; Guo et al., 2000). As would be expected from ROS reduction, E O D F also reduces lipid peroxidation (Sharma and Kaur, 2005). Additionally, EODF increases a number of antioxidants (Cu-Zn SOD, catalase, GSH) (Sharma and Kaur, 2005). Dietary restriction can also have a direct effect on the cell death pathway. Two anti-apoptotic proteins, XIAP and apoptosis repressor with a caspase recruitment domain (ARC), are increased in the brains of animals on dietary restriction, along with decreased caspase-1 and caspase-2 activation, and cytochrome c release (Duan et al., 2003; Shelke and Leeuwenburgh, 2003; Ffiona and Leeuwenburgh, 2005). The combined actions of these pathways are likely involved in the 149  positive results on neuronal survival reported with animals following dietary restriction for several months prior to neuronal insult (Bruce-Keller et al., 1999; Duan and Mattson, 1999; Anson et al., 2003; Shelke and Leeuwenburgh, 2003; Sharma and Kaur, 2005). Our results of increased density of NeuN-positive neurons in the penumbra of the lesion and the decrease in lesion size are consistent with the effect of dietary restrictions on increasing cell survival and reducing secondary damage. However, the current results are the first to report a positive effect on survival and reduction of lesion size when dietary restriction is implemented after a neural insult. While neuroprotective effects often become apparent early after injury, the time course of several behavioral improvements observed here (along with correlation of CST sprouting and behavorial outcome) suggests the additional involvement of functional plasticity. In our experiment exploring prophylactic use of EODF we failed to observe an increase of injured rubrospinal axon regeneration or sprouting, therefore we explored the possibility on the involvement of the CST (Chapter 3) (Plunet et al., 2006). The intact CST does not normally sprout in response to the loss of the rubrospinal system (Jeffery and Fitzgerald, 2001; Hendriks et al., 2006). E O D F treatment elicited an increase in CST gray matter sprouting both proximal and distal to the lesion site. Since the RST and CST have overlapping projections and co-operative functions (Raineteau et al., 2002), EODF-induced CST sprouting could compensate behaviorally for the loss of the RST. The increase of CST sprouting is the first report of dietary restriction (prophylactic or therapeutic) increasing morphologically measured axonal plasticity. After a spinal cord injury the full-length catalytic active isoform of the B D N F receptor, trkB , is downregulated in the lesion area (King et al., 2000; Liebl et al., 2001). In contrast, the fl  truncated isoform (trkB ), suggested to sequester BDNF away from full-length trkB, is increased tc  in the lesion area (Frisen et a l , 1993; King et al., 2000; Liebl et al., 2001; Widenfalk et al., 2001). Additionally, trkB is downregulated in corticospinal and rubrospinal axons after injury fl  (Lu et a l , 2001; Kwon et al., 2004). The trkB :trkB ratio influences BDNF-trkB signaling fl  tc  fl  which promotes neuronal survival and plasticity (Chao, 2003; Lu et al., 2005). At five days post injury control animals displayed higher trkB protein levels in the lesion area compared to the tc  EODF animals. Thereby, the trkB^trkB ratio was higher in the E O D F animals. Three weeks 10  after injury, distal to the injury site, there was very low expression of trkB protein in both tc  groups as would be expected in the non-damaged area of the spinal cord. However, the E O D F group displayed an increase in trkB protein resulting again in an increased trkB :trkB ratio as 11  fl  tc  compared to the control group. The increase in the trkB :trkB ratio in E O D F animals is fl  tc  150  consistent with findings in the hippocampus of EODF rats (Lee et al., 2002). The increased trkB :trkB ratio in the spinal cord of EODF animals is a likely contributing mechanism behind fl  tc  both the neuroprotection and CST plasticity observed in the E O D F animals. Improving functional recovery from spinal cord injury and other neuronal insults is a multi-obstacle challenge (Plunet et al., 2002; Filbin, 2003; Park et al., 2004). The broad spectrum of effects initiated by EODF makes it a proficient treatment for spinal cord injury and possibly other CNS trauma. The current results demonstrate for the first time that dietary restriction implemented after a neuronal insult effectively elicits molecular and morphological changes promoting functional improvements, making it a clinically viable treatment. Humans on EODF maintain their weight even while engaging in their normal activity level, suggesting the ability to acquire adequate nutrition on eating days (Halberg et al., 2005). Because E O D F is a low-risk, low-cost, drug-free regimen, this treatment should meet low regulatory hurdles for clinical translation and could also be easily combined with other therapeutic strategies.  151  Figure 4.1. EODF increased functional recovery after cervical spinal cord injury (A) The percentage of errors made with the ipsilateral forelimb while traversing a horizontal ladder was significantly lower in the EODF animals (p < 0.05, two-way repeated-measures A N O V A , EODF n = 7, control n = 8) (* , p < 0.05) and returned to baseline levels by 17 days post injury. (B) EODF animals steadily increased ipsilateral forelimb use while rearing in a transparent cylinder, and performed better than control on day 31 (*, p = 0.017). (C) Control animals (black bars), but not EODF animals (white bars), had a significantly wider base of support at 33 days post injury than prior to injury (p < 0.001), and E O D F animals had a reduced base of support compared to the control animals (p = 0.027). (D) Stride length variation was less in the EODF animals at 33 days post injury (p = 0.032) and not different from pre-operative values. A l l error bars indicate SEM.  152  153  Figure 4.2. EODF reduced lesion size and protected neurons after a cervical spinal cord injury Representative pictures of the lesion area in (A) control, (B) EODF animals (demarcated by GFAP immunostaining) (scale bar = 200 um). (C) Histogram plotting the dorsal to ventral extent of the lesion revealed significant differences between the two groups (p = 0.004, two-way repeated-measure A N O V A , n = 6 for both groups) ( *, p < 0.05). (D) The E O D F group had a higher density of NeuN-positive neurons in the intact tissue surrounding the lesion site (p = 0.012, n = 6 for both groups). All error bars indicate SEM.  154  Distance dorsal-ventral f r o m largest lesion area ( u m )  155  Figure 4.3. EODF increased corticospinal axon total length rostral and caudal to the lesion site (A, B) Fluoro-emerald labeled (digitized by hand) CST axons 1 mm rostral and 2 mm caudal to the lesion (horizontal sections) in (A) control, (B) EODF animals (scale bar = 500 pm). (C,E) Transverse sections of the C6/C7 spinal cord with digitized CST axonal profiles in the gray matter (scale bar = 100 pm) (C = control, E '= EODF). (D, F) Higher magnification of the axonal tracer (not digitized) in the boxed area of C and E respectively (scale bar =100 pm). (G-I) Quantification of total CS axonal length: (G) EODF animals (n = 6) had more CST axonal profiles than the control group (n = 6) in the gray matter rostral (1 mm) to lesion site (p = 0.0023), as well as in (H) distal to the lesion site (2 mm) (p = 0.0076). (I) E O D F animals also had more axonal length at the C6/C7 level of the spinal cord (p = 0.0028, n = 6). A l l error bars indicate S E M .  156  Normalized CST axonal profiles o  o  o  o  o  o  o  - *  k)  w  ^  b i  b)  s  01  mo O o  o 3 -n 3 1*  Normalized CST axonal profiles o  o  o  o  o  o  o  o  11  •  mo O o a 3 •n 3  —1*  Normalized CST axonal profiles J 3  O ^  O O io u  O O O O ^ cn b> s  Figure 4.4. E O D F increased the trkB" to trkB ratio tc  (A) Immunoblot of trkB (145kDa) and trkB (95kDa) from the injury site 5 days post injury. 11  tc  (B) trkB* expression did not differ at the lesion site 5 days post injury, but (C) trkB expression 1  tc  was significantly lower in the EODF group compared to the control group (p = 0.008, n = 6). (D) The trkB :trkB ratio was increased in the EODF group at the injury site 5 days post injury (p = fl  tc  0.049; n = 6). (E) Immunoblot of trkB and trkB 3 weeks post injury distal to the injury site 11  tc  (C6/C7). (F) 3 weeks post injury trkB expression was increased in the E O D F group distal to the 0  lesion site (p = 0.012, n = 9). (G) trkB expression did not differ between the two groups. (H) tc  The trkB :trkB ratio was significantly higher in the EODF group at 3 weeks post injury (p = fl  te  0.023, n = 9). A l l error bars indicate S E M .  158  5 days post injury Injury site  B  0.5  g  l =  ro 0.4  C EODF  |  145kD •  0.3  £0.2  95kD-  I 0.1 0.0  3 weeks post injury Caudal to injury EODF 145kD 95kD  F  i.o  r  Control EODF  1=  —  Control]]  D  Io 2.5 3  0  ' Control i EODF  CD  £ 1.5 1.0 0.5 0.0  b 20 g 15 2 .= 10 t5 £ 5  £ o  r  mmmm Control  c = EODF  H  .2 2 80 1  0  0  i Control *  "cO 60  *2  40  1  20 0  159  Table 4.1. Correlations of morphology with behavior Pearson's correlations coefficients test of significance between morphological measurements (Lesion size and CST sprouting) and behavioral outcome (absolute r values, p values, * = significant, n = 6).  160  Behavioral outcome % forelimb errors on ladder % use of left forelimb Forelimb base of support Stride length variation  Lesion size r = 0.744, p = 0.0055 * r = 0.579, p = 0.0486* r = 0.321, p = 0.3097 r = 0.523, p = 0.0810  C S T sprouting r = 0.669, p = 0.0174* r = 0.357, p = 0.2539 r = 0.628, p = 0.0289 * r = 0.290, p = 0.3611  161  Legends to the supplementary figures Figure S4.1. EODF animals continued to gain weight and were not overactive (A) EODF animals (n = 7) continued to gain weight but at a slower rate than controls (n = 8). The EODF group only weighed 8 % less than the control group at the end of the experiment. (B) Activity level as measured by rearing frequency in the cylinder test did not differ between groups (p > 0.05, two-way repeated-measure A N O V A ) .  162  Figure S4.2. Control animals displayed a decline in ipisilateral forelimb usage Cumulative averages of percent initial wall contact with ipsilateral limb from four independent experiments for both pre-operative and post injury periods (n = 25). We included this data to point out that a deterioration of ipsilateral limb usage to ~ 2 % by 28-31 days post-injury is typical in this model in control rats.  164  Figure S4.3. C S T branching did not differ between groups at C2 The total length of CS axons in the C2 gray matter did not differ between groups.  166  Figure S4.4. BDNF protein expression did not differ between groups BDNF protein expression did not differ between control and E O D F groups, as determined by ELISA. 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Zhu H, Guo Q, Mattson MP (1999) Dietary restriction protects hippocampal neurons against the death-promoting action of a presenilin-1 mutation. Brain Res 842:224-229.  173  Chapter 5 Conclusions and future directions  5.1. Summary of thesis  In this thesis, I examined the effects of four different treatments on functional recovery after a cervical spinal cord injury. Counter to my hypothesis, B D N F cell body treatment of rubrospinal neurons at time of injury failed to promote sprouting or regeneration (but did reduce cell body atrophy and dieback of rubrospinal tract), despite previous research indicating a growth promoting effect into a peripheral nerve graft (Kobayashi et al., 1997; Kwon et al., 2002) (chapter 2). Acute BDNF cell body treatment appears not to increase rubrospinal axonal growth within the hostile CNS environment after SCI. Additionally, BDNF-acute treatment had no effect on functional recovery. Pretreatment of rubrospinal neurons with B D N F 7 days prior to injury reduced cell body atrophy and dieback, and promoted motor recovery. However, in contrast to my hypothesis, BDNF pretreatment did not promote regeneration through, or around, the lesion site, nor did it promote sprouting of rubrospinal axons in the gray matter rostral to the lesion site. Therefore, it appears that pretreatment of CNS descending supraspinal neurons does not have the same growth promoting effect as pretreatment of PNS neurons (Neumann et al., 2002; Qiu et al., 2002). The absence of a clear morphological correlate with the behavioral improvements observed in the BDNF pretreatment group prompted me to consider the reduced weight gain (food intake) associated with BDNF treatment as a possible alternative explanation for the observed recovery. Consistent with my hypothesis, the non-invasive intervention of dietary restriction, in the form of every-other-day fasting (EODF), was neuroprotective and promoted recovery of function if started one month prior to injury (chapter 3). Prophylactic E O D F reduced glial scar formation (as measured by GFAP immunostaining). EODF was also neuroprotective as it decreased lesion size and increased the number of NeuN-positive neurons surrounding the lesion site. Remarkably, EODF also improved functional recovery if started after the injury (therapeutic EODF) (chapter 4). There was a dramatic reduction in lesion size with therapeutic EODF treatment along with better preservation of neurons surrounding the lesion site indicating a reduction in secondary damage. EODF treatment induced an increase of the intact corticospinal tract sprouting, both proximal and distal to the lesion, which could be partially responsible for motor recovery. This is the first report in the literature of CR/EODF (prophylactic or therapeutic) demonstrating an increase of morphologically measured axonal plasticity. EODF did not increase the expression of BDNF, but increased full-length trkB expression, decreased truncated trkB levels, and thus improved the full-length to truncated trkB ratio in a positive manner. The 175  increased ratio might have played a role in mediating both the neuroprotection and plasticity observed. EODF is a practical combinatorial treatment that targets neuroprotection and growth promoting pathways. This thesis is the first demonstration of CR/EODF implemented after a CNS insult improving functional outcome. Because EODF is safe (non-invasive and extends lifespan), costs very little to implement, and should face low regulatory hurdles, it could be brought into clinical testing either as a stand-alone treatment or combined with other strategies.  176  5.2. Strengths and Weaknesses  A weakness of this thesis is the lack of mechanism for the treatment-induced improvement in recovery observed after SCI (chapter 2-4). However, very few spinal cord regeneration papers reporting improved function can offer a clear mechanism either (Bradbury and McMahon, 2006). While an increase in axonal regeneration may correlate with motor recovery and may be revealed by statistical post hoc analysis, this does not establish causality. Regeneration studies often cannot analyze lesion sizes (or this parameter is not measured), synaptic efficiency or sprouting of multiple other tracts, which is next to impossible. Hence, a clear link between the gain of function and the morphological effects is often hard to prove. Neuroprotective or regeneration/plasticity inducing treatment effects of a single molecule leading to a reduction in lesion size and/or more surviving neurons and/or an increase in sprouting/regeneration are deemed acceptable explanations for behavioral benefits. BDNF treatment is such a single molecule approach. The differential effect of timing of BDNF application on behavior (pre versus acute), yet the lack of regeneration and or sprouting in both BDNF groups, suggest that the rubrospinal axons are not likely mediating the behavioral recovery observed. The two EODF chapters offer no single molecular mechanism - although the change in trkB receptors expression may be one candidate. EODF reduced secondary damage with pretreatment or post injury treatment, and in the latter case I also investigated and found positive evidence for sprouting of the intact CST. In addition to these two possible mechanisms there are likely many other yet unknown biological changes that participate in the functional gains (see section 1.7). The involvement of multiple pathways and mechanisms is a strength of this thesis rather than a weakness as the lack of return of function after SCI is a multifaceted problem (see chapter 1), which is ineffectively treated with a single pathway approach. E O D F is a combinatorial treatment addressing multiple obstacles to improve function. Hence, the strength of this thesis is the attempt to test a practical combinatorial SCI therapy. The possible multiple effects of EODF on SCI were not fully explored in this thesis. While I found a reduced lesion size, the molecules responsible for this effect were not studied. Candidate mechanisms include the reduction of free radicals and subsequent attenuation of lipid peroxidation as reported from previous work using EODF (Guo et al., 2000; Sharma and Kaur, 2005). CST sprouting was enhanced with EODF, however many other tracts could also have 177  contributed to the return of motor function and they were not fully studied. Exploring the likely multiple molecular mechanisms involved in the effect of EODF on SCI would require huge resources and time beyond the frame of this thesis. It is highly unlikely the pretreatment of people by delivering B D N F to various brain structures will be clinically established since it entails considerable local tissue damage. The BDNF pretreatment experiment was a proof of principle testing a specific hypothesis. Pretreatment had produced favorable regeneration results in vitro and in vivo for peripheral D R G neurons (see section 1.4.B), hence conceivably may have promoted CNS regeneration of descending supraspinal neurons. Moreover, in my opinion prophylactic E O D F might never be implemented clinically to promote recovery. It is extremely unlikely that a meaningful percentage of the population would abide by such a regime just in case they have an accident, even though EODF has a wide range of health benefits (see section 1.6-1.7) including extending lifespan in numerous species. Less obviously, for several reasons, I am skeptical that EODF will be implemented even after a spinal cord injury. First and foremost, a significant fraction of the acutely injured patients have some form of polytrauma and current clinical guidelines prescribe adequate caloric treatment as this seems to lessen the risk of multiple organ failure (however CR/EODF might reduce multiple organ failure - see section 5.5.B). This risk of multiple organ failure is hard to assess during the first 72 hours and hence E O D F would not be started prior to this time point in polytrauma patients. The efficacy of EODF started at 72 hours has yet to be determined. Other reasons may include the difficulty to find financial support as nobody owns a patent on dietary restriction and hence funding for a clinical trial has to come from the limited public sources. Patients are unlikely to accept this treatment, at least initially, since it is not a simple pill but rather a treatment by omission at the price of hunger. A counter argument is that patients are willing to put forth great effort undergoing extensive physiotherapy for the chance of increase in function - so why not EODF. Continuing the argument in favor of EODF implementation, the strength of this thesis is the easy implementation of the EODF treatment. It is unlikely this treatment would create harm to the patient, which is not true for many of the strategies currently being tested in the SCI field. In theory EODF could have a positive effect on many of SCI secondary complications (see section 5.5.B) including infections. EODF should not have to face years of regulatory hurdles to reach clinical treatment since EODF is not a drug. If this treatment approach could be replicated in other SCI injury models (see section 5.6) it could be easily implemented in human trials. The consistent effect of CR/EODF on lifespan in numerous species (see section 1.6. A), along with 178  cardiovascular improvements observed in human studies (see section 1.8.C and 5.5.B) suggests benefits for humans as well.  179  5.3. How the chapters of this thesis are related One consistent link in this thesis is the use of the same lesion model for all three chapters. I chose a partial cervical lesion since this is the most common form of injury seen in humans. Moreover, the loss of hand function leads to a high degree of dependency compared to the loss of locomotion. "Hand function" was by far the highest priority mentioned by tetraplegic individuals (see section 1.9). At first glance, it might be hard to see how treatment of rubrospinal neurons with BDNF to test its effect on spinal cord injury relates to EODF treatment of SCI. However, there is a direct link as BDNF infusion into the brain causes weight loss due to a B D N F induced reduction of food intake (see section 1.4.D, 5.4.B) (Lapchak and Hefti, 1992; Pelleymounter et a l , 1995; Bariohay et al., 2005). The increase in behavioral recovery observed with B D N F pretreatment could be linked to this unintended dietary restriction. While I did not test if BDNF pretreatment had an effect on CST sprouting in chapter 2, the CST might have contributed to the observed behavioral effects. EODF started prior to injury (similar to pretreatment) had a very strong effect on behavioral recovery. However, there is a discrepancy between acute B D N F treatment (no behavioral effect) and EODF acute treatment (positive behavioral effect) even though they both result in dietary restriction. A possible explanation could be the difference between constant calorie restriction (CR) induced by BDNF and the alternating fasting by EODF. Mattson's group found the EODF regimen had a stronger neuroprotective effect that a constant 40 % CR (Anson et al., 2003) and additional research suggests that EODF more effectively increased BDNF/trkB expression as compared to CR (see section 1.8.B). There is a more obvious link between chapter 3 and chapter 4. In chapter 3, EODF was started one month prior to injury while in chapter 4, EODF was started at time of injury. While there was a strong positive effect on functional recovery with prophylactic EODF treatment I wanted to test if this diet regime was still beneficial when started after the SCI, which would reflect a clinically more realistic setting (chapter 4). The CST was not labeled in the experiment reported in chapter 3 (EODF started one month prior to injury) as I wanted to examine if RST regeneration/sprouting would occur. However, the gain in function without any regeneration of the RST (but with neuroprotection and reduced glial scaring) observed in this pretreatment experiment prompted me to examine the intact CST in chapter 4. E O D F started prior to injury most likely also promoted CST sprouting. Since EODF started prior to injury is less clinically relevant than acute EODF, further resources was not dedicated to this project. 180  5.4. How this thesis relates to the literature 5.4.A.  Cell body neurotrophin treatment and pretreatment/priming of neurons for  increased axonal growth Previously our lab had shown that acute BDNF treatment of the red nucleus promotes a greater number of rubrospinal axons to grow into a free end of a peripheral nerve graft (PNG) inserted into the spinal cord (Kobayashi et al., 1997). Such stimulation of rubrospinal axon growth by BDNF delivered to the cell body of rubrospinal neurons was observed even when initiated one year after spinal cord injury (Kwon et al., 2002). However, neither of these papers examined regeneration of rubrospinal axons through a PNG bridge with re-entry into the hostile CNS environment, or growth/regeneration within the CNS without a PNG. Regenerating axons have to leave the grafted bridge and re-enter the CNS in order to make functional connections. Moreover, acute BDNF cell body treatment of rubrospinal neurons failed to promote regeneration through or around the lesion site, and the 2 fold increase in rubrospinal axon branches proximal to the injury did not reach significance (chapter 2). Moreover, no behavioral gains by cell body treatment of rubrospinal neurons were found when B D N F was given at time of injury (chapter 2). Pretreatment of neurons with neurotrophins in vitro promoted axonal growth on the normally inhibitory substrate of myelin (Cai et al., 1999). B D N F caused an increase of cAMP in the neurons which is believed to mediate the increase in growth propensity. Pretreating DRGs with cAMP in vivo promoted their growth into the hostile CNS lesion site (Neumann et al., 2002; Qiu et a l , 2002). Therefore, it was somewhat unexpected that in chapter 2 pretreatment of descending CNS rubrospinal neurons with BDNF did not increase regeneration into or around the lesion site, nor did the sprouting in the gray matter proximal to the lesion reach significance. Pretreatment of descending supraspinal neurons prior to injury with trophic factors (e.g. BDNF) had previously not been attempted. Both BDNF pre-treatment as well as post-treatment reduced red nucleus atrophy and dieback of the RST, indicating that bioactive-BDNF reached its targeted rubrospinal neurons (RSN). Hence, differences between previous work on DRGs and the present observations on RSN might be due to differences in the response to axotomy of these populations of neurons. DRGs are peripheral neurons which have a higher intrinsic growth response than CNS neurons (Fernandes, 2000). Additionally, Neumann and Qiu used cAMP to prime their neurons, while BDNF was used in the present study (Neumann et al., 2002; Qiu et 181  al., 2002). However, BDNF increases cAMP in neurons (and therefore likely to increase cAMP in rubrospinal neurons) and enhances axonal growth on myelin inhibitory substrates (Gao et al., 2003). In conclusion, it appears that descending GNS axons have a lower ability to grow in the hostile CNS compared to PNS axons, when pretreated in vivo. While acute rubrospinal cell body treatment failed to promote recovery, many researchers have reported beneficial effects of treatments at the lesion site with various neurotrophins (see section 1.3.C.ii); however, there are some complicating issues that need to be addressed, such as reduced food intake.  5.4.B. Complicating effects of exogenously applied B D N F in SCI  In the introduction (see section 1.4.D), I discussed a secondary physiological complication of neurotrophin treatment in the CNS which is a reduced food intake. Chapter 4 shows a behavioral effect of EODF started at time of injury, which raises the question whether, reduced food intake plays a role in the numerous studies that applied neurotrophin treatment to the lesion site at time of injury. In none of the studies (see section 1.3.C.ii) were the weight of the animals reported other than at the beginning of the experiments. In sheep the intrathecal application of BDNF to the intact spinal cord caused a large increase of B D N F in the cerebrospinal fluid and a 12-22 % decrease in bodyweight along with a decrease in food intake (Dittrich et al., 1996). These results are consistent with the reduced food intake and subsequent weight loss seen after infusion of BDNF into the lateral ventricles of rats (Lapchak and Hefti, 1992; Pelleymounter et a l , 1995; Bariohay et al., 2005). While most SCI studies have used some form of transfected cell lines to apply BDNF, and not intrathecal application, one can understand that an injured spinal cord allows easier diffusion of BDNF, or other neurotrophins, into the cerebrospinal fluid compared to intact tissue. Once BDNF reaches the cerebrospinal fluid it would likely elicit a change in food intake. I demonstrated that one form of calorie restriction, EODF, started prior to injury or even at time of injury improved functional recovery after spinal cord injury. These results raise the possibility that some of the enhanced functional recovery seen in the trophic factor application to the lesion site could possibly be due to the altered food intake produced by these trophic factors and the downstream physiological changes.  182  5.4.C. Need for combinatorial treatments and possible complications Since there are more than one obstacle to the return of function after SCI (see section 1.3) there is a need for combinatorial treatment (Ramer et al., 2005). However, a combined treatment approach could prove more invasive and may introduce increased risks to the patient. Therefore, it is vital to design safe combinatorial treatments that effectively address the three main obstacles to functional recovery. Additionally, the outcome of combinatorial treatments is often unpredictable. Researchers hope for an additive effect of combined treatments, but there is growing evidence of some combinations even eliminating positive effects. Methylprednisolone (MP) has been extensively reported to produce positive effects in various animal SCI experiments (though controversy exists over its effectiveness in clinical trials: see section 1.3.A.iii). Erythropoietin (EPO) was demonstrated to reduce secondary inflammation and lesion size, and to promote better recovery after SCI (Gorio et al., 2002; Brines et al., 2004). However, when EPO was combined with MP the positive effect of EPO was lost suggesting that MP interferes with the positive effects of EPO (Gorio et al., 2005). Further to this, GM1 ganglioside treatment combined with MP resulted in a reduction of MP effect along with increased post injury mortality (Constantini and Young, 1994). A third paper reported a similar negative interaction between MP and anti-CDl Id antibody (Weaver et al., 2005). The anti-CDl Id was effective in promoting recovery by itself, and while MP increased sparing it had no effect on behavior. The combination of MP and anti-CDl Id resulted in a worse functional outcome than anti-CDl Id mono-treatment. While the need of combinatorial treatment is well recognized the above three examples demonstrate the complexity of this approach. Therefore, due to the increased risk of combinatorial treatments and possible negative interactions researchers will likely continue to look for separate approaches to the three problems, which can be combined safely and effectively. The optimal treatment would be neuroprotective, decrease adult CNS inhibitory properties, and increase the intrinsic cell body growth response, with additive effects and no negative interactions. In this thesis, I have possibly found one strategy that combines all three treatments in a safe approach (see chapter 3 and chapter 4). Since EODF extends lifespan and has a positive effect on most measurements of health (see section 1.6 and 1.7), it is most likely a safe and 183  clinically useable combinatorial treatment (it appears to have a positive effect on the three obstacles to functional recovery, see chapter 3 and 4). However, it remains to be determined whether multi-trauma, patients at risk for multiple organ failures can undergo E O D F (see section 5.5.B.).  5.4.D. Problems and complications with current neuroprotective treatments One problem with most (if not all) current neuroprotective treatments is they are very time sensitive (Hall and Springer, 2004). Most of these treatments are only effective if started within the first few hours (usually within the first 8 hours) after injury. Therefore, there is the open question of whether we will be able to treat SCI patients within these small time windows. Such narrow time windows might be most easily met in urban and suburban areas. However, rural residents are 2.5 times more likely to suffer a SCI (at least for Canadian residents) (Dryden et al., 2003), therefore, in many cases patients might not receive treatment in the critical time period. The exact time window for effective treatment is still an open debate. Some research with MP in a rat model of SCI demonstrated the window to effectively reduce lesion size is within 30 minutes, with later treatments having no effect (Yoon et al., 1999). Additionally, some of these treatments have negative side effects (Hall and Springer, 2004). In some animal studies MP treatment caused an increase in lesion size (Haghighi et al., 2000) or acute corticosteroid myopathy (Qian et al., 2005). How these animal results translate to the human situation is difficult to estimate. Researchers have raised serious concern regarding the effect of MP (or other corticosteroid treatments) administered outside the limited 8 hour time window. Not only is MP ineffective outside this time window but possibly detrimental (Hurlbert, 2001; Molano Mdel et al., 2002; Sayer et al., 2006). The most recent multicenter trial of MP (NASCIS III) revealed a modest (not quite significant; p = 0.07) effect on the functional independence score however, the data also pointed to an increase in sepsis (4 fold) and pneumonia (2 fold) after 48 hours of treatment, which did not reach significance (Bracken et al., 1998). Despite these reservations, neuroprotection might be a more achievable short-term goal than long-range regeneration of axotomized tracts. Since immediately after SCI most human subjects are unlikely consuming food, they will have already started a calorie restriction treatment and hence the vital time window will unlikely be an issue. Interestingly, in my rat studies (chapter 3 and 4) the control animals did not consume food for the first 8-12 hours (unpublished observations). Therefore, the first 8-12 hours of fasting 184  are not an essential component for the positive effects observed since both groups are not eating during this period.  5.4.E. Previous work on dietary manipulation after SCI  Wise Young's group studied the effects of a short fasting period (overnight) prior to spinal cord injury (Sala et al., 1999). The rationale was based on the observations that blood glucose levels increase with severity of injury and hyperglycemia worsens neurological outcome; hence fasting would lower blood glucose levels and might positively affect the injury. Overnight fasting did not reduce lesion volume but decreased blood glucose levels during the first 3 hours after injury. This line of research suggests that the reduced lesion size I observed in chapter 3 and 4 was not due to a short-term (overnight) effect of fasting. In a clinical study Dvorak and colleagues hypothesized that early enteral feeding (initiated within 72 hours) would result in a better outcome than late enteral feeding (initiated more than 120 hours after injury) in human patients after acute cervical spinal cord injury (Dvorak et al., 2004). Continuous feeding was provided via a nasogastric tube connected to a pump delivering 1.0-1.3 g/kg of protein (average calories consumed over the first 15 days in the early feeding group 1,938.1 +/- 1,100.2 kcal, late feeding group 1,588.4 +/- 982.9 kcal). The number of infections (primary measurement) was not different between the groups with the early feeding group having 2.4 infections and the late group having 1.7 infections. Secondary measurements in this experiment were the length of stay in the hospital and the time on mechanical ventilation. Hospital stay for the early feeding group was 53.0 days while the late feeding group stayed 37.9 days, ventilation time for the early feeding group was 762.8 hours and the early feeding group was 502.1 hours. While this study was underpowered and did not find statistical differences the trend towards shorter hospitalization and ventilation times for the late feeding group along with less infections is interesting. This paper did not support the prevailing clinical practices that early feeding would help the outcome of cervical spinal cord injury, if anything it hinted that late feeding had a better outcome. This clinical study suggests that one of the concerns raised about EODF treatment that patients need an acute (and substantial) nutrient supply after SCI might not be accurate.  185  5.4.F. Previous research on CR/EODF effect on neuroprotection and plasticity Extensive research with various forms of dietary restriction started prior to injury has demonstrated beneficial effects on neuroprotection and behavioral outcomes. The seminal paper by Bruce-Keller in Mattson's lab pioneered the field of neuroprotection by dietary restriction (Bruce-Keller et al., 1999). They tested EODF implemented for 2, 4, 8 and 12 weeks prior to kainic acid induced injury. While there was a trend with 4 weeks of EODF, it was only 8 or more weeks of E O D F that significantly promoted survival of hippocampus neurons. Behavioral effects were positive after 3 months of prophylactic EODF, however earlier time points were not assessed based on their cell survival results. Subsequent papers from this group, and other researchers, typically used 3 months of EODF or CR prior to neuronal insult to ensure positive effects. In chapter 3 I found as little as 4 weeks of EODF prior to injury (and continued in the post injury period) was neuroprotective as evidenced by reduced lesion size and more NeuNpositive neurons, and promoted motor recovery. It was surprising then that (in chapter 4) with no prior exposure to any dietary restriction, EODF started at time of injury also proved beneficial in reducing lesion size, increasing density of healthy neurons, promoting CST sprouting, and increasing motor recovery. Only one other paper started E O D F after neuronal insult. Holmer and coworkers implemented EODF after MPTP insult and observed a reversal of the typically decreased extracellular level of glutamate in the striatum (Holmer et al., 2005). However, this group did not test if the increase of striatal glutamate had a behavioral effect in their animal model of Parkinson's disease. While CR/EODF has been shown repeatedly to be neuroprotective, its ability to promote plasticity is less conclusive. Since CR/EODF increases B D N F levels in the hippocampus it might facilitate memory function. BNNF plays an important role in enhancing synaptic plasticity by a number of mechanisms (for review see Nagappan and Lu 2005). Studies have reported positive effects of CR/EODF on memory in aged rats (Idrobo et al., 1987; Pitsikas and Algeri, 1992). Though other groups have found inconsistent results of CR/EODF on memory (Beatty et al., 1987; Bond et a l , 1989). A possible confounding variable is the reported increase in activity/drive levels for some forms of CR. However, Hashimoto and Watanabe specifically controlled for activity/drive levels and still found an increase in memory performance in CR animals (Hashimoto and Watanabe, 2005). Long-term potentiation (LTP), which is considered essential for memory formation, is reduced in normal aged animals (Moore et al., 1993). C R prevented the age-related decrease in LTP in concert with the prevention of the decrease in 186  N M D A receptor expression (specifically NR1) (Eckles-Smith et al., 2000). In none of the papers studying memory or any other CR/EODF papers could I find a morphological correlate for the increased functional plasticity. Therefore, chapter 4 is the first report to demonstrate increased morphological plasticity with CR/EODF.  5.4.G. Choices of behavioral tests in this thesis and implications of this thesis for neurotrauma behavioral testing It would have been desirable to include other tests for forelimb function in my experiments. However, the food pellet retrieval task (or any other food-reward dependent tasks) was deemed not suitable because even though the behaviors were tested at least 8 hours after animals had been fed, any behavioral differences could have been due to a higher motivation for food. Also, I did not use any climbing paradigms or raised angle platform because previous research in mice has shown better performance in uninjured aged CR animals compared to control aged animals (Ishihara et al., 2005). Given that the EODF fed rats are approximately 40100 g lighter (approximately 40 g for therapeutic EODF - only 8 % less than control animals, and 100 g for prophylactic EODF) by the end of the experimental period, any benefits would be difficult to interpret in part due to the generally higher strength-to-body weight ratio seen in lighter animals as well as humans. Many behavioral paradigms used in the field of SCI and traumatic brain injury (not to mention almost every animal behavioral paradigm) require food restriction to motivate the animals to perform (Herrnstein and Loveland, 1974; Petry and Heyman, 1994). One prime example in the SCI field is the reaching task (Schrimsher and Reier, 1992, 1993; Li et al., 1997; McKenna and Whishaw, 1999; Ballermann et al., 2001; Weidner et a l , 2001). Traditionally, researchers food restrict animals (both experimental and control groups) to encourage reaching behavior. Obviously, this would have been a comfounding factor when food restriction is the primary variable, as in this thesis. The typical food restriction reported is at a level that maintains the animals at only 90 % of their starting baseline weight (Metz and Whishaw, 2000; Piecharka et al., 2005; Smith and Metz, 2005). This would be a more severe food restriction than I used in the various experiments reported in this thesis. The animals on E O D F continued to gain weight, albeit at a lower rate than the controls, while the animals used for the typical behavioral paradigm used in SCI lose 10 % of their starting bodyweight. Interestingly, Smith and Metz reported that in uninjured animals, the typical food restriction used for the reaching task actually 187  caused a reduction in skilled fine motor performance, such as the reaching task, and an increase in forelimb errors (but not hindlimb) while crossing a ladder. (Smith and Metz, 2005). These authors argue that the animals are in a somewhat frantic state and therefore make more mistakes. This makes my finding of improved performance on the horizontal ladder in the EODF animals all the more impressive. I observed neither hyperactivity nor any weight loss with the EODF regimen implemented in this thesis. Increases in activity levels in animals with 30-40 % caloric restriction have been reported (Ingram et al., 1982; Y u et al., 1985; Duffy et al., 1989). However, the increased activity level reported could be simply due to the better health of the CR animals in these aging studies. EODF regimen in Sprague-Dawley rats (the same strain of rats I used) did not increase their activity levels and assured significant weight gains (Wan et al., 2003a, b). In fact, total activity level as measured by continuous telemetric monitoring showed a decrease in activity during the nighttime and no changes during the daytime on both fasting and feeding days in the EODF animals compared to ad libitum fed animals (Wan et al., 2003b). Further to this, in my experiments (chapter 3 and 4) the number of rears within the first five minutes (when the rats were most active in the rearing cylinder) did not differ between the control and E O D F groups. This suggests the increase in functional recovery after a spinal cord injury was not simply due to increased activity level. This thesis demonstrated DR in the form of EODF is neuroprotective and promotes functional recovery. It is possible that the food restriction imposed on both experimental and controls groups by various researchers in order to motivate the animals, reduces their chances to detect specific treatment effects. Their results might be masked by the neuroprotective and axonal growth promoting effects imposed by DR on both groups. I propose that the neurotrauma (SCI, TBI, stroke) field should choose alternative behaviors that do not require food restriction (rearing cylinder, horizontal ladder crossing), or provide alternative motivation for the animals.  5.4.H. Possible role of trkB on C S T sprouting in E O D F animals  One of the most robust effects of EODF is the increase of B D N F protein levels in various brain regions. Interestingly, I found no increase of BDNF protein expression (as measured by ELISAs) in the EODF treated animals in neither the spinal cord nor cortex, 3 weeks post injury. The difference between my results and others is most likely due to the length of E O D F treatment. M y experiments involved short term EODF (5 days to 3 weeks for protein expression 188  studies) while other researchers typically used at least 3 months of EODF. However, in chapter 4 I found that short term EODF did elicit changes in protein expression of full-length trkB and the truncated isoforms in the spinal cord. The expression changes resulted in increased ratio of fulllength to truncated form of trkB in the EODF animals suggesting a better signaling potential (see section 1.3. C.i). The effect of BDNF on neurons is induced by its binding to the trkB transmembrane tyrosine kinase receptors (Glass et al., 1991; Klein et al., 1991; Soppet et al., 1991; Squinto et al., 1991). When bound to trkB BDNF causes receptor dimerization and subsequent autophosphorylation. Multiple possible signaling pathways can be induced by the different adaptor and effector proteins that attach to the docking sites of the phosphorylated tyrosines in the cytoplasmic domains of trkB (Kaplan and Miller, 2000). In addition to the full-length tyrosine kinase containing isoform of trkB (trkB ), fl  alternative splicing generates three intracellularly truncated isoforms (trkB ) lacking the tyrosine tc  kinase domain. The function of the three different C-terminally truncated receptor isoforms ( T l , T2, T-Shc) is not fully known. It has been suggested that the truncated trkB isoforms inhibit BDNF signaling by taking up BDNF and sequestering it from the full-length trkB isoform (Beck et al., 1993; Biffo et al., 1995; Rubio, 1997; Alderson et al., 2000). Alternatively, the truncated isoforms could alter signaling by forming inactive heterodimers with trkB (Eide et al., 1996; fl  Ninkina et al., 1996; L i et al., 1998; Gonzalez et a l , 1999). After SCI the truncated expression of trkB was increased in the penumbra of the lesion site (Frisen et a l , 1993; King et al., 2000; Liebl et al., 2001; Widenfalk et al., 2001); and in addition, the full-length version was downregulated (Liebl et al., 2001; Lu et al., 2001). In contrast the same magnitude of altered expression was not observed at the cell body level which is the basis for the idea of cell body treatment instead of treating at the lesion site (see section 1.3.C.ii and chapter 2). The decreased ratio of trkB : trkB at the lesion site after SCI would lead fl  tc  to less trkB signaling and therefore more cell death and less axonal plasticity. The downregulation of trkB at the injury site and not at the cell body level in the midbrain could be 11  explained by the increase of hydrogen peroxide cell body level.  H2O2  (H2O2)  and ROS at the lesion site but not at the  which was increased at the injury site after SCI (Liu et al., 1999; Liu et al.,  2003) is known to increase oxidative stress, and was demonstrated to downregulate full-length trkB receptor expression in neurons (Olivieri et al., 2003). This illustrates how secondary damage can have direct molecular effects on the intrinsic axonal growth response by downregulating trkB expression. In direct contrast, oxidative stress is not increased in 189  rubrospinal neurons after a SCI (Xu et a l , 2005) and therefore it is understandable that researchers only report a small or no downregulation of trkB mRNA (Kobayashi et al., 1997; Liebl et al., 2001). TrkB protein expression is maintained in the injured red nucleus after SCI (but not on their axons) (Kwon et al., 2002; Kwon et al., 2004). EODF is known to increase the efficiency of mitochondria metabolism and result in less H 0 production (Sohal et al., 1994; Hagopian et al., 2005; Sanz et al., 2005) along with less free 2  2  radical production (Sohal et al., 1994; Guo et al., 2000; Lopez-Lluch et al., 2006) in several tissues including the brain. Therefore, reducing mitochondrial dysfunction and subsequent H2O2 production is a likely mechanism behind the effect of EODF to maintain normal levels of trkB  n  in certain regions of the spinal cord. Neurotrophins are known to downregulate peroxide production and lipid peroxidiation products which may play a role in the feedback mechanism (Mattson et al., 1995; Dugan et al., 1997; Joosten and Houweling, 2004). This could possibly play a role in the recently reported antioxidant properties of B D N F when applied to the injury site (Joosten and Houweling, 2004), as well as the positive behavioral effects on SCI when applied to the lesion site (see section 1.3.C.ii.) while having no effect when applied acutely at the cell body level (Chapter 2). Additionally, transfection of neurons with the truncated form of trkB results in a reduction of trkB cell surface expression and therefore would lead to reduced signaling potential fl  (Haapasalo et al., 2002). In animals with overexpression of trkB (thereby altering the full-length fl  to truncated ratio of trkB) there was an increase of BDNF induced downstream signaling in conjunction with an increase in memory performance (Koponen et al., 2004). More specifically, trkB +/- mice demonstrated a reduced capacity in recovery of various gait measurements after a fl  femoral nerve injury (Irintchev et al., 2005). This indicates the importance of trkB signaling 11  even in peripheral nervous system regeneration as well. The improvement I observed in gait analysis with EODF treatment could be due the altered trkB : trkB ratio and would be fl  tc  consistent with the study above. Since EODF prevented the increase of the truncated forms of trkB at the injury site (resulting in higher trkB : trkB ratio), instead of BDNF being sequestered, the neurotrophin fl  tc  could activate the full-length version of trkB leading to both increased cell survival and enhanced axonal outgrowth. Additionally, the increase in the full-length trkB distal to the injury increased the ratio of full-length to truncated trkB, which could have a positive effect on CST axonal sprouting.  190  5.5. Possible negative and positive consequences of CR/EODF on SCI patients 5.5.A. Possible negative consequences of CR/EODF on SCI patients One concern raised against treating SCI with EODF is that a multi-trauma patient needs adequate nutrition to recover (see section 5.5.B). As mentioned earlier this was examined with early or late enteral feeding in SCI patients (see section 5.4.E) (Dvorak et al., 2004). In summary, this group did not observe any positive effects of early feeding, and if anything, there was a trend towards detrimental results. I would also like to remind the readers that short-term studies of EODF in humans only reported very small weight loss or none at all (see section 1.8.C). However, the effect of CR/EODF in injured humans has not been tested. Additional research has shown that traumatic brain injury (TBI) patients undergo weight loss due to an inflammatory response (Mansoor et al., 1996). The muscle wasting is in part due to the recruitment of amino acids by the liver to produce inflammatory proteins (Vary and Kimball, 1992), and the consumption of energy by immune cells (Alexander, 1995). Since CR/EODF decreases inflammatory response (see section 1.7. A) one might expect a better outcome and a paradoxical reduction of the weight loss typically observed. Further, research on human patients with traumatic brain injury revealed that increased calorie intake did not prevent the weight loss or the nitrogen imbalance (the increased calorie treated group actually lost more nitrogen - the extra nitrogen was not absorbed and excreted) (Hadley et al., 1986). Concerns of muscle size and function when on CR/EODF are extensively addressed in section I.6.D. In summary CR/EODF animals were stronger than age matched control animals and some studies showed that aged CR/EODF animals were as strong as, or even stronger than, young animals in the prime of their life. As an addition to the EODF regimen reported in this thesis, I would argue for an increase of nutrients, particularly protein, on the feeding days of this paradigm in SCI patients (see section 5.6). A second more serious concern, which most people would be unaware of, is the possible increased susceptibility to drug addiction of patients undergoing CR/EODF. Food restriction increases self-administration of a wide range of abused drugs (Carr, 2002). It appears that food restriction sensitizes the reward system (Carr, 1996). EODF increased amphetamine induced locomotor responses (a behavioral measurement used in the drug addiction field as an indicator of dopamine response) (Mamczarz et al., 2005). This would suggest that EODF sensitizes the reward system to be more responsive to the rewarding effects of dopamine. One may speculate 191  that the system is responding to the reduced food by tuning the reward system to be more responsive so that the organism is 'enticed' to seek out the rewarding effects of food, because apparently it is not adequately performing at present. The offshoot of this effect is the organism becomes more sensitive to all rewards including the detrimental effect of addictive drugs that exist in our current society. Therefore, it would be prudent if E O D F were implemented clinically that the use of prescribed and recreational drugs be carefully monitored. Looking at the glass half full, with a sensitized reward system 'everything' in life would be a little sweeter since the reward system would activate more quickly and often to the "little pleasures" in life.  5.5.B. Possible additional positive benefits of C R / E O D F for SCI patients  While researchers have traditionally concentrated on increasing regeneration and/or gains in motor function, SCI patients suffer from a large variety of dysfunctions and complications which are not well studied (Noreau et al., 2000; Walter et al., 2002; Krassioukov et al., 2003; Ackery et al., 2004). The exact order of prevalence of the specific secondary complications might differ between papers, however, the lists usually contain: spasticity, pain, pressure ulcers, bladder conditions (urinary tract infections, problems with bladder emptying, urinary inconsistence), bowel problems (from constipation to diarrhea), autonomic dysreflexia, metabolic problems (diabetes, weight management, nutrition, exercise), respiratory problems (pneumonia, shortness of breath, bronchitis, asthma), cardiovascular conditions (hypertension, hypotension, angina, myocardial infarction), and psychiatric disorders (depression, posttraumatic stress disorder). While I report an increase of motor function with EODF treatment either started prior to or after SCI, I propose that this form of treatment could also help many of the additional complications that occur in this patient population. A major acute complication after SCI is sepsis. Bacterial infections are the predominant initiator of sepsis, but may also occur after injury and after surgery, even without evidence of bacterial infection (Vega et al., 2004). Hallmarks of sepsis are a hypermetabolic state and an overactive inflammatory response (Livingston and Deitch, 1995). A systemic inflammatory response occurs (systemic inflammatory response syndrome - SIRS) which can result in acute respiratory distress syndrome and multiple organ dysfunction (multiple organ dysfunction syndrome - MODS). MODS is the leading cause of death in surgical intensive care units (Meakins, 1990).  192  Clinicians who have heard the results of my E O D F experiments are quick to point out that CR/EODF cannot be used in SCI because of sepsis. I will offer scientific evidence to point out that it is possible CR/EODF might have positive effects on sepsis rather than making the condition worse. First off, after experimentally induced ulcerous skin lesions 8 9 % of control animals died from infection/septicemia while only 3 3 % of CR ( 4 0 % ) animals died, and they survived twice as long as the control group (Perkins et al., 1998). Another group using Escherichia coli lipopolysaccharide (LPS) to examine cytokine-stimulated production by peritoneal macrophages as a model of sepsis, found that animals on EODF had a reduced production of TNF-a (Vega et al., 2 0 0 4 ) . The authors concluded the altered dietary regimens could be used to treat trauma patients. When mice were injected intraperitoneally with bacteria the animals on CR (with equal percent of protein in their diet) had a greater survival rate (Peck et al., 1992). While these are only a few examples, there has not been any direct evaluation of CR on sepsis after SCI. However, we have a good molecular understanding of why CR/EODF might be effective against the development of sepsis. Septic animals and humans show a marked increase in the nuclear factor ( N F ) K B in every organ studied (Liu and Malik, 2 0 0 6 ) . This nuclear factor plays a key role in the immune and inflammatory responses. Higher levels of N F K B are associated with worse clinical outcomes and higher mortality rates. Additionally, inhibiting the activation of N F K B reduces multiple organ injury and increases survival in animal models. CR reduced the age related increase in N F K B due to its antioxidative action (Kim et al., 2 0 0 2 ) . More specifically, CR also decreased N F K B in the cortex while at the same time increased the N F K B inhibitor, I F K B (Lee et al., 2 0 0 0 ) . The wellestablished effect of DR reducing inflammatory response and the specific decrease of N F K B , lead to the possibility that D R could positively affect sepsis in human SCI patients. There is a body of research pointing to the importance of mitochondrial function in the mechanisms of sepsis (Crouser, 2 0 0 4 ) . Sepsis can damage mitochondria which then negatively feedbacks into the condition by producing more ROS. The increase in ROS can further damage mitochondria to the point that the organ/system suffers from a serious energy crisis and/or apoptotic cell death. CR enhances mitochondrial functions by inducing mitochondrial biogenesis, decreasing oxygen consumption, maintaining or increasing A T P content, and drastically reducing ROS production (Nisoli et al., 2 0 0 5 ; Lopez-Lluch et al., 2 0 0 6 ) . Hence, CR is likely to offer the possibility of improving mitochondrial health and function, which could help the outcome of sepsis.  193  While not reported as direct secondary complications of SCI, two additional problems encountered by SCI patients are CNS injury-induced immune deficiency syndrome (CIDS) (Meisel et al., 2005) and chronic inflammation (Manns et al., 2005). It is argued that CNS injuries, including SCI, lead to secondary immunodeficiency termed CIDS, and subsequently increase probability of infections (Meisel et al., 2005). CR has been shown to reduce infections. CR protected rats from mortality after intraperitoneally injections with Salmonella typhimurium, an intracellular pathogen (Peck et al., 1992). In addition to the SCI associated immunodepression, patients can also suffer from a subclinical chronic inflammation (Manns et al., 2005). One possibility of rectifying these divergent lines of research is that the CIDS leaves the system vulnerable to chronic infection to which the body eventually responds with low levels of inflammation. However, the depressed immune system may not sufficiently respond to a more serious infection. The incidences of infections (pneumonia and kidney infection) in SCI patients are life threatening complications (Meisel et al., 2005). Chronic inflammation in SCI patients is accompanied by increased levels of IL-6 and C-reactive protein (CRP) (Rebhun et al., 1991; Segal et a l , 1997; Manns et al., 2005). Human CR studies have shown reduced serum levels of C-reactive and TNF-a protein suggesting reduced inflammation (Fontana et al., 2004; Meyer et al., 2006). Along with the altered levels of proteins linked to inflammation, SCI patients also display high levels of fasting glucose and lower levels of high-density lipoprotein cholesterol (HDL-C) (the good form of lipoprotein) which are associated with metabolic syndrome, diabetes, and heart disease in the general population (Manns et al., 2005). The above mentioned SCI serum profiles might partially explain why SCI patients have a 3-5 times higher risk of developing type 2 diabetes mellitus (Duckworth et a l , 1980; Duckworth et al., 1983; Bauman and Spungen, 1994, 2001). The SCI induced metabolism changes leading to metabolic syndrome/diabetes could be the underlying mechanism leading to the failure of the circulatory system and subsequent death The leading cause and contributor to death of people with SCI is due to circulatory complications (40 %) (Garshick et al., 2005). Therefore, people with SCI need to be proactive in reducing their chances of developing diabetes. CR and EODF have been shown to reduce glucose and insulin levels while improving glucose tolerance (Wang et a l , 1997; Anson et a l , 2003). Both EODF and fasting only two days per week reduced diabetes in Brattleboro rats (Pedersen et al., 1999). In Zucker diabetic rats CR prevented the development of diabetes (prevented hyperglycemia) and improved the glucagon/insulin ratio (Colombo et al., 2006). Oxidative stress and inflammation are associated with causing, and/or a byproduct of, diabetes. These combined 194  phenomena are implicated in detrimental effects on brain function. In experimentally induced diabetic rats the CR group displayed reduced triglycerides, ROS, IL-6 and TNF-a (Ugochukwu et al., 2006). Therefore, CR appears to reduce the possibility of developing diabetes and as well as reducing some of the negative effects of established diabetes. Further to this, PNS regeneration in diabetic animals and humans is hampered (Kennedy and Zochodne, 2005). This has implications for any form of treatment for PNS and CNS chronic injuries. If human SCI patients become insulin resistant to fully diabetic their response to treatment strategies would likely be greatly reduced. Therefore, CR or E O D F might be implemented to prevent the insulin resistant/diabetic condition that would reduce the regenerative capacity of the system. SCI leading to the secondary complications of metabolic syndrome and diabetes may contribute to the cardiovascular complications in this patient group. As far as we know, the cardiovascular systems of individuals with SCI suffer from many of the same complications that occur in inactive, sedentary non-injured people (leading cause of death in people with SCI is circulatory based). EODF reduces heart rate, blood pressure and insulin levels similar to, or to a greater amount, than regular exercise (Wan et a l , 2003b). This could be particularly important if the SCI patient is unable to perform a high enough level of exercise. More recent research shows that EODF and CR increase the variability of beat to beat heart rate and diastolic blood pressure variability within one month of onset of the diet regime (Mager et a l , 2006). Lower heart rate variability is associated with risk of coronary heart disease and therefore the increase in heart rate variability induced by E O D F could be indicative of a lowered risk for heart disease (Liao et al., 1997). Atherosclerosis (fibrosclerosis: hardening of the heart arteries) is one cause of cardiovascular problems that is at least partially induced by the oxidative process that is greatly attenuated with CR or EODF (Castello et al., 2005). Human studies with CR have also shown positive effects. A study examining risk factors for atherosclerosis in individuals practicing long term CR found a marked reduction compared to age matched controls (Fontana et al., 2004). Total cholesterol, triglycerides, fasting glucose and insulin, along with C-reactive protein (CRP) and blood pressure was lower in the CR group. The intima-media thickness of the carotid artery was 40% less in the CR group. Interestingly, the vast majority of the effects in the CR group for blood measurements and blood pressure occurred within one year of starting the CR diet. Follow up research found that diastolic function (measured by Doppler flow) is improved in the CR group, along with reduced blood pressure, serum CRP, and TNF-a (Meyer et al., 2006). The evidence strongly supports that CR/EODF affects a host of parameters that reduces the risk of heart disease. 195  While CR is likely to reduce the probability of developing cardiovascular disorders it will not eliminate the possibility of cardiovascular events. The question becomes whether CR/EODF can also reduce the damage from a cardiovascular incident. Ischemic preconditioning (PC) of the heart is a phenomenon known to protect the heart from a more serious ischemic incidence. However, as organisms age the PC effect is either lost or reduced (Tani et al., 1997). Prophylactic CR preserves PC in aged rats resulting in an increase in aortic flow and cardiac output after an ischemic incident (Long et al 2002 cite). Prophylactic E O D F was found to reduce the myocardial infarct lesion size by close to 50 % (Ahmet et al., 2005). Cardiovascular disorders include stroke and CR/EODF reduces many of the cardiovascular risk factors (see table 1.1 in chapterl). Specifically, in a rat strain that is prone to stroke, CR (40%), even if started in adulthood, reduced blood pressure and the incidence of stroke along with a subsequent increase in lifespan (Stevens et al., 1998). If EODF is started in rats 3 months prior to an experimentally induced stroke; the lesion size, heat shock protein 70 expression, and behavioral outcome are improved (Yu and Mattson, 1999). Therefore, CR/EODF not only reduces the risk of developing cardiovascular complications (heart disease and stroke) but also protects the system if they occur (heart and brain). Since the leading cause of death of SCI patients is due to cardiovascular complications, EODF might provide a treatment that would extend the lifespan in this group of individuals. Another common complication is pressure ulcers which have a 38 % frequency in SCI patients (Walter et al., 2002). CR has shown benefits in reducing skin ulcers by more than one half in chemically induced skin ulcers (Perkins et al., 1998). While the difference between chemically induced skin ulcers and pressure induced ulcers could be argued, since CR also appears to have beneficial effect of blood flow in humans (Meyer et al., 2006) this could by itself reduce skin ulcers. Additionally one statistically significant risk factor for the development of skin ulcers is abdominal girth (Walter et al., 2002) and EODF could help manage weight/girth. SCI patients also suffer from an increased risk of bladder cancer related to the use of indwelling catheters (Groah et al., 2002). CR and EODF has been extensively shown to reduce the probability of developing cancer in general (for review see, (Hursting et al., 2003)), and specifically decrease bladder cancer (Dunn et al., 1997). Pain is reported by 44 % of SCI patients (Walter et al., 2002) and pain dramatically reduces the quality of life. A reduced pain response was observed in CR (40% reduction) mice after injections of formalin into the paw, and of collagen into the joints (arthritis model) (Hargraves and Hentall, 2005). More specifically, EODF showed a marked reduction in pain 196  responses to thermal (hot-plate) and visceral noxious stimuli (i.p. acetic acid injections) along with a reduced c-fos expression in the sensory interneurons of the dorsal spinal cord (de los Santos-Arteaga et al., 2003). Interestingly, spinal cord levels of prodynorphin and K-opioid receptors are increased in EODF animals and the transcriptional repressor D R E A M (downstream regulatory element antagonist modulator) which is the main regulator of prodynorphin levels is decreased. These EODF-induced changes suggest that this diet regime could reduce chronic pain suffered by some SCI patients. Beyond physical complications, SCI patients also suffer from psychological problems which include post-traumatic stress disorder and depression (Krassioukov et al., 2003; Chung et al., 2006). EODF increased both BDNF and neurogenesis in the hippocampus (Lee et al., 2002a; Lee et al., 2002b), which have been directly linked with decreasing depression (Duman, 2004; Newton and Duman, 2004; Duman, 2005; Filus and Rybakowski, 2005; Duman and Monteggia, 2006; Warner-Schmidt and Duman, 2006). Therefore, it is possible that EODF reduces the development and/or helps treat depression in SCI patients. While scientists in the spinal cord regeneration field might be excited by the possibility of EODF increasing plasticity and improving motor function after SCI, this diet regimen might prove more valuable in its potential ability to reduce the multiple secondary complications after SCI. This treatment could increase the length and quality of life for these patients, even if it fails to improve motor function.  197  5.6. Future experiments  The positive molecular, morphological, and behavioral results of therapeutic EODF open many new research questions. One interesting avenue would be to examine a dose response curve of EODF. One could test if instead of every second day fasting, every third day or every fourth day has an effect on neurotrauma. We already know that even one day of fasting in rats reduced the probability of developing cancer (Berrigan et al., 2002). Since, it had been suggested that D R works via hormesis (see section 1.6.B), one could argue that a consistent stress is predictable and its effect would be abated over time. One possibility would be three weeks of EODF followed by a week of ad libitum consumption before switching back to EODF. The one week of a normal eating pattern would hopefully reset the system to be more sensitive to the DR. This line of reasoning is quite extensively followed in the athletic world (though not well documented scientifically). Or some alternating schedule of 1, 2, or 3 days of ad libitum eating followed by varied lengths of fasting that could range from 12 hours to 36-48 hours. The principle would be to keep the body and nervous system "guessing". This could lead to a stronger hormesis response. Instead of measuring recovery from spinal cord injury one would need a simpler, faster assay. It would also be of interest to examine if a shorter fasting period is effective. Is 20 hours of fasting followed by 28 hours of ad libitum eating also beneficial? Various combinations could be tested with the rationale that humans are more likely to be able to emotionally handle slightly shorter fasting periods compared to longer ones. Additionally, we do not know if the first 24-hour period of fasting after injury is essential for the downstream behavioral effects. It was noted that the control animals do not eat for the first 8-12 hours (unpublished observations). Combined with the findings by the Wise Young's group (Sala et al., 1999) that overnight fasting had no effect on lesion size, this would either suggest that the first day of fasting might not be that important; or alternatively, the first 8-12 hours of fasting (instead of 24 hours) are not sufficient to produce a favorable outcome. Beyond figuring out the optimal dose of fasting that would prove most beneficial, the efficacy of EODF should be tested in other spinal cord injuries (i.e. contusion model), or in a host of other brain injuries. Additionally, the effects of EODF on SCI could be tested in several other species before human clinical trials. Traumatic brain injury would be an obvious next target along with stroke. Mattson's group has already shown E O D F produces better functional outcome in a stroke model if started 8 weeks prior to the onset of the stroke (Yu and Mattson, 1999), but would it be effective if started after injury? 198  Another important line of follow up experiments would concern the effect of EODF on older animals. The age of SCI onset has increased over the years with a growing population of older adults also suffering SCI (Sekhon and Fehlings, 2001; Pickett et al., 2006). As organisms age there is an increase of free radical production that could induce greater damage than in younger organisms. Since DR reduces free radical production we might expect an even stronger neuroprotective effect of DR when tested in older animals. However, it would most likely be important to gradually induce CR/EODF in older animals based on adulthood implemented CR in the longevity research field (Weindruch and Walford, 1982; Dhahbi et al., 2004; Rae, 2004) Numerous additional experiments could be performed to fully understand therapeutic effects of DR on not only spinal cord injuries but also additional brain insults. The majority of researchers would be primarily interested in the mechanisms behind the effects of DR. Many years, men/women hours, and animals would be needed and required. And what would we gain from this extensive endeavor: that pathway A , B, C,  Z  , to mention a few, are involved in  EODF effect on SCI. And what would this lead to? We would spend many more years developing small molecules that can affect each of these pathways in the appropriate direction then turn around and give all these small molecules only in the hope of getting a positive effect. These multiple molecules could interfere (since they are rarely that selective) with each other in multiple pathways or the treatment might be overly invasive (surgery, transfections, etc). Why not just use the original treatment of EODF, which is safe, affordable, non-invasive, and ready for implemenation? The direct parallel to this current story is that for 90 years, we have known that DR increases lifespan but the biotechnology world is spending millions and millions of dollars trying to replicate the powerful effects of DR on lifespan with specific molecules. The interesting part is none of the knockout animals with targeted deletions in specific pathways involved in longevity live as long as the wildtype mice on DR (Barger et al., 2003). In humans we will most likely not be able to fully knockout a particular pathway. At best, we can hope for a partial block with whatever molecule we derive and plan to sell to the public. Of course there is the counter argument that CR/EODF by itself is not as powerful as we would like and hence if we can discover all the pathways involved we could further enhance the appropriate response (with the above caveats). One specific target from this thesis would be the BDNF/trkB pathway. There could be some problems associated with using knockout animals to elucidate this pathway's role in the effect of EODF after SCI. Homozygous trkB knockouts die due to lack of feeding response which highlights the confounding variable of BDNF and trkB when examining the role of EODF 199  (Klein et al., 1993). Homozygous BDNF -/- are also not viable (Ernfors et al., 1994; Jones et al., 1994). Heterozygous B D N F knockouts are hyperphagic, become obese, and have high levels of leptin and insulin (Lyons et al., 1999; Kernie et al., 2000; Dluzen et al., 2001; Rios et a l , 2001). The obesity phenotype can be eliminated with exogenous application of B D N F (Kernie et al., 2000). Additionally, these animals are more anxious, aggressive and hyperactive. Even if BDNF is conditionally knocked out in early embryonic development in the forebrain the animals display abnormal eating behavior that results in obesity (but not to the same level as the above mentioned BDNF +/-), and a shortened lifespan (Gorski et al., 2003). In summary, it would be difficult to interpret the results of BDNF or trkB +/- animals when used in EODF experiments due to direct effects of these genetic manipulations on eating behavior. The use of trkB-IgG (containing the extracellular domain of trkB) to inhibit trkB signaling by trapping B D N F and preventing it from activating trkB (Shelton et al., 1995) is a more promising approach, however fl  there could still be complications. If trkB-IgG molecules circulate to either the dorsal vagal complex or the hypothalamus it could cause a reduction in food intake and possibly be a confounding variable. Instead of investing a large number of years to chase down all of the exact mechanisms of EODF on SCI, I would suggest investigating this diet regime to examine if it can reduce some of the secondary complications of SCI as outlined above (see section 5.5.B). I believe this line of research has the possibility of contributing to the quality of life of SCI patients. The testing of EODF's effect on chronic pain after SCI is one obvious experiment that could be conducted in a rat model. While it might be a more difficult and unexplored terrain, it would be interesting to examine EODF's effects on many other of secondary complications human SCI patients suffer from (e.g. diabetes, cardiovascular, chronic inflammation, etc) (see section 5.5.B). I do believe we should look at ways of augmenting the already beneficial effects of DR, but not necessarily along traditional molecular biology lines of thinking. These augmenting techniques would include further dietary alterations. As I mentioned in section 5.5.A, I think that the EODF regimen should be supplemented on feeding days with specific nutrients including an increase of proteins. A n increase in branch-chained amino acids (BCAA) increased cognitive recovery in human TBI patients (Aquilani et al., 2005). Recent work suggests very positive functional recovery effects with the addition of omega-3 after SCI (King et al., 2006). Both omega-3 and curcumin (a phenolic yellow curry pigment) prevented the decrease of BDNF in the brain after traumatic brain injury (Wu et al., 2004, 2006). Additionally, CR/EODF combined with other nutritional interventions (fish-oil, olive-oil) has synergistic (additive) effects on 200  increasing antioxidants and decreasing inflammatory response, ROS, and lipid peroxidation (Faine et al., 2004; Kim et al., 2006). These various lines of nutritional interventions suggest we have a great deal more work to explore the optimal combination that would be most advantageous to neurotrauma victims. I envision the combination of EODF with the addition of increased B C A A protein and omega-3 on feeding days might additively affect the functional outcome after neurotrauma.  201  5.7. Final thoughts  A not patentable treatment might never reach clinical trials. EODF is not patentable, it is an open source solution that is owned by nobody, and the treatment has absolutely no cost. I suggest that this free treatment, while not providing a full solution to the problem of SCI, can at least provide the possibility of some enhanced functional recovery. Think of the difference between clumsily moving your hands to increased precise control of hand movement that allows one to lift up a cup or spoon and feed yourself. I challenge the readers to find another treatment that has such universal positive health effects. Many, if not most, of our medical interventions usually have a host of negative side effects ranging from trivial to life threatening. If someone came out with a drug that could affect multiple targets leading to a reduced lesion size, increased neuron density, decreased inhibitory nature of adult CNS myelin/glial scar, increased intrinsic cell body response by increasing appropriate neurotrophin signaling, and increased CST sprouting, it would be heralded as a phenomenon. I will leave the readers to draw their own conclusions of what scientists will actually think of this idea and why these scientists come to their conclusions. The majority of follow up research that is possible from this thesis would be the further testing of therapeutic EODF. However, the remarkable effects of prophylactic EODF in SCI along with the numerous other papers consistently finding neuroprotection along with functional gains, not to mention increased lifespan, should make one ponder for a moment. I would argue that this is the true doctor of philosophy part of my PhD. Someone tosses onto your coffee table something that will cost you nothing (might even save money) but will increase your lifespan, decrease the chances of suffering cancer, stroke, heart disease, diabetes, Alzheimer's disease, Parkinson's disease, and protect your CNS in case of an accident. Will you pick up this free gift off the coffee table? Interesting question isn't it? If you are like most you will walk away from the table muttering some excuses disguised as criticisms like, "too good to be true", or "I don't believe it" or "who wants to be hungry all the time?". 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