"Applied Science, Faculty of"@en . "Mechanical Engineering, Department of"@en . "DSpace"@en . "UBCV"@en . "Choo, Anthony Min-Te"@en . "2011-02-14T20:02:35Z"@en . "2007"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "There remains no cure for traumatic spinal cord injury (SCI). Pre-clinical research typically models spinal cord transection, contusion, and compression, although in humans, other injury mechanisms such cord shearing from vertebral dislocation, and stretching from distraction occur frequently. This creates a potentially important disparity between experimental paradigms and clinical injuries. The objective of this thesis was to develop and compare three biomechanically distinct, yet clinically relevant SCI animal models. A multi-mechanism SCI system was developed to deliver high-speed (~100cm/s) injuries along any direction vector. A new vertebral clamping strategy with enhanced clamping strength (64.7\u00C2\u00B110.2N) and stiffness (83.6\u00C2\u00B118.9N/mm) was designed for modelling cervical vertebral dislocation (2.Smm), distraction (4.1mm), as well as contusion (1.1mm) injuries. The pattern of primary mechanical injury (n=36 rats) was found to differ between the three injury models. Contusion and dislocation produced intramedullary hemorrhage whereas overt vascular damage was not detected following distraction injury. Vertebral dislocation consistently sheared axons in the lateral columns. Plasma membrane disruption was detected by assessing the intracellular penetration of 10kDa dextran. Following contusion, membrane compromise of neuronal somata and axons was localized near the lesion epicentre whereas following dislocation and distraction, membrane damage extended several vertebral segments rostrally. At 3 hours post-trauma (n= 39 rats), damaged cell membranes were found to reseal especially in the white matter. In spite of this recovery, extensive loss of neurofilaments and accumulation of \u00CE\u00B2-amyloid precursor protein was observed following dislocation injury. In the gray matter, staining for cytochrome c and the oxidative stress marker 3-nitrotyrosine was similar following contusion and dislocation but less pronounced after distraction. Reactive astrocytes and activated microglia extended over a greater rostro-caudal zone only in the dislocation model. These rostro-caudal patterns demarcate disparate populations of primary and secondary injury suggesting that therapies developed in contusion paradigms may not translate to other SCIs. Neuroprotection and repair strategies might favour contusion and distraction injuries, whereas axonal regeneration across a lengthy lesion may inevitably be required for restoring function following dislocation. This interaction between the primary mechanism of injury and secondary neuropathology suggests treatment paradigms may be guided in a mechanism-specific manner."@en . "https://circle.library.ubc.ca/rest/handle/2429/31276?expand=metadata"@en . "CLINICALLY R E L E V A N T MECHANISMS OF SPINAL CORD INJURY: CONTUSION, DISLOCATION, AND DISTRACTION by ANTHONY M I N - T E C H O O B . A . S c , The University of Toronto, 1998 M . A . S c , The University of British Columbia, 2001 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Mechanical Engineering) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A August 2007 \u00C2\u00A9 Anthony Min-Te Choo 2007 Abstract There remains no cure for traumatic spinal cord injury (SCI). Pre-clinical research typically models spinal cord transection, contusion, and compression, although in humans, other injury mechanisms such cord shearing from vertebral dislocation, and stretching from distraction occur frequently. This creates a potentially important disparity between experimental paradigms and clinical injuries. The objective of this thesis was to develop and compare three biomechanically distinct, yet clinically relevant SCI animal models. A multi-mechanism SCI system was developed to deliver high-speed (~100cm/s) injuries along any direction vector. A new vertebral clamping strategy with enhanced clamping strength (64.7\u00C2\u00B110.2N) and stiffness (83.6\u00C2\u00B118.9N/mm) was designed for modelling cervical vertebral dislocation (2.5mm), distraction (4.1mm), as well as contusion (1.1mm) injuries. The pattern of primary mechanical injury (n = 36 rats) was found to differ between the three injury models. Contusion and dislocation produced intramedullary hemorrhage whereas overt vascular damage was not detected following distraction injury. Vertebral dislocation consistently sheared axons in the lateral columns. Plasma membrane disruption was detected by assessing the intracellular penetration of lOkDa dextran. Following contusion, membrane compromise of neuronal somata and axons was localized near the lesion epicentre whereas following dislocation and distraction, membrane damage extended several vertebral segments rostrally. At 3 hours post-trauma (n = 39 rats), damaged cell membranes were found to reseal especially in the white matter. In spite of this recovery, extensive loss of neurofilaments and accumulation of p\u00C2\u00B0-amyloid precursor protein was observed following dislocation injury. In the gray matter, staining for cytochrome c and the oxidative stress marker 3-nitrotyrosine was similar following contusion and dislocation but less pronounced after distraction. Reactive astrocytes and activated microglia extended over a greater rostro-caudal zone only in the dislocation model. These rostro-caudal patterns demarcate disparate populations of primary and secondary injury suggesting that therapies developed in contusion paradigms may not translate to other SCIs. Neuroprotection and repair strategies might favour contusion and distraction injuries, whereas axonal regeneration across a lengthy lesion may inevitably be required for restoring function following dislocation. This interaction between the primary mechanism of injury and secondary neuropathology suggests treatment paradigms may be guided in a mechanism-specific manner. ii T A B L E O F C O N T E N T S Abstract ii Table of Contents iii List of Tables vi List of Figures vii Acknowledgements ix Dedication . x Co-Authorship Statement xi Chapter 1 Introduction 1 1.1 Overview 1 1.2 Spine and Spinal Cord Anatomy 2 1.2.1 Orientation 2 1.2.2 Spinal Column 2 1.2.3 Spinal Cord 4 1.2.4 Neurons and Glia _ 6 1.2.5 Cytoskeleton _10 1.3 Human Spinal Cord Injuries 11 1.3.1 Epidem iology 11 1.3.2 Vertebral Column Injuries 13 1.3.2.1 Burst Fractures 15 1.3.2.2 Dislocations & Fracture-Dislocations 16 1.3.2.3 Distraction 17 1.3.2.4 Other Vertebral Column Injuries 18 1.3.3 Spinal Cord Injury Mechanisms 18 1.3.4 Pathology of Human Spinal Cord Injury 19 1.3.5 Clinical Spinal Cord Treatment 21 1.3.5.1 Decompression _21 1.3.5.2 Pharmacotherapy 22 1.3.6 Summary of Human Injuries ; 23 1.4 Biomechanics of Neurotrauma . 24 1.4.1 Mechanical Properties of Neural Tissue 24 1.4.2 Constitutive Models 28 1.4.3 Primary Mechanical Injury 29 1.4.3.1 Tissue Failure 30 1.4.3.2 Vascular Damage _31 1.4.3.3 Electrophysiological Deficits 32 1.4.3.4 Cellular Injur/ 33 1.4.3.5 Plasma Membrane Compromise 34 1.4.4 Spinal Cord Inj ury Models 3 6 1.4.4.1 Allen's Weight-Drop 36 1.4.4.2 Controlled Displacement 37 1.4.4.3 Controlled Force 39 1.4.4.4 Residual Compression ; 40 1.4.4.5 Transection . 40 iii 1.4.4.6 Cervical Injury Models 40 1.4.4.7 Vertebral Fracture-Dislocation 41 1.4.4.8 Vertebral Distraction . 41 1.4.4.9 Comparison of Models 42 1.4.5 Summary of Biomechanics ; 43 1.5 Neurobiology of-Spinal Cord Injury _ 43 1.5.1 Secondary Injury & Neuroprotection _ _ _ 43 1.5.1.1 Hemorrhage & Ischemia 44 1.5.1.2 Inflammation , 44 1.5.1.3 Oxidative Stress 45 1.5.1.4 Calcium & Excitotoxicity 46 1.5.1.5 Apoptosis . 47 1.5.2 Repair & Regeneration 48 1.5.2.1 The Inhibitory Environment 49 1.5.2.2 Growth Factors 50 1.5.2.3 Axonal Repair . 50 1.5.3 Summary of Neurobiology 51 1.6 Thesis Objectives and Hypotheses 52 1.6.1 Scope ; 52 1.7 References . 54 Chapter 2 A Novel SCI Device 69 2.1 Introduction 69 2.2 Materials and Methods . 71 2.2.1 Multi-Mechanism Injury System 71 2.2.2 Part 1: Vertebral Clamps 73 2.2.3 Part 2: Injury Models 75 2.2.4 Hemorrhage Analysis . 78 2.2.5 White Matter Immunohistochemistry 79 2.3 Results 79 2.3.1 Part 1: Vertebral Clamps 79 2.3.2 Part 2: Injury Models 81 2.4 Discussion 88 2.5 References 92 Chapter 3 Distribution of Primary Injury 97 3.1 Introduction .97 3.2 Materials and Methods 98 3.3 Results 103 3.4 Discussion . 110 3.5 References . 116 Chapter 4 Initiation of Secondary Injury 121 4.1 Introduction 121 4.2 Materials and Methods 122 4.2.1 Animal Models 122 4.2.2 Immunohistochemistry 125 iv 4.2.3 General Image Acquisition & Analysis 127 4.2.4 Changes in Membrane Permeability 128 4.2.5 Cell Bodies in the Gray Matter 128 4.2.6 Axons in the White Matter 129 4.2.7 Reactive Astrocytes 130 4.2.8 Microglial Activation 130 4.3 Results 133 4.3.1 Changes in Membrane Permeability 133 4.3.2 Cell Bodies in the Gray Matter 137 4.3.3 Axons in the White Matter 139 4.3.4 Reactive Astrocytes 144 4.3.5 Microglial Activation 146 4.4 Discussion 148 4.5 References 153 Chapter 5 Discussion & Conclusion 159 5.1 Overview 159 5.2 Modelling Considerations 159 5.3 Biomechanics of Primary Injury Patterns 162 5.3.1 Contusion _ 162 5.3.2 Dislocation 165 5.3.3 Distraction 169 5.4 Association of Primary and Secondary Injury 171 5.4.1 Contusion _ 171 5.4.2 Dislocation 173 5.4.3 Distraction 174 5.4.4 Summary of Primary and Secondary Injury 174 5.5 Clinical Relevance 176 5.6 Limitations 177 5.7 Recommendations 178 5.8 Contributions 180 5.9 Conclusion 181 5.10 References 182 Appendix A SCI Test System _ _ _ 187 5.11 Test System Assembly 188 5.12 Test System Drawings 189 5.13 Sensors 190 Appendix B Image Analysis 192 5.14 Image Analysis Tools 193 Appendix C ANOVA Tables 194 Appendix D Ethics Board Certificates of Approval 199 v L I S T O F T A B L E S Number Page Table 2.1: Vertebral clamping characteristics 80 Table 2.2: Mechanical parameters for graded injuries 81 Table 3.1: Injury parameters for primary injury study 103 Table 4.1: Immunohistochemical markers for secondary injury 126 Table 4.2: Mechanical injury parameters in secondary injury study 133 Table C . l : A N O V A summary - Acute dextran penetration into axons 195 Table C.2: A N O V A summary - Acute dextran penetration into neuronal somata 195 Table C.3: A N O V A summary - Evolution of dextran penetration at 3 hours post-trauma 196 Table C.4: A N O V A summary - 3NT-positive cells j 197 Table C.5: A N O V A summary - Neurofilament degradation in ventro-medial white matter 197 Table C.6: A N O V A summary - pAPP accumulation in axons 197 Table C.7: A N O V A summary - Reactive astrocytes 197 Table C.8: A N O V A summary - Microglial activation 198 vi LIST OF F I G U R E S Number Page Figure 1.1: Orientation in humans and quadrupeds 2 Figure 1.2: The spinal column 3 Figure 1.3: Anatomy of a vertebra 3 Figure 1.4: Spinal cord anatomy . 4 Figure 1.5: Spinal cord gray and white matter 5 Figure 1.6: Segments of Neurologic Deficits 6 Figure 1.7: The neuron 7 Figure 1.8: Synapse 7 Figure 1.9: Micrographs and illustrations of glia cells 9 Figure 1.10: Cytoskeletal proteins 11 Figure 1.11: SCI epidemiology 12 Figure 1.12: Denis's three column spine _ _ 14 Figure 1.13: Vertebral column fractures 15 Figure 1.14: Spinal cord injury mechanisms 19 Figure 1.15: Anisotropy of neural tissue 27 Figure 1.16: Thesis scope 53 Figure 2.1: Multi-mechanism injury system 72 Figure 2.2: Cervical vertebral clamps and dimensions 74 Figure 2.3: Illustrations and photos of experimental injury configurations 76 Figure 2.4: Failure and stiffness of Allis clamps and novel vertebral clamps 80 Figure 2.5: Representative curves for graded injuries 83 Figure 2.6: Mortality rates for dislocation and distraction injuries 84 Figure 2.7: Morphology stained with H & E 85 Figure 2.8: Regression curves for hemorrhage versus injury displacement and force 86 Figure 2.9: White matter damage following three injury mechanisms 87 Figure 3.1: Classification of membrane permeability into neuronal somata 101 Figure 3.2: Dextran accumulation at nodes of Ranvier 102 Figure 3.3: Representative mechanical injury curves 104 Figure 3.4: Primary hemorrhage volumes following three injury mechanisms 105 Figure 3.5: Axonal membrane compromise detected with fluorescein-dextran 106 Figure 3.6: Quantitative measurements of dextran-positive axons 108 Figure 3.7: Quantitative measurements dextran-positive cell bodies ; 109 Figure 4.1: Illustration of three injury mechanisms 124 Figure 4.2: Novel microglial ramification index 132 Figure 4.3: Representative micrographs showing evolution of membrane permeability 134 Figure 4.4: Quantitative analysis of dextran penetration into somata and axons 136 Figure 4.5: Photomicrographs and quantitative counts of 3-NT immunostaining 137 Figure 4.6: Cytochrome c immunostaining 139 Figure 4.7: Neurofilament degradation in the ventro-medial white matter 141 Figure 4.8: PAPP accumulation in white matter ; 143 Figure 4.9: Distribution of reactive astrocytes 145 vii Figure 4.10: Microglial activation 147 Figure 5.1: Central damage pattern in contusion injuries 163 Figure 5.2: Injury patterns following fracture-dislocation 167 Figure 5.3: Ventro-medial axolemma compromise following dislocation injury 169 Figure 5.4: Dorsal axolemma compromise following distraction injury 170 Figure 5.5: Parallel patterns of primary and secondary injury 172 Figure A . l : Test system assembly sequence 188 Figure A . 2 : Accelerometer housing 189 Figure A . 3 : Rotary detent 189 Figure A.4 : Load cell calibration model 31: 22.5N 190 Figure A . 5 : Load cell calibration model 31: 225N 190 Figure A .6 : Load cell calibration model 208C02: 444N 191 Figure B . l : StereolTools interface and screen images 193 v i i i A C K N O W L E D G E M E N T S I would not be here without the support of Drs. Thomas Oxland and Wolfram Tetzlaff. Tom has given me an opportunity to venture into a new and inspiring field while W o l f has welcomed me\u00E2\u0080\u0094a naive engineer\u00E2\u0080\u0094into his neuroscience laboratory. I am forever indebted to them for their mentorship. I owe heartfelt thanks to Hanspeter Frei, Ward Plunet, Clarrie Lam, Jie L i u , Derek Wilson, David Tsai, Joey Doherty, Lin-P ' ing Choo-Smith, and my parents for their perpetual encouragement. Thanks also to Bryan Sniderman and Raymond L i at A T I Technologies for enabling me and my wife to keep our family together in Vancouver. Most importantly, I am forever grateful to my wife, Ngoc Luu, for all her patience and love during this long journey, and to my children, Cianna and Braeden, for always reminding me of what is most precious in life\u00E2\u0080\u0094I owe many thanks to God for blessing me with this family. Many others have contributed in some way to my growth over these years. Sincere thanks to, D O U G L A S B O U R N E , E D W A R D C H E U N G , E L I Z A B E T H C L A R K E , PETER CR IPTON, M A R C E L D V O R A K , C O R I N A FREI , J OHNSON G O, C A R O L Y N G R E A V E S , Z HUOWE I L I U , L I N A M A D I L A O , C A R O L Y N S PARREY , J U A Y S E N G T A N , Q I N G A N Z H U This work was supported by the Canadian Institutes of Health Research, Canada Foundation of Innovation, the Canada Research Chairs Program, the British Columbia Neurotrauma Fund, the R ix Family Leading Edge Endowment Fund, and the George W . Bagby Research Fund. ix D E D I C A T I O N for my ever supportive parents Lip Kung and Keow Geen & Ngoc and the children, my Cianna and little Braeden C O - A U T H O R S H I P S T A T E M E N T The research contained within this thesis draws on the expertise from the biomedical engineering laboratory o f Dr. Thomas R. Oxland and the neuroscience laboratory o f Dr. Wolfram Tetzlaff. The doctoral candidate designed the experiments and their outcome measures under the combined mentorship of Drs. Oxland and Tetzlaff. The research was performed by the candidate. The candidate designed and built the novel spinal cord injury apparatus and the associated stereotatic devices. The candidate conducted the animal experiments with microsurgical assistance from Dr. Jie L i u . Tissue processing and immunohistochemistry were conducted by the candidate with advice, where necessary, from M s . Clarrie Lam. The candidate wrote the image analysis software and analysed the data. The candidate led the preparation o f the manuscripts with revisions from the senior co-authors Drs. Oxland, Tetzlaff, and Dvorak. x i Ch apter 1 I N T R O D U C T I O N 1.1 Overview A clinically viable cure for traumatic spinal cord injury (SCI) remains elusive. Pre-clinical research has relied on spinal cord transection, contusion, and compression injury models, though they are representative of only some human SCI cases. Other types of vertebral column fracture result in different biomechanical injury mechanisms to the spinal cord. Comparisons between distinct injury mechanisms has received little attention because of the prevailing consensus that the pathophysiology of SCI is dominated by the myriad biochemical processes which are thought to be independent of the initial mechanical injury. Given that experimental therapies have met with only modest success in human clinical trials, this thesis examines the influence o f distinct, yet clinically relevant biomechanical injury mechanisms on the characteristics of acute SCI. This introductory chapter overviews the clinical, biomechanical, and neuropathological aspects of SCI. The chapter begins with an outline of human SCI pathology with a focus on the different types of vertebral column fractures which cause SCI by differing injury mechanisms. A review of biomechanical aspects of neurotrauma describes the behaviour of neural tissue, how biomechanical damage is characterized at the tissue and cellular levels, and how mechanically controlled injuries are currently modelled experimentally. Next, the main avenues of secondary degeneration that are initiated by primary mechanical injury are highlighted, along with a brief discussion of spinal cord regeneration. The chapter concludes by presenting the thesis objectives and hypotheses that are the focus of chapters 2 to 4. Chapter 5 discusses the role of biomechanical injury mechanisms in SCI and concludes with recommendations for future directions. 1 C h a p t e r 1 I n t r o d u c t i o n 1.2 Spine and Spinal Cord Anatomy 1.2.1 Orientation The position of anatomical structures discussed in this thesis depends on whether they refer to humans or animals. In humans (Figure 1.1 A ) , superior and cranial refer to the direction of the head while inferior and caudal refer to the direction of the feet. Anterior refers to a direction towards the front of the body while posterior denotes the direction towards the back. In quadruped animals (Figure L I B ) , rostral and caudal refer to the direction of the head and tail respectively. Dorsal refers to the animal's back while ventral refers to the front. In both humans and quadrupeds, left and right are divided by a mid-sagittal plane from which lateral denotes a direction away from this plane while medial indicates a direction towards the mid-sagittal plane. Figure 1.1: Orientation in humans and quadrupeds Cardinal planes and direction vectors used to describe anatomical positions in humans and quadrupeds. (Graphics from: Swanson LW, 2003. The architecture of nervous systems. In: Fundamental Neuroscience, 2nd Edition [Squire LR et al. eds], p.24. San Diego, CA: Academic Press. \u00C2\u00A9 Elsevier Science [USA], 2003, adapted by permission.) 1.2.2 Spinal Column The osteoligamentus vertebral column (Figure 1.2) encases the spinal cord and protects it from traumatic loads. In humans, the spinal column consists of a series of vertebrae that can be divided into cervical, thoracic, lumbar, sacral and coccygeal regions. Intervertebral discs separate the vertebral bodies to cushion loads and to allow for intersegmental motion between vertebrae. A typical vertebra (Figure 1.3 A & B) consists of a vertebral body, which supports the majority of the axial loads applied to the spinal column, and posterior elements that are attached to the vertebral body via the pedicles to form an arch that encloses the vertebral foramen through which the spinal cord courses. Intervertebral discs separate the vertebral bodies (Figure 1.3B) to cushion loads and allow for intersegmental motion. 2 Chapter 1 Introduction 7 Cervical Vertebrae 12 Thoracic Vertebrae 5 Lumbar Vertebrae 5 Sacral Vertebrae 4 Coccygeal Vertebrae Figure 1.2: The spinal column Lateral view of the spinal column and its sub-divisions: cervical, thoracic, lumbar, sacral, coccygeal. Each vertebral body is separated by an intervertebral disc that cushions loads and allows for intersegmental motion. Illustration adapted from Moore KL and Dalley AF, 1999. Clinically oriented anatomy, 4th Edition, p.433. Philadelphia, PA: Lippincott Williams & Wilkins.) Inferior articular process and facet Spinous process Lamina Transverse^*^r-process Superior articular facet Vertebral body Vertebral arch Q ace tv . HI Superior view B Superior vertebral notch Pedicle Vertebral body Inferior vertebral notch Lamina Lateral view Figure 1.3: Anatomy of a vertebra Superior (A) and lateral (B) view of a vertebra with anatomical features labeled. The spinal cord travels through the vertebral foramen visible in A. 3 Chapter 1 Introduction 1.2.3 Spinal Cord The spinal cord is covered by several membrane layers (Figure 1.4). The outermost membrane that is in direct contact with the vertebral foramen is the dura mater. The arachnoid mater lines the inner surface of the dura. Beneath the arachnoid mater, the subarachnoid space contains the cerebrospinal fluid that circulates through both this space and the cerebral ventricles in the brain. The innermost membrane is the pia mater which directly covers the spinal cord. The spinal cord is suspended within the dural sac via the denticulate ligaments which extend laterally from the pia to dura. Dorsal gray horn Ventral gray horn Dorsal rootlets Denticulate ligament Dorsal root Spinal ganglion Dorsal primary ramus Ventral primary ramus Spinal nerve Ventral root Ventral rootlets Gray Matter White matter Pia mater (ensheaths cord and vessels) Arachnoid mater Dura mater Spinal meninges Figure 1.4: Spinal cord anatomy Illustration of the spinal cord shows the gray and white matter with sensory fibers entering the cord dorsally and motor fibers exiting the cord ventrally. The spinal cord is covered in three membrane layers: pia mater, arachnoid mater, and dura mater. The cerebrospinal fluid lies between the pia and arachnoid. Nerve roots enter and exit the spinal cord between each vertebral segment. Sensory fibers enter posteriorly (dorsal rootlets Figure 1.4) while motor fibers exit anteriorly (ventral rootlets Figure 1.4). The central region of the spinal cord is occupied by gray matter while the peripheral region contains ascending and descending axons that are ensheathed in myelin giving them a white appearance (Figure 1.5A). Gray matter is highly vascularized and contains neuronal cell bodies 4 Chapter 1 Introduction (Figure 1.5B) as well as glial cells which support neuronal functions (described below). The white matter axons (Figure 1.5C) transmit signals between the neurons in the brain and brainstem, and the neurons in the spinal cord gray matter. The cells in the gray matter mediate motor and sensory functions primarily at the local segment in which they lie and hence damage to gray matter results in localized segmental deficits. In contrast, interruption of ascending and descending white matter tracts wi l l compromise the transmission of signals between the brain and segments below the level of injury (Figure 1.6). Figure 1.5: Spinal cord gray and white matter Photomicrographs o f rat cervical spinal cord cross-section immunostained for non-phosphorylated epitopes o f heavy neurofilament (SMI-32, Sternberger Monoclonals). A . Gray matter is located in the central region of the spinal cord surrounded by ascending and descending white matter tracts. B . Higher magnification image o f ventral gray matter in tile A Arrowhead highlights an example o f neuronal cell body with positive neurofilament staining in the cytoplasm. C. Higher magnification image o f ventral white matter in tile A . Arrowhead marks example o f large caliber axon whose cytoskeleton stains positive for heavy neurofilaments. Arrow marks ventral exit o f an axon from a motoneuron. Scale bars 500um in A , 50um in B and C . 5 Chapter 1 Introduction Figure 1.6: Segments of Neurologic Deficits Illustration shows injury to the spinal cord (star graphic) can result in neurologic deficits at the segment of injury and segments below (shaded red) due to interruption of ascending and descending white matter fibres. (Illustration adapted from asia-spinalinjury.org by permission). 1.2.4 Neurons and Glia Neurons possess a complex architecture (Figure 1.7) that stems from the specialized functions of its different components. Dendritic processes extend from the cell body to receive neurotransmitters at synaptic clefts (Figure 1.8A). These neurotransmitters open channels embedded in the cell's membrane, allowing the exchange of ions across the electrochemical gradient between the extracellular and intracellular space (Figure 1.8B). This exchange of ions can transiently raise (excitatory influx of positive charges) or lower (inhibitory influx of negative charges) the voltage across the cell membrane. The cell body integrates repeated signals over time and integrates spatially separate signals over space. If the membrane potential rises above an excitatory threshold, the cell body fires an action potential that propagates down its axonal process to the axon's terminal where neurotransmitters are released to the next target. 6 Chapter 1 Introduction Figure 1.7: The neuron Pyramidal neuron from the cerebral cortex demonstrates the specialized components of the nerve cell. Dendritic arborizations receive signals. The cell body transmits action potentials via the axon. (Graphics from: Hof PR et al, 2003. Cellular components of nervous tissue. In: Fundamental Neuroscience, 2nd Edition [Squire LR et al. eds], p.49. San Diego, CA: Academic Press. \u00C2\u00A9 Elsevier Science [USA], 2003, adapted by permission.) presynaptic element vesicle neurotransmitter ion channel Figure 1.8: Synapse Illustration of an excitatory synapse. A. Neurotransmitters released from vesicles in the pre-synaptic element stimulate channels on the post-synaptic element. B. Zoomed illustration of ion channel embedded in the plasma membrane. For an excitatory neurotransmitter, cation channels are opened allowing positively charged ions to enter the intracellular space resulting in a depolarization of the membrane potential. 7 Chapter 1 Introduction Glia l cells coexist with neurons and support their survival and function. Astrocytes are the most numerous cells in the central nervous system. Their stellate morphology (Figure 1.9A) is visualizable by immunostaining with antibodies directed against glial fibrillary acidic protein (GFAP) which is an intermediate filament of the astrocyte cytoskeleton. Astrocytes perform numerous supporting functions including buffering neurotransmitters such as glutamate and converting it to glutamine to be recycled by axon terminals. Following injury or disease, an increase in G F A P expression is a commonly used indicator of reactive astrocytes (Figure 1.9B). Microglia are resident immune cells of the central nervous system. In their resting state, microglia have small cell bodies and a network of highly branched\u00E2\u0080\u0094ramified\u00E2\u0080\u0094processes (Figure 1.9C). Under pathological conditions, microglia become activated whereupon their processes thicken and retract (Figure 1.9D) as they evolve into phagocytes. Oligodendrocytes (Figure 1.9E) extend their cell membranes to ensheath axons in compacted concentric layers of myelin (Figure 1.9F). Myel in electrically insulates the axon thereby allowing action potentials to travel more quickly between exposed segments of the axon called nodes of Ranvier (arrowhead Figure 1.9G and I). Nodes of Ranvier are identifiable by immunostaining for voltage-gated sodium and potassium channels which are highly organized in the nodal region (Figure 1.9H and I). 8 Chapter 1 Introduction Astrocytes Reactive r Astrocytes -B oligodendrocyte myel in J0\u00C2\u00A33^W l amel lae M'- VJV jar node of B / ^ Ranvier A x o n \u00E2\u0080\u00A2 G Kv 1.2 i I H Merge Figure 1.9: Micrographs and illustrations of glia cells Immunostaining with anti-glial fibrillary acid protein (GFAP) reveals stellate morphology of astrocytes (arrow in A). Following neurotrauma, reactive astrocytes express greater GFAP (arrow in B). Immunostaining with anti-Ibal shows the morphology of resting microglia which exhibit small cell bodies with highly ramified processes (arrow in C). Following activation, microglial processes thicken and retract (arrow in D) following disease or neurotrauma. E. Illustration of oligodendrocyte which ensheath the axon in myelin lamellae. The axon is exposed at periodic gaps in the myelin sheath called nodes of Ranvier. F. Electronmicrograph of axon shows electron-dense layers of myelin. G. An axon partially filled with a molecular tracer (dextran) which also accumulates at the axon's node of Ranvier (arrow in G) thereby highlighting this gap in the myelin sheath. H. Immunostaining for voltage-gated potassium channels (Kvl.2) highlights the juxtaparanodal region. I. Merged image of G and H. Scale bars 50um in A to D, lOum in G to I. (Graphics in E from: Quarles RH et al. 2006. Myelin formation, structure and biochemistry. In: Basic Neurochemistry: Molecular, Cellular and Medical Aspects, 7th Edition [Siegel GJ et al. eds], p.53. Burlington, MA: Elsevier Academic Press. \u00C2\u00A9 American Society for Neurochemistry, 2006, adapted by permission. Electronmicrograph in F reproduced from Journal of Experimental Medicine, 1947, 86:175-184. \u00C2\u00A9 1947 Rockefeller University Press, adapted by permission. Remaining micrographs were taken from thesis work.) 9 Chapter 1 Introduction 1.2.5 Cytoskeleton Cytoskeletal proteins form the internal structure of cells which allows them to exhibit diverse morphologies such as those found in neural tissue. Microtubules, intermediate filaments, and microfilaments are the three basic classes of cytoskeletal proteins (Figure 1.10) in eukaryotic cells. Microtubules are hollow tubes approximately 25nm in diameter. Microtubules consist of alternating units of a- and P-tubulin and serve to provide structural stiffness to the cytoskeleton. In addition, microtubules serve as tracks for the transport of proteins and organelles to and from the cell body. A n example o f this cargo is P-amyloid precursor protein which is assessed later in this thesis. Intermediate filaments are approximately 1 Onm in diameter. Gl ia l fibrillary acidic protein is a specific intermediate filament of astrocytes while neurofilaments are specific to neurons. There are three subtypes of neurofilaments which are distinguished by molecular weight. The molecular weight of heavy neurofilaments (NF-H) is 200kDa while medium ( N F - M ) and light neurofilaments (NF-L) are approximately 160kDa and 68kDa respectively. Medium and heavy neurofilaments possess protein sidearms that project outward from a core domain (Hirokawa et al., 1984). These projections maintain the spacing between neurofilaments and this has been shown to be necessary for the development of large caliber axons (Elder et al., 1998). Microfilaments are the smallest of the cytoskeletal proteins and they have a diameter of approximately 5nm. Microfilaments such as actin are highly labile structures which are found in areas such as the axonal growth cone and beneath the plasma membrane where they maintain the distribution of membrane bound proteins. 10 Chapter 1 Introduction Figure 1.10: Cytoskeletal proteins Cytoskeletal proteins form the internal structure of cells enabling them to exhibit a diverse range of morphologies. Microtubles consists of repeating units of a- and B-tubulin. Neurofilaments consist of a core domain with sidearm projections that are believed to maintain the separation distance between these intermediate filaments. Microfilaments such as actin are the smallest of the three basic cytoskeletal proteins. (Graphics from: Pigino G et al. 2006. The cytoskeleton of neurons and glia. In: Basic Neurochemistry: Molecular, Cellular and Medical Aspects, 7th Edition [Siegel GJ et al. eds], p. 125. Burlington, MA: Elsevier Academic Press. \u00C2\u00A9 American Society for Neurochemistry, 2006, adapted by permission.) This brief overview highlights some of the anatomical structures that arise later in this thesis. The structure and function of neural tissue and its cellular constituents are rather more intricate than can be covered in this introduction, though these additional details are readily available elsewhere (Squire et al., 2003; Siegel et al., 2006). 1.3 Human Spinal Cord Injuries 1.3.1 Epidemiology The overall incidence of traumatic SCI has remained relatively unchanged over the last thirty years (Wyndaele and Wyndaele, 2006). In the developed world, the annual incidence rate has been reported between 11.5 and 53.4 per million (Sekhon and Fehlings, 2001). Within the United States, this rate translates to approximately 11,000 new SCI cases per year with an estimated prevalence of nearly 250,000 individuals currently living with SCI impairments. The mortality rate ranges between 48.3 and 79% prior to hospital admission and between 4.4 to 16.7% during hospital care. Complications are predominantly due to respiratory failure, pneumonia, pressure sores, urinary tract infections, and thrombosis. Many of the injuries occur in a youthful adult population where individuals between the ages of 20-40 years of age account for approximately 40% of SCI cases (Pickett et al., 2006). The elderly (>65 years) account for approximately 10% of SCI cases with falls being the dominant cause in this group. Cases of SCI in males outnumber those in females by a ratio of 3:1. Overall, motor vehicle accidents consistently account for the majority of traumatic SCI with falls, work and sports related injuries, and violence accounting for 10-20%o each (Figure 1.11 A ) . 11 Chapter 1 Introduction Traumatic SCI is frequently associated with vertebral column injuries (Figure 1.1 IB). Two of the most common vertebral fracture patterns are burst fractures and fracture-dislocations; each having a reported incidence rate of 30-40% in the human population (Sekhon and Fehlings, 2001; Pickett et al., 2006). SCI without radiographic abnormality ( S C I W O R A ) and SCI without obvious radiographic evidence of trauma (SCIWORET) have also been reported though these cases have declined because the use of magnetic resonance imaging has improved the sensitivity of detecting cord injuries that were previously missed with x-ray radiography (Sekhon and Fehlings, 2001). Dislocations without fracture of the facets are also encountered clinically and it is hypothesized that a dynamic flexion-extension force cause these vertebrae to jump over each other resulting in dislocation with locked facets (Allen et al., 1982). Causes Ver tebra l Injuries Ver tebra l Level N e u r o l o g i c Def ic i t Thoracic 15% Thoraco-lumbar 15% Lumbosacra 15% Cervical 55% ASIA D 30% ASIAC 10% ASIA A .45% ASIA B 15% Figure 1.11: SCI epidemiology Causes compiled from Pickett et al. 2006. Spine 31:799-805. Vertebral injuries, vertebral level, neurologic deficit compiled from Sekhon LH and Fehlings MG, 2001. Spine 26:S2-S12. 12 Chapter 1 Introduction SCI is most common in the cervical region (55%) with the incidences in the thoracic, thoracolumbar, and lumbar spines estimated at 15% each (Figure 1.11C). The extent of neurological deficit is frequently assessed by the American Spinal Injury Association (ASIA) impairment scale. A S I A A denotes a complete loss of motor and sensory function in the sacral segments while A S I A E indicates normal motor and sensory function (Figure 1.1 ID). Between these two extremes, A S I A B represents the preservation of sensory function but no motor function below the level of injury (includes sacral segments S4-S5), A S I A C indicates the presence of motor function below the lesion with more than half of the key muscles exhibiting a grade less than 3/5 (where 0/5 denotes total muscle paralysis), while A S I A D denotes the presence of motor function with at least half of the key muscles exhibiting a grade of 3/5 or better. Almost half of all SCI patients (45%) are A S I A A while the remaining patients are distributed between A S I A B to A S I A D . Several trends have been reported in SCI subpopulations. Incidences due to motor vehicle accidents have been decreasing due to improved seat belt usage. This decrease, however, has been offset by an increase in SCI in the elderly population. Thoracic injuries are more frequently (73%) associated with complete SCI (Tator, 1983). Vertebral dislocation has been also more frequently associated with complete injuries (Kiwerski, 1991). Isolated SCI only occurs in 20% of cases while other types of trauma such as brain and thoracic injuries are present in the remaining cases. SCI without fracture occurs more frequently in the cervical region (92%) in the older population (mean age 58) and is often associated with cervical spondylosis or spinal canal narrowing. 1.3.2 Vertebral Column Injuries SCI in humans usually occurs when the osteoligamentus vertebral column dynamically traumatizes the spinal cord. Injuries due to progressive canal stenosis, tumor growth, and ischemia subsequent to vertebral artery occlusion wi l l not be addressed here. Different patterns of vertebral column injuries are readily visible on radiographs and magnetic resonance images (MRI). Numerous classification systems have been proposed for describing the different types of vertebral injuries in order to standardize terminology and assist in the development of treatment algorithms. Denis introduced the terminology of the three column spine (Denis, 1983) which is convenient for describing structural damage in spinal injuries (Figure 1.12A). The anterior column encompasses the anterior longitudinal ligament, the anterior vertebral 13 Chapter 1 Introduction body and the anterior portion of the annulus fibrosus (Figure 1.12B). The middle column includes the posterior wall of the vertebral body, the posterior longitudinal ligament, and the posterior segment of the annulus fibrosus (Figure 1.12C). The posterior column is comprised of the posterior bony complex, ligamentum flavum, interspinous and supraspinous ligaments, and the joints capsules (Figure 1.12D). Note that Al len and Ferguson have proposed mechanistic classifications for cervical (Allen et al., 1982) and thoracolumbar (Ferguson and Al len , 1984) spine fractures, though these mechanisms refer to vertebral column injuries rather than spinal cord injuries. No classification system, however, has gained unanimous acceptance (Schweitzer et al., 2006). The following sections wi l l focus on three stereotypical vertebral injuries\u00E2\u0080\u0094burst fractures, fracture-dislocations, and distractions\u00E2\u0080\u0094as these vertebral column injuries account for over 80% of the column injuries observed in humans (Sekhon and Fehlings, 2001; Pickett et al., 2006) and therefore, they wi l l form the basis of the three idealized mechanisms of spinal cord injury compared in this thesis. ISLLF f Figure 1.12: Denis's three column spine A. Labels for structures of the three column spine. B. The anterior column is comprised of the anterior longitudinal ligament (ALL), the anterior half of the annulus fibrosus (AF), and the anterior wall of the vertebral body. C. The middle column which consists of the posterior longitudinal ligament (PLL), the posterior annulus fibrosus, and the posterior wall of the vertebral body. D. The posterior column consists of the posterior bony complex (posterior arch) and the posterior ligamentous complex (supraspinous ligament [SSL], interspinous ligament [1SL], ligamentum flavum [LF], capsule [C]). 14 Chapter 1 Introduction 1.3.2.1 Burst Fractures The burst fracture (Figure 1.13 A ) is characterized by comminution of one, or sometimes both vertebral endplates, that results in the collapse of vertebral body height with retropulsion of bony fragments into the spinal canal (Denis, 1983; Magerl et al., 1994). In this type of vertebral fracture, the exploding bony fragments contuse the spinal cord from the anterior to posterior direction. After this dynamic contusion, the bone fragments may come to rest in a position that occludes the spinal canal and often results in residual compression of the cord. These fractures occur most often in the thoracolumbar spine where they account for approximately 50% to 80% of the spine fractures in this region (Petersilge and Emery, 1996; Bensch et al., 2006). In this region, the narrower spinal canal increases the likelihood of SCI (Bohlman, 1985). Burst Fracture Fracture-Dislocation Distraction Figure 1.13: Vertebral column fractures Three clinically relevant types of vertebral column injuries. A. Burst fracture is characterized by the retropulsion of bony fragment into the spinal canal. B. Fracture-dislocation is characterized by the translation of one vertebrae over another. C. Distraction injuries are characterized by a distractive tensile component that results in an increased intersegmental space between the vertebral bodies or posterior elements. Biomechanically, burst fractures occur by axial compression to the vertebral bodies (Oxland, 1992). In the cervical and thoracolumbar spines, Al len and Ferguson classified these injuries as resulting from compressive flexion and vertical compression mechanisms. In compressive flexion, anterior vertebral wedging is observed with the posterior elements injured in tension. In contrast, vertical compression uniformly collapses the vertebral height without wedging and hence, no tensile loads are applied to the posterior elements. O f prime importance in the acute assessment of the burst fracture is the extent of spinal column instability that may exacerbate neurologic damage. Vertical compression fractures, where 15 Chapter 1 Introduction the posterior elements are intact, are generally thought to be clinically stable meaning that the neurologic status of the patient wi l l not deteriorate. In severe wedge fractures, however, tensile rupture of the posterior ligamentus complex may render these injuries mechanically unstable and susceptible to progressive kyphosis that can further displace bone fragments into the spinal canal (Denis, 1984). A 50% reduction in the height of the anterior vertebral body suggests a risk of posterior element failure and instability (Ferguson and Al len , 1984). Stable burst fractures can be treated non-operatively and the retropulsed bone usually resorbs over time (Petersilge and Emery, 1996). 1.3.2.2 Dislocations & Fracture-Dislocations Vertebral dislocations and fracture-dislocations (Figure 1.13B) are characterized by the relative displacement\u00E2\u0080\u0094subluxation\u00E2\u0080\u0094of one vertebral body over another (Denis, 1983). The post-traumatic misalignment of the adjacent vertebrae suggests the spinal cord is injured by a shearing mechanism; however, the precise trajectory of the vertebrae during the injury is uncertain. In some injuries, dislocation occurs with facet fracture whereas in other injuries, dislocation occurs with jumped facets indicating a dynamic distractive component during the dislocation. In Al len and Ferguson's mechanistic classification of lower cervical fractures, dislocations with and without fracture may occur by three types of vertebral injury mechanisms - distractive flexion, compressive extension, and distractive extension (Allen et al., 1982). In distractive flexion, the posterior ligamentous complex is injured by distractive tensile forces enabling facet dislocation while the vertebral body rotates and translates in the anterior direction over the caudal vertebra. In a compressive extension mechanism, the posterior elements are loaded in compression which may result in fracture of the vertebral arches, lamina, and articular processes. In the distractive extension mechanism, the anterior column is placed under tension and there is excessive widening of the intervertebral disc space with possible displacement of the cephalad vertebrae posteriorly shearing the spinal cord between the inferior margin of the moving vertebral body and the lamina of the caudal vertebra. Dislocation injuries can also be caused by torsion (Magerl et al., 1994) or pure translations (Ferguson and Al len , 1984) and this highlights the broad range of injuries grouped under this type of vertebral injury. Fracture-dislocation involves disruption of Denis's anterior, middle, and posterior columns rendering these vertebral injuries mechanically unstable (Denis, 1984). Anatomical repositioning 16 Chapter 1 Introduction of dislocated vertebrae can be achieved with traction (Reindl et al., 2006). Posterior instrumentation, anterior plating, or both can be used to stabilize the fractured segments. Rapid reduction is likely favourable for neurologic recovery, however, anatomical reduction may cause retropulsion of a herniated disc into the spinal canal. The presence of a herniated disc can be ruled out with pre-operative M R I but the delay introduced (1-2 hrs) may compromise the potential for neurologic recovery afforded by the rapid vertebral reduction and decompression of the spinal cord (Hart, 2002). 1.3.2.3 Distraction Distraction injuries are characterized by tensile forces acting on vertebral bodies, facet capsules, or posterior ligaments (Figure 1.13C). Although pure axial distraction of the vertebral column is routinely used clinically to restore vertebral alignment, this type of injury is not typically encountered in accidental trauma (Iencean, 2003). Rather, distraction between vertebrae usually accompanies other types of injury. In the mechanistic classification proposed by Al len and Ferguson (Allen et a l , 1982), distractive tensile forces are present in mechanisms such as distractive flexion, compressive extension, and distractive extension. In the more recent classification proposed by Magerl et al. (Magerl et al., 1994), flexion-distraction and hyperextension-shear injuries both incorporate aspects of tensile distraction forces on the vertebral column. Due to these combinations, the role of distraction in traumatic SCI has been difficult to isolate and remains poorly understood. In contrast to burst fractures and vertebral dislocations, vertebral distraction does not necessarily occlude the spinal canal. A s such, the biomechanics of force transfer between the vertebral column and spinal cord is unclear. A distraction injury of 3cm between C5 and C6 has been associated with complete spinal cord rupture (Schauer and Sokolove, 2003). Spinal cord stretching has been proposed as the underlying cause of non-contiguous SCI that extends over several vertebral levels (Silberstein and McLean, 1994). In tethered cord syndrome, vertebral distraction does not occur but thickening of the filum terminalis results in stretching of the lumbosacral cord which causes motor and sensory deficits of the lower limbs (Yamada et al., 2004). In the cervical and thoracic region, tension may occur between the brain and nerve roots (Cusick et al., 1982). During surgical distraction, somatosensory evoked potentials are frequently monitored as an indication of spinal cord integrity. Excessive distraction results in the loss of these 17 Chapter 1 Introduction evoked potentials due to a combination of ischemia and direct mechanical stretching of axons (Cusick et al., 1982). The biomechanics of distraction injuries and their clinical treatment are similar to that already outlined above for dislocation injuries with a tensile component. 1.3.2.4 Other Vertebral Column Injuries There are other types of spinal column injury than the three highlighted above. Vertebral compression fractures may occur without the retropulsion of bone into the spinal canal. Lateral flexion injuries are a distinct classification in both the cervical and thoracolumbar spines (Allen et al., 1982; Ferguson and Al len , 1984). Upper cervical injuries such as atlato-occipital dislocations are also important, though they are usually lethal (Horn et al., 2007) and hence, less relevant for SCI treatment. A problem of utilizing the mechanism of injury to classify spinal fractures is that the mechanism needs to be inferred from post-traumatic imaging and some have advocated that a morphological classification may remove the need to make this inference (Schweitzer et al., 2006). 1.3.3 Spinal Cord Injury Mechanisms The different types of vertebral column failure result in distinct spinal cord injury mechanisms. Vertebral burst fracture results in a contusion injury mechanism where the cord is transiently compressed in a direction that is transverse to its cranial-caudal axis (Figure 1.14A). Vertebral dislocations and fracture-dislocations shear the spinal cord between adjacent vertebral segments. The term dislocation injury mechanism w i l l be used in this thesis to denote the spinal cord shear injury caused by the anterior-posterior subluxation of vertebrae (Figure 1.14B). The term shear has not been selected in this thesis as there wi l l come a day when it is possible\u00E2\u0080\u0094indeed preferable\u00E2\u0080\u0094to discuss the local shear stress between gray and white matter, the shear strain near a blood vessel, or the shear strength of myelin lamellae; quantities that wi l l be present in all types of injuries. Vertebral distraction results in a distraction injury mechanism that imparts tensile forces to the spinal cord (Figure 1.14C). Local tensile forces w i l l also be present in other injury mechanisms hence the use of the term distraction. In this way, contusion, dislocation, and distraction, SCI mechanisms are defined here to tie-in with vertebral injuries that may be readily classified via radiographs and M R I . 18 Chapter I Introduction Contusion Dislocation Distraction Figure 1.14: Spinal cord injury mechanisms A. Vertebral burst fracture injures the cord by a transverse contusion mechanism. B. Fracture-dislocation injures the cord by a shearing dislocation mechanism. C. Vertebral distraction injures the cord by a tensile distraction mechanism. 1.3.4 Pathology of Human Spinal Cord Injury Acute human SCI is typically characterized by hemorrhage, necrosis, and edema (Hayes and Kakulas, 1997) that is detectable with magnetic resonance imaging (MRI). On T2-weighted MRIs , hemorrhage appears dark while edema appears bright (Bondurant et al., 1990; Hackney et al., 1994). Blood-borne monocytes infiltrate the SCI lesion where they transform into phagocytic macrophages that have still been found at the lesion five months post-trauma where their cranial-caudal distribution follows the protracted course of Wallerian degeneration of disconnected axons (Croul and Flanders, 1997). Over time, necrosis evolves into a cystic cavity surrounded by a fibrous astrocytic scar (Croul and Flanders, 1997; Hayes and Kakulas, 1997) though the extent of astrogliosis has been reported to be milder in humans than animals (Norenberg et al., 2004). On occasion, a fluid filled syrinx may also form that requires drainage. Evidence of spared peripheral white matter has been consistently reported even in cases of complete neurological deficits (Hayes and Kakulas, 1997). These spared fibers are often demyelinated, and consequently dysfunctional, though remyelination from Schwann cells originating from the peripheral nervous system has been observed (Norenberg et al., 2004). Functional recovery following SCI is frequently assessed using the American Spinal Injury Association (ASIA) impairment scale. Approximately half of all patients exhibit incomplete neurological damage with residual motor and sensory function (i.e. A S I A B-D) . Intramedullary hemorrhage is predictive of greater neurologic deficits (Schaefer et al., 1989; Bondurant et al., 1990; Ramon et al., 1997). Spinal cord edema without concomitant hemorrhage has been 1 9 Chapter 1 Introduction associated with better outcomes unless the edema spans more than one spinal segment (Schaefer et al., 1989). The cranial-caudal extent of the lesion holds greater functional significance in the cervical region (Hedel and Curt, 2006) where the gray matter contains the phrenic nuclei that control respiration and the motoneurons that innervate the upper limbs. Thoracic lesions between T l and T12 result in similar A S I A motor scores (-50/100) whereas each ascension in lesion level from C8 ( A S I A -50/100) to C4 ( A S I A -10/100) results in a decline on 10 points per level (Hedel and Curt, 2006). In humans, some have reported the cranial-caudal extent of the lesion spans several vertebral segments (Hayes and Kakulas, 1997), while others have reported a relatively narrow lesion that would be more favourable for axonal regeneration (Tuszynski et al., 1999; Norenberg et al., 2004). The influence of the mechanism of SCI on the ultimate functional outcome of patients remains unclear. In a series of 1687 cervical spine injuries, Kiwerski (Kiwerski, 1991) found that neurologically complete lesions were more strongly associated with a flexion mechanism (364/651 or 56% of complete injuries) which was partitioned into two groups: flexion with fracture (n = 96 complete injuries) and flexion with dislocation (n = 268 complete injuries). Within injury mechanisms, however, burst fractures (referred by Kiwerski as crushing compression) were more likely to result in neurologically complete injuries (149/195 or 76% of all burst fractures were complete). B y comparison, flexion injuries (with fracture or dislocation) resulted in complete lesions in 364 of 817 (45%) patients. In a post-mortem analysis of 20 sports related injuries, Hayes and Kakulas described two fracture-dislocation injuries that resulted in a lesion extending over several vertebral segments (Hayes and Kakulas, 1997). In one C3/4 fracture-dislocation, the patient died at 14 days post-trauma and the lesion was found to span approximately 10cm between C I and C6. In a C4/5 fracture-dislocation where the patient died at 9 days post-trauma, a central hematoma was observed over 4 to 5 segments. In a more recent study, neurologic deterioration due to ascension of the SCI lesion was observed in 12 of 186 (6.0%) patients (Harrop et al., 2001). Nine of the patients had a subluxation or facet dislocation. The neurologic deterioration for these patients was attributed to traction and hypotension rather than the mechanism of injury. 20 Chapter 1 Introduction 1.3.5 Clinical Spinal Cord Treatment A n understanding of the characteristics of vertebral column injury has been used to specifically target treatment paradigms towards repairing the damaged osteoligamentus structures. Bone grafts, interbody cages, and anterior plates can be used to repair fractures of the vertebral body. Similarly, posterior instrumentation such as wires and rods can restore the stability that is lost by the rupture of posterior ligaments and the fracture of posterior elements (Pitzen et al., 2003; K w o n et al., 2006). Hence treatment for the vertebral column differs to accommodate the pattern of structural failure, however, regardless of the mechanism of spinal cord injury treatment for the cord remains essentially the same. 1.3.5.1 Decompression Spinal cord injury in humans is frequently accompanied by persistent spinal canal occlusion from fractured or dislocated vertebral elements which can compress the spinal cord. Intuitively, this residual compression should exacerbate cord injury due to factors such as protracted ischemia and therefore should be rapidly relieved via anatomical reduction or decompressive surgery. Clinically, however, no definitive evidence exists to prove the benefit of early decompression (Fehlings et al., 2001). Some have argued the neurologic damage is caused by the initial mechanical insult and is thus irreversible regardless of the timing of decompression (Boerger et al., 2000). Indeed, the maximum dynamic spinal canal occlusion during a burst fracture has been reported to be 85% greater than that measured from post-traumatic radiographs (Panjabi et al., 1995) indicating that radiographs may vastly underestimate the extent of the initial mechanical injury. In experimental animals, early decompression has consistently improved post-traumatic locomotor function (Dolan et al., 1980a; Guha et al., 1987; Delamarter et al., 1995; Dimar et al., 1999) and somatosensory evoked potentials (Delamarter et al., 1995; Carlson et al., 1997b; Carlson et al., 1997a). Delamater et al. (1995) found recovery in dogs i f decompression was performed within one hour, but no recovery i f decompression was delayed until 6 hours after the onset of compression. Dimar et al. inserted spacers of different sizes into the spinal canal of Sprague-Dawley rats following a weight-drop contusion injury and found improved locomotor scores for decompression within 2 hours and poorer function for canal occlusions of 35% or greater. 21 Chapter I Introduction The disparity between the experimental and clinical literature remains unresolved. The clinical definition of early decompression may be 24 hours whereas in the experimental setting, shorter time-periods have typically been analysed. In addition, it is possible that the current animal models do not reflect the high injury velocities and broader spectrum of injury mechanisms encountered clinically. In burst fractures that are stable, a canal encroachment greater than 50% may benefit from decompression whereas smaller intrusions may be treated conservatively (Agus et al., 2004). In bilateral facet dislocations, retrospective evidence suggests possible neurological improvements with early reduction (La Rosa et al., 2004). 1.3.5.2 Pharmacotherapy To date, the only treatment that has been widely used clinically is high dose methylprednisolone (MP) though its application has declined amidst intense controversy regarding its efficacy (Hurlbert, 2000; Sayer et al., 2006). Hemorrhagic necrosis, inflammation, and oxidative stress had long been postulated to exacerbate the primary spinal cord lesion (Hall and Braughler, 1982). Several animal studies have demonstrated the effectiveness of M P in attenuating the secondary inflammatory response thought to exacerbate the primary SCI lesion (Means et al., 1981), however, the first National Acute Spinal Cord Injury Study (NASCIS) found no benefit of M P in a prospective series of patients (Bracken et a l , 1984; Bracken et a l , 1985). Further experimental work then suggested higher M P doses were necessary to achieve lasting benefits (Hall et al., 1984; Braughler et al., 1987). In 1992, N A S C I S II reported a beneficial effect of M P i f it was administered within 8 hours of acute SCI (Bracken et al., 1990; Bracken et al., 1992). N A S C I S III refined the treatment protocol. Patients treated within 3 hours would receive one high dose bolus of M P , followed by M P over the next 24 hours (Bracken et al., 1997; Bracken et al., 1998). Patients treated between 3 and 8 hours would receive one high dose bolus followed by M P over the next 48 hours. N A S C I S II originally included patients admitted up to 12 hours post-trauma. The post hoc change to 8 hours has been criticized for its lack of statistical merit (Hurlbert, 2000; Sayer et al., 2006). Likewise, in N A S C I S III the 3 hour versus 3 to 8 hour stratification seemed arbitrary and again lacked statistical rigor (Sayer et a l , 2006). In both cases, however, the partitioning of patients emphasized the need for examining very early intervention within a few hours of trauma. This early therapeutic time-window is consistent with meta analyses of the clinical timing of 22 Chapter 1 Introduction decompression (Fehlings et al., 2001; L a Rosa et al., 2004). Moreover, many animal studies have shown the benefit of pharmacotherapies administered within minutes to a few hours following injury (Faden et al., 1988; M u et al., 2000; Gris et al., 2004), while studies comparing both early and late drug administration have found the latter to be ineffective (Wrathall et al., 1996; Rosenberg and Wrathall, 2001). Opponents of M P continue to argue that the neurological benefits are extremely modest while the increased risks of sepsis and pneumonia are unacceptable (Hurlbert, 2000; Sayer et al., 2006). Although Young and Bracken have argued that numerous alternative analyses show the same trends (Young and Bracken, 1992), the issue can likely only be settled with another prospective trial that identifies the optimal treatment population a priori. The amount of tissue available for pharmacological sparing is controlled by the extent of primary mechanical damage, the degree and duration of residual compression, and the timing of intervention. Other therapies have also been tested. In addition to M P , N A S C I S II tested the opiate antagonist naloxone which is thought to reduce edema and post-traumatic allodynia (Baptiste and Fehlings, 2006) but found no significant benefit (Bracken et a l , 1990). In N A S C I S III, M P was compared with Tirilazad\u00E2\u0080\u0094a 21-aminostereoid, which was hoped to reduce lipid peroxidation without the glucocorticoid side-effects of M P . Tirilazad did not show improved benefits over M P (Bracken et al., 1997). Gangliosides are thought to promote the growth of neurites and hence restore post-traumatic neurologic function (Geisler et al., 1991). A pilot clinical trial comparing monosialotetrahexosylganglioside (GM-1) to placebo showed a significant improvement of motor deficits in the lower limbs between 2 and 12 months after trauma (Geisler et al., 1991), but a larger trial failed to confirm the results (Geisler et al., 2001). 1.3.6 Summary of Human Injuries Promising experimental therapies have yet to demonstrate clear benefits when applied to human SCI. In humans, the spinal cord is injured by differing mechanisms that result from various types of vertebral column trauma. A s wi l l be discussed later in this thesis, the current animal models do not reflect this broader range of injury mechanisms encountered in the human population. In addition, evidence from clinical trials suggests earlier pharmacological treatment improves neurological recovery, thus indicating the importance of the first few hours following mechanical injury. 23 Chapter 1 Introduction 1.4 Biomechanics of Neurotrauma The rationale for comparing biomechanical mechanisms of injury stems, in part, on the understanding that most materials and structures exhibit different patterns of mechanical damage in response to the application of different forces. In neurotrauma, one of the most relevant measures is that of injury or damage. Injury, however, can manifest itself in different contexts. Damage can be defined as overt gross tissue tearing, though this only represents one extreme end of the injury continuum. Cells may be dysfunctional or die in the absence of overt mechanical tissue failure. Neural tissue is inhomogeneous and consists of an intricate architecture of cells such as neurons, astroglia, oligodendroglia, and microglia, embedded in an extracellular matrix that is interwoven with a network of capillaries. Neurons themselves are complex structures with dendritic arbors extending from neuronal somata (cell bodies) that transmit action potentials (nerve impulses) via their axonal processes, many of which are ensheathed in myelin lamellae that are punctuated by the unmyelinated nodes of Ranvier. Each cell's plasma membrane encapsulates a cytoskeleton of organized microtubules, intermediate filaments, and microfilaments. Hence, the following section outlines some of what is known about the gross mechanical response of neural tissue, then progresses to discuss the myriad injuries that occur at the tissue, cellular, and subcellular levels. The section concludes with an overview of existing methods used to model spinal cord injuries. 1.4.1 Mechanical Properties of Neural Tissue The characterization of the macroscopic mechanical behaviour of neural tissue has long confounded the experimentalist. The inherent softness of the tissue complicates attempts to grip the tissue for tests such as tensile loading while post-mortem degradation potentially alters the material properties. Consequently, the mechanical properties that have been reported vary widely and is an ongoing limitation in the field (Prange and Margulies, 2002; Kohandel et al., 2006). Neural tissue can be partitioned into gray and white matter components. The gray matter consists of neuronal cell bodies, their dendrites and axonal processes as well as supporting glia cells such as astrocytes and microglia. White matter consists of organized tracts of axons, as well as astrocytes, microglia and the oligodendrocytes which myelinate the axons and gives them their white appearance. A network of vasculature permeates neural tissue and these capillaries are more numerous in the gray matter. 24 Chapter 1 Introduction In general, the modulus is a quantity that characterizes the material's response to force and deformation. The modulus is defined by the ratio of stress (force per unit area) to strain (ratio of the change in length to the original length). Hence, a tensile modulus characterizes the relation between force and deformation to tension. Likewise, the bulk modulus is measured under confined compression, and the shear modulus is the response under shear. The modulus is an intrinsic property of the material that is independent of its macroscopic geometry. Doubling the cross-sectional area of a rod wi l l double its stiffness under axial tension, however, the modulus of the rod's material would remain the same. For neural tissue, the modulus has usually been measured grossly and hence these values represent some aggregate behaviour of the individual cellular components. Neural tissue possesses fluid components, thereby rendering it viscoelastic. Consequently, the modulus, is characterized by an elastic component and a viscous component. Deformations imparted to the elastic component are recoverable (i.e. energy stored). In contrast, viscous deformations are not recoverable unless additional force is applied (i.e energy lost). The viscous property also exhibits a dependence on the rate of deformation. Higher rates usually result in greater losses in energy. To detect these properties of energy stored, energy lost, and the rate dependence, sinusoidal excitations at different frequencies are used to cyclically displace the material and measure the resultant forces which wi l l be offset in sinusoidal phase due to viscous energy losses. This type of material test yields a complex modulus, G = G ' + iG\", where G ' is the storage modulus, G \" is the loss modulus, and i is the complex variable *J-1 . Complex numbers are a tool for representing the phase relationship of sinusoids. The in vivo tensile properties of the spinal cord has been reported by Hung et al. (Hung and Chang, 1981). After removal of the canine dura, stainless steel rings were bonded with cyanoacrylate to the lumbar spinal cord and the rings were coupled to an actuator. A t a loading rate of 0.021 mm/s, the in vivo Young's modulus of the spinal cord was found to range from 200 kPa to 300 kPa (where 1 Pascal = 1 Newton per m 2 per strain, where strain is the dimensionless ratio of the change in length to the initial length) and similar values were found by the same group in the cat lumbar spine (Chang et al., 1988). In vitro testing of human cervical spinal cords examined within 24 hours of death indicated a Young's modulus of 1020 kPa at a tensile loading rate of 0.068s\"1 that increased to 1370 kPa at a loading rate of 0.21s\"1 (Bilston and Thibault, 1996). 25 Chapter 1 Introduction Ex vivo compressive tests of unconfined cylindrical specimens obtained from human brains exhibited a modulus of -70 kPa at a sinusoidal displacement frequency of 34 H z (Galford and McElhaney, 1970). Neural tissue is usually considered to be incompressible with a bulk modulus that is much higher than either its tensile or shear modulus (Mendis et al., 1995b; Lippert et al., 2004). To determine the shear moduli, Fallenstein et al. (Fallenstein et al., 1969) applied sinusoidal (9-10 Hz) shear to in vitro human brain white matter and found the storage modulus to be in the range of 0.6 kPa to 1.1 kPa while the loss modulus ranged between 0.3 to 0.6 kPa. In a higher frequency test (up to 350 Hz), the storage and loss moduli of gray and white matter in post-mortem brains were found to be on the order of 7 kPa and 2 kPa respectively. More recently, in vitro tests of porcine brainstems tested dynamically in shear showed the anisotropic behaviour of white matter (Arbogast and Margulies, 1998). The shear modulus transverse to the direction of axonal fibers (G' - 2 kPa, G \" - 2.5 kPa at 200Hz) was significantly greater than in the other two directions ( G -1.75 kPa; G \" - 2.2 kPa at 200Hz) demonstrating the anisotropy of white matter tracts (Figure 1.15). This anisotropy is lost, however, in regions such as the corona radiata (in the brain) where the white matter tracts are not aligned along a single orientation (Prange and Margulies, 2002). 26 Chapter 1 Introduction lateral v iew anterior posterior v iew Figure 1.15: Anisotropy of neural tissue Arbogast and Margulies examined the anisotropy of neural tissue using oscillatory shear tests. A. Illustration of core samples taken from the brainstem. B. Schematic of axonal fibre orientations in the directions tested. C. Magnitude of complex shear modulus (G*) shows a greater modulus when shear is applied transverse to the parallel direction of the fibres (PT). (Graphics compiled from: Arbogast KB and Margulies SS, 1994. Material characterization of the brainstem from oscillatory shear tests. J Biomech 31:801-807. \u00C2\u00A9 Elsevier, 1994, adapted by permission.) In addition to anisotropy, the heterogeneity of gray and white matter characteristics remains unresolved. In 2001, two studies addressed this issue using different techniques (Ichihara et al., 2001; Ozawa et al., 2001). Ichihara and colleagues applied tensile tests to 2.5mm diameter biopsy specimens obtained from bovine cervical spinal cords. Over a range of strains (5% to 55%), they reported a higher tangent modulus for gray matter (~100kPa at 15% strain) compared to white matter (~38kPa at 15% strain). In addition, their gray matter specimens failed at a lower strain level (-50%) compared to white matter (-126%). In contrast, Ozawa et al. utilized a pipette aspiration technique with rabbit spinal cords and found no difference in the elastic modulus of gray and white matter (~3.5kPa). 27 Chapter 1 Introduction The estimated material properties for neural tissue span a wide range due to the experimental differences such as in vivo testing versus in vitro testing and gray matter versus white matter. A shear modulus on the order of 1 to 10 kPa appears consistent between several reports (Fallenst.Gt et al., 1969; Shuck and Advani, 1972; Arbogast and Margulies, 1998). In addition, the organized architecture of white matter results in transverse anisotropy (Arbogast and Margulies, 1998) in contrast to gray matter and regions of less organized white matter (Prange and Margulies, 2002). 1.4.2 Constitutive Models Empirical determination of the material properties of neural tissue can ultimately be used to solve for the parameters of mathematical models that relate force and deformation\u00E2\u0080\u0094the constitutive model. These constitutive relations can then be used in computational models which predict the internal stresses and strains within the neural tissue under a more general range of conditions than the simple paradigms used to initially determine the material properties. For example, in a numerical simulation of traumatic brain injuries, high shear strains were detected in the corpus callosum\u00E2\u0080\u0094the white matter tract that bridges the two cerebral hemispheres\u00E2\u0080\u0094while high tensile strains were detected in some of the bridging veins which could lead to hemorrhage (Zhou et al., 1995). Although the quantitative values generated by numerical simulations are highly dependent on the validity of the material properties (shown above to be problematic), these simulations can still provide insight into the behaviour of the brain and spinal cord during trauma that would be difficult to obtain experimentally. Several material models have been fit to experimental data of neural tissue. In linear viscoelastic models, the elastic component is proportional to the strain and the viscous component is proportional to the strain rate. Fung's quasilinear viscoelastic formulation proposes tensile experimental data can be fit to a linear elastic component that is multiplied by a relaxation function that is parameterized by exponentials (Fung, 1993). The model has been widely used (Bilston and Thibault, 1996; Darvish and Crandall, 2001). Hyperelastic models have also been used which utilize the experimental data to determine the exponents in a strain-energy equation (Mendis et al., 1995a; Meaney, 2003). More recently, biphasic models have also been applied to describe the flow of fluid out of a viscoelastic solid matrix for neural tissue under compression (Cheng and Bilston, 2007). Other modified formulations have also been reported (Mendis et al., 1995b; Davis 28 Chapter 1 Introduction et al., 2006; Kohandel et al., 2006; Velardi et a l , 2006), however, no single constitutive model has been agreed upon as the standard. The constitutive equation provides a valuable mathematical tool to describe the gross behaviour of neural tissue, however, the formulations do not capture the underlying behaviour of the tissue's individual cellular components (Fung, 1993). Microstructural models have thus been developed to provide an anatomically more meaningful sense of the tissue's behaviour. Arbogast and Margulies applied a fiber-reinforced composite formulation to model the brainstem (Arbogast and Margulies, 1999). Myelinated axons were treated as fibers in a gray matter matrix. Combining the known distribution of fibers and matrix in the brainstem (i.e. from histology) with mechanical data from shear tests (Arbogast and Margulies, 1998), they determined the relative complex moduli of myelinated axons and gray matter. The method was validated by empirically determining the mechanical properties of myelinated axons from the optic nerve which is devoid of gray matter. The innovative approach indicated a shear modulus on the order of 2kPa for fibers and \u00E2\u0080\u00940.050 \u00E2\u0080\u0094 45 0.015 0.313-60\u00C2\u00B0 Figure 2.2: Cervical vertebral clamps and dimensions Clamps closed (A) to support cervical vertebrae beneath the transverse processes (B) for dislocation (C), distraction (D), and contusion (E). Block arrows denote direction of injury vector. Dimensions shown in inches (B, F, G) were suitable for C3 to C6 of 300-350g male Sprague-Dawley rats. Suggested tolerance <0.002in. The mechanical characteristics of the novel clamping strategy were evaluated in the (weaker) dorso-ventral direction using the version designed for contusion (Figure 2.2E) with failure loads applied using the new SCI system. Eighteen cadavers of freshly euthanized (without paraformaldehyde fixation) Sprague-Dawley rats (mean \u00C2\u00B1 S.D. weight = 300 \u00C2\u00B1 42g) were used. Specimens were evenly divided into three load-to-failure groups: cervical fast ramp, thoracic fast ramp, and thoracic slow ramp. The prescribed ramp loading rates were lOOcm/s (fast) and 3cm/s (slow) applied on the intact laminae of C4 or T10. High rates were used to simulate traumatic injury speeds (Panjabi et al.. 1995; Nightingale et al., 1996) while slow rates were used to facilitate comparisons with A l l i s clamp testing. A preload of 0.1N was used to establish lamina contact. In the cervical tests, the clamp was centered at C4 with support extending to C3 and C5. In the thoracic tests, the clamp was centered at T10 with support extending to T9 and T l 1. T9 was not selected as the centre because the narrower T8 would not have been supported. 74 Chapter 2 A Novel SCI Device For all mechanical tests, the failure load was manually identified and the stiffness was defined by the steepest regression line fitted to 5 consecutive data points within the initial portion (*0.8 15 TO 0.6 - Q o \u00C2\u00B1 0.4 ro _C OJ 0.2 0 0 B o oco 0 n -23.31+5.18387* e / fm Vilioil ' < , \u00E2\u0080\u009E-23.31+5.18387i 1 + e - \" J D oo 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 Displacement (mm) Figure 2.6: Mortality rates for dislocation and distraction injuries Logistic regression analysis curves for dislocation (A) and distraction (B). Data points at 0 denote non-lethal injuries whereas lethal injuries are plotted at 1.0. Logit fit was significant for both dislocation (p=0.011) and distraction (p=0.001). The 10% acute mortality rate was found to be 2.6mm of dislocation and 4.1mm of distraction. Morphology on H & E stained sections showed primary hemorrhage predominantly localized to the gray matter following both contusion (Figure 2.7A) and dislocation (Figure 2.7B). Following dislocation, the hemorrhage volumes ranged from 0.18mm 3 to 2.49mm 3 in the different severities and were observed to extend rostrally from the C4/5 epicentre. In addition, dislocation resulted in shearing of the lateral columns (Figure 2.7C). H & E sections from distraction injuries did not show any obvious hemorrhage (Figure 2.7D). 84 Figure 2.7: Morphology stained with H & E Central gray matter damage in contusion (A) and fracture-dislocation (B). Lateral column damage was consistently observed following dislocation (C). No obvious pathology following both lethal and non-lethal distraction injuries (D). Scale bars 500um. Quantitative analysis of hemorrhage following 1.1mm contusion indicated a mean volume of 0.82 \u00C2\u00B1 0.52mm 3. The variation in the initial touch position (0.7 \u00C2\u00B1 0.2mm) used to displace the C S F between the dura and spinal cord did not significantly correlate with differences in the contusion hemorrhage volume (r2 = 0.27, p = 0.190; Figure 2.8A). Instead, the contusion force exhibited a significant positive correlation with the hemorrhage volume (r2 = 0.51, p = 0.048; Figure 2.8B). In the dislocation model, no significant correlation was found between the displacement and hemorrhage volume (r = -0.45, r 2 = 0.21, p = 0.119, Figure 2.8C). For dislocations greater than 2.6mm, decreased hemorrhage volumes were observed. There was no correlation between the dislocation force applied to the vertebrae and the hemorrhage volume (r = 0.26, r 2 = 0.07, p = 0.391). 85 Chapter 2 A Novel SCI Device r2 = 0.51, p = 0.048 2.4 26 28 3.0 3,2 3.4 3.6 Dislocation Displacement (mm) 16.0 180 20.0 220 24.0 26.0 28.0 Peak Dislocation Force (N) 32C Figure 2.8: Regression curves for hemorrhage versus injury displacement and force A. Contusion severity was fixed at 1.1 mm but variation was caused by viscoelastic relaxation of the spinal cord when determining the initial touch position on dural surface. This variation in touch position did not significantly account for variability in acute hemorrhage volumes. B. Significant correlation was found between hemorrhage volume and peak contusion force. C. As dislocation severity increased beyond 2.5mm, hemorrhage volume decreased. D. The dislocation force applied to the vertebrae was not correlated with the intramedullary hemorrhage severity. Dotted lines mark 95% confidence intervals. Different patterns of white matter damage were observed on sections immunostained for the neuronal cytoskeleton with neurofilament 200 and B-Tubulin III. A t low levels of dislocation (Figure 2.9A), the dorsal and ventral columns were predominantly spared while damage was localized to the gray matter (asterisk Figure 2.9A) and the axons bordering this region. At higher magnitudes of dislocation, primary axotomy was observed to extend outward from the gray matter into the dorsal column (Figure 2.9B). Dislocation injury also produced axotomy of fibers in the lateral funiculi (Figure 2.9C & arrowheads in D). In contrast, after distraction injuries there were only minor alterations in the neurofilament and B-Tubulin III staining patterns without prominent localized tissue tears or axotomies (Figure 2.9E). In the contusion model, the ventral and dorsal funiculi in most animals were largely spared with damage concentrated centrally (asterisks Figure 2.9F). With deeper touch positions, axotomies were also observed in dorsal funiculi following contusion (data not shown). 86 Chapter 2 A Novel SCI Device Dislocation 2.4 mm Dislocation 3.0 mm caudal Dislocation Lateral Column caudal Distraction 4.1 mm caudal \u00E2\u0080\u0094 ID \u00C2\u00A7\u00E2\u0080\u00A2 Contusion jjj 1.1 mm caudal caudal Figure 2.9: White matter damage following three injury mechanisms Representative parasagittal photomicrographs of white matter immunostained simultaneously for neurofilament 200 (large axons) and P-Tubulin III (fine axons). Near the midsagittal plane, lower magnitudes of dislocation (A) grossly sparred the dorsal and ventral white matter with damage originating centrally in the gray matter (asterisk in A). At higher magnitudes of dislocation (B), primary damage extended outward into the deep dorsal column. Fracture-dislocation at C4/5 sheared axons in the lateral funiculi (C). Arrowheads in D highlight axotomy line. Distraction injuries did not produce gross primary axotomy (E). Peripheral white matter was generally sparred following contusion (F) with damage focused in the gray matter (asterisks F). Scale bars 250um except in D, 50um. 87 Chapter 2 A Novel SCI Device 2.4 Discussion The objective of this study was to develop a novel device for the experimental modelling of distinct clinically relevant mechanisms of SCI. In part 1, novel vertebral clamps were developed and shown to have a high stiffness (i.e. low vertebral slippage or deflection when force was applied) thereby demonstrating the devices' utility for delivering accurate injury displacements. In addition, the clamping strength (i.e. high failure load) was found to be sufficient to reliably produce high-speed cervical injuries. In part 2, the new devices were used to model contusion, dislocation, and distraction injuries. Injuries were parameterized by controlled displacements rather than forces because in the dislocation and distraction models, forces were applied to the vertebral column rather than to the spinal cord. Moderate-severe injury severities were determined for the dislocation (2.6mm) and distraction (4.1mm) models based on 10% mortality rates that are comparable to rates reported for 0.95-1.1mm cervical contusions (Pearse et al., 2005). In our contusion model, variation in the impactor's contact position on the dura was found to be small (SD = 0.2mm), and did not correlate with intramedullary hemorrhage volumes, suggesting this variation is an acceptable drawback of displacement controlled spinal cord contusion. Primary axotomy in the lateral funiculi was a distinctive feature of dislocation injuries, while contusion and dislocation shared similar patterns of central gray matter damage. Although several reliable injury models are already in widespread use, the methods reported here enable the investigation of distinct biomechanical injuries encountered clinically and hence provide additional pre-clinical paradigms for assessing promising therapies for specific types of injury. Although the new vertebral clamp design was shown to have optimal mechanical characteristics for producing cervical injuries, we could not draw direct comparisons with Al l i s clamps. The mechanical testing was performed without the typical contusion model laminectomy. Consequently, the measured failure loads could potentially be higher than with the laminectomized vertebrae. In our hands, A l l i s clamps tested on intact spinal columns resulted in early failure (<10N) thereby demonstrating the necessity for novel clamps for the dislocation and distraction models. Tests of the new clamp in the cervical spine resulted in fracture of the loaded lamina rather than the pedicle (clamping axis) indicating a conservative estimate of the clamping strength. These failure loads (~65N) were found to be sufficiently high to enable the production of fracture-88 Chapter 2 A Novel SCI Device dislocation (<31N) and importantly, suggest this clamping method may be adapted as a viable post-traumatic stabilization strategy for longer-term survival studies. Clinically, intramedullary hemorrhage is a strong predictor of poor neurological outcome (Bondurant et al., 1990; Ramon et al., 1997), though in this study, hemorrhage volumes only correlated with the contusion force. The lack of correlation between contusion displacement and hemorrhage (Figure 2.8A) stems from the relatively narrow variation in the impactor's contact position (0.7 \u00C2\u00B1 0.2mm) and thus is a favourable finding in this context - suggesting this undesirable variation has minor effects. In the dislocation model, the injury displacement controls mortality (Figure 2.6A, p=0.011) but unexpectedly did not linearly correlate with hemorrhage volumes. Post hoc observations indicate, counter-intuitively, that hemorrhage declines at greater severities (Figure 2.8C) but this may reflect hypotension and hypoperfusion caused by the more severe SCIs (Guha and Tator, 1988; Guha et al., 1989). Hence, while hemorrhage is a clinically useful indicator of SCI severity, its utility can potentially be confounded by a reduction in vascular perfusion. The vertebral dislocation force did not correlate with the hemorrhage volume suggesting the model could be improved with additional intramedullary pressure sensors. The lack of direct visualization of cord-column interaction is a limitation of the closed vertebral column models (i.e. dislocation and distraction). Radiographic studies in the cat suggest a cord-column coupling ratio of approximately 0.5 to 0.8 between the cord and cervical vertebral column (Maiman et al., 1989a) however it is unclear whether this ratio is the same in the rat. The spinal canal diameter and the initial spinal cord tension both likely contribute to variability. In this study, the animal weights varied less than 10% and their position within the stereotactic frame was standardized to minimize these sources of variation. In addition, the surgery time was longer than that typically required for a contusion injury (20-25 minutes vs. 10-15 minutes). The primary shearing of the lateral columns (Figure 2.7C & 2.9C-D) after fracture-dislocation is distinct from the lateral white matter sparing characteristic of contusion lesions (Blight, 1988; Bresnahan et al., 1991). Both dislocation and contusion severely affect the gray matter of the spinal cord, which wi l l likely lead to the demise of motoneurons and interneurons. Our 5 minute time-window, selected to facilitate comparisons between lethal and non-lethal injuries, was too short to allow for the detection of oxidative stress and apoptotic markers (data not shown) which might be expected to evolve in the dislocation model given the similarity in central 89 Chapter 2 A Novel SCI Device hemorrhage with the contusion model (Hall and Braughler, 1986; Shuman et al., 1997; Springer et al., 1999; Beattie et al., 2002). The high hemorrhage volume reported in our dislocation injuries differs from the diffuse hemorrhage reported in lateral thoracolumbar dislocation (Fiford et a l , 2004). The difference may be attributed to the higher traumatic speed modelled here (95cm/s vs. <15cm/s) as well as the disparity in the vertebral levels (dislocation between two vertebrae [C4 and C5] versus three vertebrae [T12 and T13 and LI] ) . In addition, although the mechanical tests of the vertebral clamps suggest vertebral compliance of 0.48 \u00C2\u00B1 0.11mm at 30N, the loads at maximum displacement were substantially lower (10.9 \u00C2\u00B1 3.5N) yielding an estimate of <0.2mm of compliance, reflecting the accuracy of the dislocation magnitude. Although pure distractive injuries are less common clinically than contusion and dislocation, local tensile forces at the cellular level are widely believed to play an important role in neurotrauma (Breig, 1970; Blight, 1988; Myklebust et a l , 1988; Dabney et al., 2004; Henderson et al., 2005). It has often be posited that mechanical failure occurs along principal tensile axes in gray and white matter (Maiman et al., 1989b) producing a central cavitation following contusion (Raynor and Koplik , 1985; Blight, 1988). A distraction model could hypothetically provide a more uniform mechanical loading paradigm for modelling tensile deformation (Myklebust et al., 1988; Dabney et al., 2004; Greaves, 2004) similar to optic nerve stretch methods (Maxwell et al., 1991; Jafari et a l , 1998; Bain and Meaney, 2000). However, the distraction modelled here was lethal in the absence of overt mechanical tissue failure and hemorrhage. This appears consistent with reports of cat spinal distraction where it may be interpreted that hemorrhage was rare and cellular injury was not detected until after 24 hours (Myklebust et al., 1988; Maiman et al., 1989b). Although gross disruptions in the cytoskeleton were not detected here, other methods such as electron microscopy might reveal subtler damage (Balentine, 1978; Banik et al., 1982; Maxwel l et a l , 1991; Anthes et a l , 1995; Maxwel l and Graham, 1997; Maxwel l et al., 1997). Hence, the morbidity reported here is likely a consequence of traumatically induced depolarization of phrenic nuclei spanning C3 through C5 (el-Bohy et al., 1998). The choice of controlling contusion injuries using force or displacement feedback remains controversial (Scheff et al., 2003; Ghasemlou et al., 2005). Both parameters have correlated with experimental SCI severity (Anderson, 1985; Noyes, 1987a; Behrmann et al., 1992; Scheff et al., 90 Chapter 2 A Novel SCI Device 2003; Ghasemlou et a l , 2005). In a behavioural analysis of displacement controlled contusions, a force window of \u00C2\u00B11.3 standard deviations was necessary to isolate distinct biomechanical groups resulting in an exclusion rate of approximately 30% (Kloos et al., 2005). A n analogous displacement window has also been advocated for force controlled injuries (Ghasemlou et al., 2005). In the contusions reported here, a new method of vertebral clamping eliminated the vertebral slippage (Stokes et al., 1992; Stokes and Jakeman, 2002) that reduces impact accuracy (Gruner, 1992), and enabled us to isolate the variability of the impactor's initial contact position which we found to be minor. Instead, the relation between force and displacement is likely dominated by variation in the dura, cerebrospinal fluid, spinal cord, vascular anatomy, and perfusion pressure. Given this biological variation, numerous trials over wider severity ranges (i.e. greater than the 0.2mm variation in contact position) are required to clearly see the relation between force and displacement (Scheff et al., 2003; Ghasemlou et a l , 2005). Our contusion force, however, significantly correlated with hemorrhage (Figure 2.8B), thereby lending support for the force controlled injury paradigm and its ability to accommodate for potential differences in perfusion pressure, vascular anatomy, and i f it occurs, vertebral slippage from the clamps. In summary, we have developed a novel SCI device and demonstrated the utility of an innovative vertebral clamping method for the production of three biomechanically distinct cervical SCI models. Although cervical models are functionally more severe (Schrimsher and Reier, 1993; el-Bohy et al., 1998), regaining hand function has been identified as by far the highest priority of tetraplegic individuals (Anderson, 2004) and cervical contusions have only recently been addressed (Pearse et al., 2005; Baussart et a l , 2006; Gensel et al., 2006). The functional significance of neurons in the cervical enlargement allows for the assessment of both gray and white matter protective strategies utilizing forelimb tests which may have a higher sensitivity than hindlimb locomotor scoring (Onifer et al., 2005). The multi-mechanism system and methods are adaptable for modelling a broad spectrum of clinically relevant injuries in the cervical (Choo et al., 2004), thoracic (Sjovold et al., 2005), and thoraco-lumbar (Clarke et al., 2006) spines. 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Young W (2002) Spinal cord contusion models. Prog Brain Res 137:231-255. 96 Ch apter 3 DISTRIBUTION OF PRIMARY INJURY 3.1 Introduction The tissue damage in spinal cord injury (SCI) is typically described as a central cavitating lesion surrounded by a peripheral r im of spared white matter (Bresnahan et al., 1991). Although transection models are highly prevalent, particularly in regeneration research where spared fibers can introduce ambiguity, contusions are felt to be biomechanically similar to vertebral burst fractures and thus provide the most realistic experimental setting in which to test potential neuroprotective and regenerative strategies (Kwon et al., 2002; Rabchevsky et al., 2002; Pearse et al., 2004; Teng et al., 2004). The pattern of central damage has been consistently reported in numerous experimental animal models that injure the spinal cord by contusion and compression injury mechanisms (Noyes, 1987a; Anderson and Stokes, 1992; Gruner, 1992; Jakeman et al., 2000; Young, 2002; Scheff et al., 2003). Alternative models to contusion have lagged in development due in part to the current understanding that the secondary biochemical cascade overwhelmingly dominates the neuropathology of SCI. However, the pattern of primary mechanical injury to the spinal cord has rarely been examined in other clinical injury mechanisms such as vertebral fracture-dislocation and distraction. Spinal distraction has been studied by some (Dolan et al., 1980; Myklebust et al., 1988; Maiman et a l , 1989a; Maiman et a l , 1989b; Dabney et a l , 2004) as well as slow distraction for scoliosis correction (Salzman et al., 1988; Sarwark et a l , 1988; Salzman et al., 1991), but only one study has modelled dynamic vertebral dislocation (Fiford et al., 2004). Fracture-dislocation shears the spinal cord between adjacent vertebral levels whereas distraction injuries hypothetically stretch the cord axially. These traumatic loading conditions are mechanically distinct from the \" A version of this chapter has been published. Choo AM, Liu J, Lam CK, Dvorak M, Tetzlaff W, Oxland TR (2007) Contusion, dislocation, and distraction: Primary hemorrhage and membrane permeability in distinct mechanisms of spinal cord injury. J Neurosurg Spine 6(3): 255-266. 97 Chapter 3 Primary Injury transient transverse compression of a contusion injury. The spinal cord is comprised of heterogeneous cellular populations that are organized into a tissue structure that is mechanically anisotropic (Arbogast and Margulies, 1998). Biomechanically, one would expect the characteristics of the tissue damage to depend on the direction of trauma. It has previously not been possible to experimentally compare clinical injury mechanisms in order to identify any mechanism-specific characteristics which may be exploited to enhance treatment strategies. The multi-mechanism devices and animal models profiled in chapter 2 of this thesis enabled the comparison of primary mechanical injuries that are addressed in the current chapter. The objective of this study was to compare the primary damage in three distinct models of cervical SCI - contusion, fracture-dislocation and flexion-distraction. We focused on vascular damage and compromised membrane integrity as injuries to these structures are key primary events in the neuropathology of SCI. Hemorrhage has been widely correlated in the clinical literature with poor functional outcomes (Bondurant et al., 1990; Flanders et al., 1990; Flanders et al., 1996; Ramon et al., 1997). Meanwhile, traumatically induced increases in membrane permeability and the subsequent loss of ionic homeostasis results in a range of pathologic sequelae including signal conduction failure (Galbraith et al., 1993; Shi and Blight, 1996) and the influx of calcium ions which has been linked to myriad degenerative processes (Young, 1992; Stys, 2004; Sullivan et al., 2005). 3.2 Materials and Methods A l l procedures were approved by our institution's Animal Care Committee in accordance with the guidelines published by the Canadian Council on Animal Care. Thirty-six male Sprague-Dawley rats (mean \u00C2\u00B1 SD weight = 319 \u00C2\u00B1 26g) were divided into three injury groups (n = 9 contusion, n = 10 dislocation, n = 9 distraction) and three sham surgical control groups (n = 2 contusion, n = 3 dislocation, n = 3 distraction). Animals were anesthetized with an intraperitoneal injection of ketamine (80mg/kg) and xylazine (lOmg/kg) and maintained under deep anesthesia throughout the tests. Fluorescein-dextran with a molecular weight of lOkDa (0.375mg diluted in 15uL dH20, Molecular Probes, Eugene, OR) was infused into the cisterna magna 1.5 hours prior to injury in order to detect increases in membrane permeability (Pettus et al., 1994; Singleton and 98 Chapter 3 Primary Injury Povlishock, 2004; Stone et al., 2004). Custom designed stereotactic clamps were used to rigidly hold the cervical vertebrae between C3 and C6 laterally beneath the transverse processes. A l l injuries were produced under displacement feedback control using the SCI multi-mechanism system described in chapter 2 of this thesis (Figure 2.1). The surgical procedures are described in chapter 2 and summarized briefly here. For contusion, C4 to C5 were held as a single continuously supported unit while partial laminectomies on the caudal side of C4 and rostral side of C5 allowed a 2mm spherical head indenter to strike the cord to a depth of 1.1 \u00C2\u00B1 0.06mm (SD) to produce a moderate-severe injury (Behrmann et al., 1992; Kloos et al., 2005; Pearse et al., 2005). A touch force of 0.03N on the dural surface was used to establish the initial position (Stokes et al., 1992; Pearse etal., 2005). For fracture-dislocation, the dorsal ligaments and facets at C4/5 were removed to reduce the possibility of residual dislocation following injury. C3 and C4 were clamped together and held stationary while the vertebral clamp holding C5 and C6 was coupled to the actuator, dislocated dorsally 2.5 \u00C2\u00B1 0.16mm (SD), and then returned to its initial position. Trials over a range of displacements indicated 2.5mm was severe, but not lethal (Figure 2.6A), and the likelihood of residual dislocation was negligible. For flexion-distraction, the facets at C4/5 were removed. The animals were secured on the stereotactic frame with 15\u00C2\u00B0 of flexion at C4/5 measured using a digital inclinometer (0.1\u00C2\u00B0 resolution). C3 and C4 were clamped and held stationary while the clamp holding C5 and C6 was distracted caudally to a displacement of 4.1 \u00C2\u00B1 0.01mm (SD) and held for one second before being returned to its initial position. Preliminary trials indicated the chosen severity was severe but not lethal (Figure 2.6B). The one second dwell was introduced into the protocol to allow visual confirmation under stereomicroscopy that the vertebral clamps did not slip. In contusion and dislocation, dorso-ventral clamp slippage was not possible without fracture of the transverse processes. Immediately following dislocation and distraction, the vertebral clamps were themselves clamped together to prevent further injury at the now unstable C4/5 joint. Sham injured controls underwent identical surgical procedures and were secured within the stereotactic frame. For contusion, the same touch load was applied then removed. For dislocation and distraction the caudal vertebral clamp was coupled to the actuator without the injury stroke being applied. 99 Chapter 3 Primary Injury Animals were transcardially perfused with 250mL phosphate buffered saline followed by 500mL of freshly hydrolyzed cold 4% paraformaldehyde at 5 minutes post-trauma in order to analyse the primary injury. Spinal cords were post-fixed overnight in 4% paraformaldehyde and cryoprotected in graded sucrose (12, 18, 24% in phosphate buffer) before being frozen in isopentane cooled with dry ice. Cords were cryosectioned at 20pm parasagittally. One set of slides from all animals was stained with hematoxylin and eosin (H&E) for hemorrhage analysis. Fluorescein-dextran was immediately visualizable under epifluorescent microscopy and slides from all animals were also immunostained with mouse anti-NeuN (Serotec, Raleigh, N C ) to colocalize dextran-positive neuronal somata. A subset of slides was immunostained with rabbit ant i-Kvl .2 (Alomone Labs, Jerusalem, Israel) to detect potassium channels at the nodes of Ranvier. For immunohistochemistry, sections were washed 3 X 5 minutes in 0.01M phosphate buffered saline (PBS). Sections were blocked for 30 minutes in normal donkey serum before being incubated for 3 hours at room temperature in the primary antibody diluted (1:100 mouse anti-NeuN; 1:500 rabbit anti-Kvl.2) in 0.01M PBS with 0.1% Triton X-100). Sections were then washed 3x5 minutes in 0.01M PBS and incubated in Cy3-conjugated donkey anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, P A ) . Sections were washed 3x5 minutes in 0.01M PBS then mounted in ProLong anti-fade (Molecular Probes) and sealed with nail polish. For hemorrhage analysis, sections were imaged using a 5 X objective on an AxioPlan2 microscope (Carl Zeiss, Thornwood, N Y ) equipped with monochrome camera with R G B unit (Retiga E x i , Qlmaging, Burnaby, B C ) using Northern Eclipse acquisition software (Empix Imaging, Mississauga, ON). Volumes were calculated using Cavaleri's method(Howard and Reed, 1998) with a 50x50um 2 spacing of point probes on sections (14 to 18 per animal) spaced 160pm apart. The area root-mean-square measurement accuracy of this probe spacing was found to be 5.6% compared to manual tracing of 20 sections. Fluorescein-dextran and NeuN were imaged with a 20x objective. The ventral gray matter and four white matter columns (dorsal, lateral, ventro-lateral, ventro-medial) were imaged at 1mm intervals from (-) 5mm caudal to (+) 5mm rostral to the apparent lesion epicentre using a motorized scanning stage (Scan 100x100, Marzhauser, Wetzlar-Steindorf, Germany; M A C 5 0 0 0 controller, Ludl , Hawthorne, N Y ) . For each specimen, 13 series (3xventral gray, 2xdorsal, 100 Chapter 3 Primary Injury 2> v-\u00C2\u00BB , | V \ 1 . 1 r n m \ ^ contusion * * Dura impact \u00E2\u0080\u0094 \u00E2\u0080\u00A2 1 1 \u00E2\u0080\u0094t\u00E2\u0080\u0094 Cord impact j i \u00C2\u00AB \u00E2\u0080\u00A2 Disp mm Possible f4 > endplate failure/ , 1 \u00E2\u0080\u0094 Force N \" \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 VelxlOcm/s - Accel xlOG / l 2.5mm dorsal dislocation / X / o f C5 relative to C4 ft\" \u00E2\u0080\u0094 \u00E2\u0080\u00A2V\" * # V V \ y 15.005 5.015 t ime (s) Distraction 5.02 B 3.004 3.006 3.008 3.01 3.012 3.014 3.016 3.018 3.02 time (s) Distraction Disp mm Force N Vel xlO cm/s Accel xlOG one second dwell to verify no rostro-caudal clamp slip 3.004 3.006 3.008 3.01 3.012 3.014 3.016 3.018 3.02 time (s) 3.4 3.6 t ime (s) Figure 3.3: Representative mechanical injury curves A) Dashed line at 0 shows initial touch (0.03N) position which dimpled the cord. The actuator was retracted from the dimpled position and began its downward acceleration from 6mm above the cord. The compressive load prior to Omm shows impact with dura. B) Fluctuations in force curve during dislocation reflected the failure of different soft-tissues and structures such as the C4 endplate. C) Highest forces were observed in distraction. The initial decline in force at peak displacement was due to the continuing caudal motion of tissue producing compression after the actuator had stopped. Increasing the timescale (D) shows the load increased again before exhibiting viscoelastic decay. Note, y-axis units are listed beside labels. \u00C2\u00A9 Journal of Neurosurgery, 2007, by permission. Contusion and dislocation injuries produced similar patterns of hemorrhage concentrated in the gray matter while no appreciable hemorrhage was observed following distraction (Figure 3.4). Contusion resulted in a mean hemorrhage volume of 1.03 \u00C2\u00B1 0.3mm 3 (SEM) while a similar volume was found for dislocation (1.08 \u00C2\u00B1 0.2mm 3 [SEM]). 104 Chapter 3 Primary Injury Distraction Figure 3.4: Primary hemorrhage volumes following three injury mechanisms Bar graph and H&E photomicrographs showing that hemorrhage was concentrated in the gray matter following contusion and dislocation but not distraction. Mean hemorrhage volumes and SEMs (nine animals per injury group; eight combined surgical shams) were calculated using point probes and Cavalieri's method. Scale bar 250um. \u00C2\u00A9 Journal of Neurosurgery, 2007, by permission. Qualitatively, different patterns of membrane compromise were observed between the three mechanisms. Following contusion (Figure 3.5, top panel), the extent of increased membrane permeability to fluorescein-dextran was generally localized to the lesion epicentre in the gray matter (Figure 3.5E arrowhead) and white matter (Figure 3.5B, H arrowheads). Distal to the contusion lesion, dextran was observed to be excluded from neuronal somata (Figure 3.5D, F arrows) and axons (Figure 3.5A, C , G , I). In this region, dextran accumulated at the nodes of Ranvier (Figure 3.5, top panel, vertical arrows) and in the vascular endothelium (Figure 3.5, top panel, \"v \") similar to uninjured tissue (data not shown). A transition zone between 1mm and 2mm distal to the lesion was observed where neither dextran-positive axons, nor dextran accumulations at the nodes of Ranvier, were clearly visible suggesting that the myelin sheath had been compromised without axonal damage (Anthes et al., 1995). 105 Chapter 3 Primary Injury Caudal Lesion Rostral c o \u00C2\u00ABS1 3 c o U \ A T B V \ C V D E ~ * V F _ T G H 1 T T T J K 1 M \" P * N T Q V 0 _ T R c u re Q V 1 \u00E2\u0080\u00A2 \ ? s T U V W ^ \u00E2\u0080\u00A2 X _ T T Y Z A A c o \u00E2\u0080\u0094* o o Q rf 3 < \u00E2\u0080\u0094 Figure 3.5: Axonal membrane compromise detected with fluorescein-dextran Photomicrographs demonstrating that membrane compromise evidenced by permeability to fluorescein-dextran was limited to the lesion segment following contusion and extended rostro-caudally following dislocation and distraction. Representative parasagittal images taken from 3mm caudal, the lesion epicentre, and 3mm rostral in the dorsal column, ventral gray matter, and ventro-medial column. Arrows and arrow heads mark one example per image tile. Dextran was observed to accumulate at the nodes of Ranvier of axons with intact membrane integrity (vertical arrows). Neuronal somata excluding dextran appeared dark relative to the background (horizontal arrows). Dexfran-filled axons (vertical arrow heads) and neuronal cell bodies (horizontal arrow heads) indicated membrane compromise. Immediate necrosis (*) was observed at the lesion following contusion and dislocation. Dextran also enveloped the vascular endothelium highlighting blood vessels (v). Scale bars 50um. \u00C2\u00A9 Journal of Neurosurgery, 2007, by permission. 106 Chapter 3 Primary Injury The dislocation lesion epicentre appeared similar to that of contusion with areas of necrosis in the gray matter (Figure 3.5N asterisk) and some dextran-positive neuronal somata (Figure 3.5N arrowhead) as well as many dextran-positive axons in the white matter (Figure 3.5K, Q arrowheads). In contrast to contusion, membrane compromise after dislocation was qualitatively observed to extend several vertebral levels rostrally (Figure 3.5L, O, R arrowheads) and caudally (Figure 3.5J, M , P arrowheads) to the apparent epicentre. In addition, consistent primary axonal damage was observed in the lateral columns. Membrane compromise following distraction appeared more diffuse than in either of the other two mechanisms. Necrosis was not observed at the lesion epicentre (Figure 3.5W) in contrast to the other two models (Figure 3.5E, N) . Some neuronal somata at the epicentre were dextran-positive (Figure 3.5W, arrowhead) and similar to dislocation, elevated membrane compromise was encountered to extend rostro-caudally in the gray matter (Figure 3.5X, V , arrowheads). We noticed fewer dextan-positive axons, in the dorsal and ventral white matter at the distraction centre (Figure 3.5T arrow) in contrast to contusion and dislocation (Figure 3.5B and K ) . Similar to dislocation, axonal membrane compromise in the ventral and ventro-lateral columns was observed to extend further rostrally (Figure 3.5U, A A arrowheads), however, not caudally (Figure 3.5S, Y arrows). Interestingly, in the distraction model we found regions without dextran-positive axons yet lacking the typical dextran accumulations at the nodes o f Ranvier reminiscent o f the transition zone flanking the contusion lesion. Quantitative analysis detected statistically significant interactions between the three mechanisms of injury and the rostro-caudal profile of membrane compromise in all gray and white matter columns (p<0.001 for interaction in all A N O V A s ) . Comparisons in the two ventral columns (Figure 3.6C, D) indicated contusion and dislocation exhibited significantly more axolemma compromise than distraction at the lesion epicentre (p<0.015). However, the pattern was different at 5mm rostral where more dextran-positive axons were detected following dislocation and distraction than resulting from contusion (p<0.026). In the dorsal column (Figure 3.6A), more dextran-filled axons were counted at the contusion and dislocation epicentres than in distraction (p<0.001). Distraction resulted in a greater loss of membrane integrity at 5mm rostral in the lateral column (Figure 3.6B) relative to the other two mechanisms (p<0.001). 107 Chapter 3 Primary Injury Dorsal Lateral Contusion -m- Dislocation Distraaion Sham - 2 0 2 << Caudal - (mm) - Rostral >> - 2 0 2 << Caudal - (mm) - Rostral >> Figure 3 .6 : Quantitative measurements of dextran-positive axons Line graphs showing the asymmetric rostro-caudal counts of dextran-positive axons. Zero denotes lesion epicentre. Systematic random sampling was used to quantify axons within 250um of the superficial tissue border. Fluorescein-dextran diffusion past this boundary was inconsistent. Insets highlight sampled regions. Whiskers denote SEM. \u00C2\u00A9 Journal of Neurosurgery, 2007, by permission. Post-hoc analysis revealed distinct rostro-caudal boundaries of axolemma compromise in the ventral white matter (Figure 3.6C-D). In contusion, a significant increase in dextran-positive axons was detected spanning -1mm to + lmm when compared to boundaries at \u00C2\u00B12mm (p<0.001). In dislocation, the degree of axolemma compromise increased between -3mm and -2mm (p=0.001) and remained significantly elevated, although declining, towards 5mm rostral of the lesion (p<0.008 for all comparisons relative to -3mm). Following distraction, significant membrane permeability was seen between -1mm and the lesion epicentre (p=0.028) and maintained a similar level out to 5mm rostral. In the dorsal column (Figure 3.6A), axonal damage after distraction appeared to have a bimodal profile with peaks at + lmm (11 \u00C2\u00B1 2axons/mm [SEM]) and +3mm (5 \u00C2\u00B1 2axons/mm [SEM]). However, post hoc analysis of the depression at +2mm (2 \u00C2\u00B1 laxons/mm [SEM]) was not detected as significantly lower than at + lmm (p=0.061). Sham animals exhibited no membrane compromise in the white matter (Figure 3.6). 108 Chapter 3 Primary Injury In the gray matter, contusion and dislocation resulted in greater plasma membrane compromise than distraction at the lesion epicentre (p<0.036, Figure 3.7A). Dextran-positive neuronal somata were detected rostrally following dislocation and distraction but the counts were not statistically greater than those in contusion (p>0.139). Some increased membrane permeability was found in the gray matter extending from 3 mm caudal to 1mm rostral of the sham lesion at C4/5 (Figure 3.7A). Cel l bodies morphologically appearing like neurons or larger astrocytes filled with dextran but immunostaining negative for NeuN (Figure 3.2D & H) generally constituted less than 5% of total counts (Figure 3.7B). Peaks of 10.2 \u00C2\u00B1 3.5% (SEM) and 5.6 \u00C2\u00B1 2.9% (SEM) were found at 2mm rostral to the dislocation and contusion lesion respectively. Contusion and dislocation exhibited peaks at \u00C2\u00B12mm while the peak in distraction was observed at the lesion epicentre. Neurons that were not distinguishable from the background (Figure 3.2C & G) and hence classified as neutral accounted for 42.1 \u00C2\u00B1 7.3% (SEM) to 55.8 \u00C2\u00B1 9.1% (SEM) of the counts in sham control animals. This ambiguous population was considered a result of random sectioning of background tissue above and below neuronal somata and hence was not analysed further. Dext ran Posi t ive & N e u N Posi t ive Dext ran Posi t ive & N e u N Nega t i ve 100 ao 2 60 U o # 40 20 -\u00E2\u0080\u00A2- Contusion 14 -m- Dislocation Distraction 12 i A Sham - 2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral >> - 4 - 2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB F i g u r e 3 .7 : Q u a n t i t a t i v e m e a s u r e m e n t s dex t r an -pos i t i ve ce l l bod ies Line graphs showing the distinct rostro-caudal patterns of dextran-positive neuronal somata. Zero denotes apparent lesion epicentre at C4/5. Counts were normalized to total number of cells counted (NeuN positive + cells with neuronal/large astroglial morphology negative for NeuN) per animal per rostro-caudal location. A) colocalization of dextran-positive cells and NeuN immunostaining. B) dextran-positive cells with neuronal morphology but negative for NeuN. Whiskers denote SEM. Insets show example classifications. \u00C2\u00A9 Journal of Neurosurgery, 2007, by permission. 109 Chapter 3 Primary Injury 3.4 Discussion The objective of this study was to experimentally compare three clinically relevant mechanisms of primary spinal cord injury at 5 minutes post-trauma in order to identify differences which may potentially be exploited to guide future clinical paradigms. Hemorrhage and cellular membrane compromise were used as indicators of primary damage since their central role in the initiation of subsequent neuropathology has been well established. The results showed each injury mechanism produced distinct rostro-caudal distributions of primary membrane compromise in both the gray and white matter. Contusion caused localized increases in membrane permeability while fracture-dislocation and flexion-distraction resulted in asymmetric membrane compromise with increased rostral damage. Hemorrhage was similar between contusion and dislocation although not evident following distraction. There were several limitations to this study. First, given the short survival time, we were not yet able to assess the evolution of the ensuing secondary degeneration nor to determine convergence or divergence o f the injury patterns observed. Since the variable under control was the biomechanical injury paradigm, early sacrifice was necessary to enable us to map the initial spatial distribution of mechanically induced damage while limiting confounding secondary events. In addition, without long-term survival data, it remains unclear whether identical injury severities were produced in the three models. Ideally, behavioural measures showing similar functional deficits would be the most clinically relevant demonstration of comparable severities. However, as dislocation and distraction in the rat cervical spine were novel models, a survival study at this stage was considered premature without first establishing that meaningful differences existed to ethically warrant longer survival times. Instead, the lethal thresholds for dislocation and distraction were used as surrogate measures of function and the injury magnitudes in this study were reduced to a level estimated as moderate-severe. Although hemorrhage was not observed following distraction, lethal injuries in graded severity studies also did not exhibit obvious hemorrhage. The use of techniques such as the detection of molecular tracer extravasation across the compromised blood-brain-barrier could have increased the sensitivity of detecting vascular injury following distraction (Noble and Maxwel l , 1983; Noble and Wrathall, 1988; Popovich et al., 1996). Recognizing these limitations of a short survival, the study design and analysis focused on generating a detailed rostro-caudal profile in several regions to detect consistent patterns rather than focusing on the 110 Chapter 3 Primary Injury absolute count magnitudes. Alterations in severity would change the individual number of dextran-positive neurons or axons, but the rostro-caudal distributions and differences between tracts would likely remain similar. Second, the diffusion depth of the lOkDa fluorescein-dextran used in this study prevented analysis of deeper axonal populations. A smaller molecular-weight dextran may have increased the penetration depth, however, in vitro data in the literature had indicated the ability of 1 OkDa to discriminate moderate-severe stretch levels whereas smaller dextrans were able to penetrate uninjured control cells (Geddes et al., 2003). Third, the counts of dextran-positive neuronal somata and axons have a limited resolution. Extracellular tissue above and below axons and cell bodies appears to obscure the fluorescent dextran signal. This phenomenon likely introduced some bias by reducing the count sensitivity of small neurons, fine axons, and cells with \"milder\" dextran penetration. In preliminary analysis, dextran penetration or uptake into the fine processes of astrocytes and microglia were found to be difficult to reliably distinguish from background levels while dextran-positive oligodendrocytes were usually detected only at the lesion epicentre in all models ( A . M . Choo et al. unpublished observations). Consequently, glial membrane compromise was not quantified. The use of cryosections thinner than the 20pm used here could potentially improve the sensitivity of dextran measurement. The injuries in the three mechanisms reported here showed similarities as well as differences compared to those in the literature. The pattern of central damage in contusion was similar to that reported by many groups (Bresnahan et al., 1991; Gruner et al., 1996; Scheff et al., 2003). The 2.5mm dislocation examined in this study produced more severe hemorrhage than the 3.2mm to 7.5mm injuries modelled in lateral thoraco-lumbar dislocation (Fiford et al., 2004). The differences may be attributed to the slower speed used in that study (5.7-12.7cm/s) as well as the lateral dislocation being distributed across three vertebral segments in contrast to the adjacent vertebrae (C4/5) dislocated here. The distraction magnitude o f 4.1mm used in this study was lower than the moderate (5mm) to severe (7mm) injuries reported in a rat thoracic distraction model utilizing Harrington rods (Dabney et al., 2004). Pilot trials at 5mm indicated this to be a lethal severity in the cervical spine. In addition, the injury velocity used in the current study (91.9 \u00C2\u00B1 2.9cm/s [SD]) was aimed to model traumatic injury as opposed to surgical distraction (0.9cm/s). I l l Chapter 3 Primary Injury The pivotal nature of hemorrhage and increased membrane permeability has been well described in the literature. Hemorrhage and hemorrhagic necrosis have been shown to increase with injury severity and are predictive of functional outcomes experimentally and clinically (Anderson, 1985; Noyes, 1987b; Bondurant et al., 1990; Flanders et al., 1990; Boldin et al., 2006). In addition, the associated ischemia and infiltration of inflammatory cells both precipitate free radical generation causing oxidation of lipid membranes, proteins and D N A thereby exacerbating damage (Young, 1987, 1992; Popovich, 2000; Bao et a l , 2004; Park et al., 2004). Concurrently, the release of glutamate from depolarized or necrotic cells initiates the influx of calcium ions through N M D A and A M P A glutamate receptor channels resulting in excitotoxicity (Choi et al., 1987). Although antagonists to these channels have been explored to halt calcium influx (Shi et al., 1989; LaPlaca and Thibault, 1998; L i and Tator, 1999; Lipton, 2006), alternate routes through larger membrane pores permeable to macromolecules have also been detected with in vitro biaxial neuronal stretch models (Geddes et al., 2003; Geddes-Klein et al., 2006), ex vivo white matter preparations (Shi and Borgens, 2000; Luo et al., 2002; Shi and Pryor, 2002), in vivo spinal cord compression injuries (Shi, 2004), and in vivo traumatic brain injury (Pettus et al., 1994; Singleton and Povlishock, 2004; Stone et al., 2004). Evidence in the literature suggests changing the characteristics of the primary mechanical trauma alters the fate of the injured neurons. In vitro cell stretching has been shown to result in both calpain and caspase-3 mediated necrotic and apoptotic cell death (Pike et al., 2000). However, at sub-lethal stretch levels, D N A fragmentation and apoptosis has been found to result from mitochondrial dysfunction leading to excessive reactive oxygen species rather than from caspase-3 mediated pathways (Arundine et a l , 2004). In a recent in vitro study, biaxial stretching of neurons caused significantly greater increases in acute intracellular calcium compared to uniaxial stretching. However, by twenty-four hours post-trauma, the uniaxially stretched population exhibited heightened calcium influx through N M D A channels (Geddes-Klein et al., 2006). In addition, our unexpected observation of membrane compromise in the gray matter (Figure 3.7A) of sham controls in contrast to the sham white matter (Figure 3.6) also reinforces the mechanical and biological anisotropy of these two tissue components. The increased susceptibility of gray matter to damage, particularly hemorrhagic necrosis, has commonly been attributed to its increased capillary network (Tator and Koyanagi, 1997). The observations here suggest that in addition to the vascular differences, the susceptibility to membrane compromise between cell 112 bodies and myelinated axons may also be a distinguishing factor in their susceptibility to injury. The subtler relations between the injury biomechanics and the neuronal response remain unclear, but together, these observations suggest a more sophisticated role for primary injury than simply initiating an independent secondary cascade. The patterns of primary injury found at 5 minutes provide some insight into the injury biomechanics of the three models. The strain-field in contusion has been previously modelled with gel demonstrating maximal axial displacement occurring in the central region of the cord (Blight, 1988). A t the speed and impact depth used in this study, this field appears to be contained within two to four millimeters ( \u00C2\u00B1 l m m to \u00C2\u00B12mm rostro-caudal to lesion) corresponding to one to two impactor diameters (2mm). In fracture-dislocation, a consideration of the direction o f vertebral motion (C4 translating ventrally relative to C5) indicates the rostral injury field in the ventral tracts stems from the transduction of traumatic loads, perhaps tensile in nature, from the lesion epicentre rather than due to rostral vertebral contact. In flexion-distraction, the biomechanics of load transfer between the column and the cord remains unclear. Qualitatively, the depression in dextran-positive dorsal axons at -2, 2, and 5mm may imply the coupling takes place via the nerve roots which are roughly spaced at this interval. In the ventral tracts, it may be more likely that injury occurred due to contact with translating vertebrae as opposed to any sort of axial tensile coupling. The slight increasing slope in rostral membrane compromise could be related to a narrowing of the cord cross-section resulting in an increase in stress (force/unit area), however, horizontal sections along the longitudinal axis would have been better suited to detect this. Hence, the distinct biomechanics of each model produced mechanism-specific injury regions with potential therapeutic implications. Our finding after dislocation showed a similar zone of hemorrhagic necrosis at the lesion epicentre as previously described after contusion (Anderson, 1985). This similar primary damage likely triggers analogous pathomechanisms including ischemia, excitotoxicity, and inflammation giving rise to secondary damage (Tator and Koyanagi, 1997; Popovich, 2000; Dumont et al., 2001; Park et al., 2004; Starkov et al., 2004). However, the primary shearing of lateral axonal tracts by fracture-dislocation reduces the available rim of spared white matter (Blight, 1983b; Blight and Decrescito, 1986; Bresnahan et al., 1991) normally targeted for neuroprotection, thereby potentially reducing clinical treatment efficacy in some of these injuries. In addition, dislocation and distraction appeared to establish a distinct 113 Chapter 3 Primary Injury rostral region of gray and white matter damage which at this early acute stage appears to be spared of major vascular compromise but exhibited extensive membrane compromise. The optimal treatment for this sub-population remains to be determined and appears complex. I f the permeability changes in neuronal somata are transient and sublethal, the neurons may exhibit heightened susceptibility to delayed excitotoxicity (Arundine et al., 2004) originating from the growing lesion. On the other hand, calcium activation of calpain appears to be a necessary component of endogeneous membrane repair (Shi et al., 2000; Prado and LaPlaca, 2004). The widespread membrane compromise in white matter tracts following dislocation or distraction may ultimately result in a wider gap for repair and regeneration. The membrane resealing rate of in vitro white matter strips appears to be slower when subjected to forceps compression relative to a discrete transection (Shi et al., 2000; Shi and Pryor, 2000; Luo et al., 2002) and therefore axolemma recovery could be further delayed by an extended rostro-caudal injury. Abnormal axonal permeability to horseradish peroxidase has been reported to persist for up to 7 days following an in vivo compression injury (Shi, 2004). In optic nerve stretch models, ultrastructural evidence suggests axonal damage initiates at the nodes of Ranvier (Maxwell et al., 1991) with the subsequent loss of nodal microtubules detected within minutes of injury (Maxwell and Graham, 1997). Similarly, in traumatic brain and spinal cord injury, the loss of axolemma integrity has been associated with extensive cytoskeletal degradation (Banik et al., 1982; Pettus et al., 1994). This deterioration may be ameliorated by therapies aimed at limiting ionic dysregulation (Rosenberg et al., 1999; Rosenberg and Wrathall, 2001; Schwartz and Fehlings, 2001; Hains et al., 2004) and calcium mediated protease activation (Springer et al., 1997; Schumacher et al., 2000). Even for the surviving axons, however, secondary alterations to ion channels in the nodal region (Maxwell et al., 1999; Nashmi et al., 2000) combined with damage of myelin lamellae (Balentine, 1978; Bresnahan, 1978; Banik et al., 1982; Blight and Decrescito, 1986; Anthes et al., 1995) may still result in residual conduction deficits (Blight, 1983a; Blight and Someya, 1985; Waxman et a l , 1991; Nashmi and Fehlings, 2001). Thus, the pattern of dextran entry may indicate that the biomechanics of dislocation and distraction could heighten the rostro-caudal field of degeneration and chronic dysfunction relative to that previously observed in contusion models. 114 Chapter 3 Primary Injury In summary, this study profiled differences in the primary damage created by three clinically relevant mechanisms of spinal cord injury. Different distributions of acute hemorrhage and membrane compromise were detected reflecting distinctions between the injury biomechanics of these mechanisms. 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In contrast, vertebral dislocation\u00E2\u0080\u0094occurring in 29-45% of human cases (Sekhon and Fehlings, 2001; Pickett et al., 2006)\u00E2\u0080\u0094shears the cord between adjacent segments. Hyper-flexion of the vertebrae can cause SCI by tensile stretching (Breig, 1970; Silberstein and McLean, 1994) and this mode of trauma is widely studied in vitro (Shi and Pryor, 2002; Geddes et al., 2003; Lusardi et al., 2004). Neuroprotective strategies designed to mitigate the biochemical cascade that exacerbates the lesion (Tator and Fehlings, 1999), have been extensively tested in pre-clinical models of spinal cord contusion (Gruner, 1992; Stokes et al., 1992; Scheff et al., 2003) and compression (Rivlin and Tator, 1978). Clinical translation of these strategies has been modest, and suggests that current animal models may not reflect the spectrum of human SCI (Tator, 2006). Since the spinal cord is rarely transected (Norenberg et al., 2004), neuroprotective sparing of tissue from secondary degeneration holds the promise of enabling functional recovery. Myriad strategies have been explored including: the management of blood flow to minimize ischemia (Guha et al., 1989; Tator, 1992), the attenuation of inflammation to limit bystander damage ( X u et al., 1998; Gris et al., 2004; Stirling et al., 2004), the inhibition of free radicals to reduce oxidative stress (Diaz-Ruiz et al., 1999), and the blockade of glutamate receptors to prevent excitotoxicity (Faden et al., 1988; Wrathall et al., 1996; Gaviria et al., 2000b). Although methylprednisolone A version of this chapter has been submitted for publication. Choo AM, Liu J, Dvorak M, Tetzlaff W, Oxland TR. Contusion, dislocation, and distraction: Evolution of membrane permeability and early secondary pathology of neuronal somata, axons, astrocytes, and microglial in clinically relevant mechanisms of spinal cord injury. 121 Chapter 4 Secondary Injury (Bracken et al., 1990; Bracken et al., 1997) initially gained widespread clinical application, its efficacy remains intensely controversial (Hurlbert, 2000; Hal l and Springer, 2004). The interaction between neurodegenerative processes confounds the identification of treatment priorities. Some pre-clinical investigations have favoured early intervention within a few hours of injury (Wrathall et a l , 1997; Diaz-Ruiz et al., 1999; Gaviria et al., 2000a; Rosenberg and Wrathall, 2001; Gris et al., 2004). Given that pre-clinical therapies have had limited success against the broader spectrum of clinical injuries, it seems imperative to understand i f the early secondary cascades are modified by different injury mechanisms. Although mechanical trauma triggers secondary pathomechanisms, few have addressed the influence of different primary injury mechanisms on secondary pathophysiology. In neuronal cultures, biaxial stretching initiates greater calcium influx than uniaxial stretching, however counter-intuitively, delayed excitotoxicity is heightened in the uniaxial injury model\" (Geddes-Kle in et al., 2006). Biomechanically, compression, tension, and shear produce different deformations (Fung, 1993). Neural tissue is mechanically anisotropic (Arbogast and Margulies, 1998); meaning it is not homogeneous and its susceptibility to traumatic deformation depends on the direction of force application. In chapter 3 it was found that contusion, dislocation, and distraction established distinctive patterns o f primary damage when analysed immediately after injury. Here, the early secondary pathology that evolves from these three clinically relevant SCI mechanisms was compared. 4.2 Materials and Methods 4.2.1 Animal Models A l l procedures were approved by our institution's Animal Care Committee in accordance with the guidelines published by the Canadian Council on Animal Care. Thirty-nine male Sprague-Dawley rats (weight 347 \u00C2\u00B1 28 g [SD]) were divided into three injury groups (n = 10 contusion, n = 10 dislocation, n = 10 distraction), three sham surgical control groups (n = 2 contusion, n = 2 dislocation, n = 3 distraction) and one group of controls for dextran incubation (n = 2, described below). Animals were anesthetized with an intramuscular injection o f ketamine (80mg/kg) and xylazine (lOmg/kg) and maintained under deep anesthesia throughout the tests. Fluorescein-dextran with a molecular weight of lOkDa (0.375mg diluted in 15pL dH20, Molecular Probes, Eugene, OR) was infused into the cisterna magna 1.5 hours prior to injury in order to 122 Chapter 4 Secondary Injury detect increases in membrane permeability (Pettus et al., 1994; Singleton and Povlishock, 2004; Stone et al., 2004). Custom designed stereotactic clamps were used to hold the cervical vertebrae between C3 and C6 laterally beneath the transverse processes as described in chapter 2. Moderate-severe contusion, fracture-dislocation and flexion-distraction injuries were produced as previously described using an SCI multi-mechanism system equipped with a electromagnetic linear actuator and sensors to measure force, displacement, and acceleration (refer to chapter 2). Briefly, for contusion, a circular laminectomy window was produced between C4 and C5 . A 2-mm spherical head impactor (Figure 4.1 A ) was lowered in 50pm step-increments on to the dural surface until a touch force of 0.03N was detected (Stokes et al., 1992). The actuator was retracted and the touch force was re-verified. The actuator was then retracted to 6mm above the dural before being accelerated downward to strike the cord to an injury depth of 1.1 \u00C2\u00B1 0.02 mm (SD). To model vertebral fracture-dislocation, the dorsal ligaments and facets between C4 and C5 were removed. The C3 and C4 vertebrae were held stationary, while a vertebral clamp holding C5 and C6 together was coupled to the actuator and translated dorsally to produce a transient 2.5 \u00C2\u00B1 0.12 mm (SD) C4/5 dislocation (Figure 4.IB). For flexion-distraction, the facets between C4 and C5 were removed and the animals were secured within a stereotactic frame with 15\u00C2\u00B0 of flexion at C4/5. With the C3 and C4 vertebrae held stationary, C5 and C6 were held with a vertebral clamp and were distracted caudally 4.1 \u00C2\u00B1 0.03 mm (SD), held for one second, then returned to their initial position (Figure 4.1C). For sham contusions, the animals were secured within the stereotactic frame and underwent identical procedures, however, only the touch force was applied. For sham dislocations and distractions, the procedure was halted after the vertebral clamp holding C5 and C6 was coupled to the actuator. 123 Chapter 4 Secondary Injury Contusion Dislocation Distraction Figure 4.1: Illustration of three injury mechanisms A: Contusion injury was produced with a 2mm diameter impactor that struck the cord through a laminectomy between C4 and C5. B: Dislocation was modelled by holding C3 and C4 stationary while C5 and C6 were displaced together dorsally. C: Distraction injuries were generated by translating C5 and C6 caudally. Proportions and perspectives in illustrations are approximations. Instrumentation used to constrain stationary vertebral clamps is not shown. Following dislocation and distraction injuries, the rostral and caudal vertebral clamps were clamped together to prevent further movement at the now unstable C4/5 joint. Animals were removed from the stereotactic frame and the vertebral clamps were carefully removed under stereo microscopy. For animals in the dislocation and distraction groups, 0.8mm stainless steel rods were bonded (Vetbond, 3 M , St Paul, M N ) to the C4 and C5 laminae to prevent additional movement. The wound was covered with saline-soaked gauze and closed. Animals were carefully maintained under anesthesia in a warmed chamber with temperature monitored via rectal probe (RET-2, K o p f Instruments, Tujunga, C A ) and heart rate and blood oxygenation were monitored using a pulse-oximeter (8600V, Nonin, Plymouth, M N ) . At two hours post-trauma, lOkDa cascade blue-dextran (0.75mg diluted in 20u.L dH^O) was infused into the cerebrospinal fluid to detect secondary changes in membrane permeability. A higher concentration of cascade blue-dextran was used compared to fluorescein-dextran in order to accommodate for its weaker photostability. For three animals (one per injury mechanism), the order of dextran infusion was reversed (i.e. cascade blue-dextran used 1.5 hours prior to trauma and fluorescein-dextran used 2 hours post-trauma) in order to confirm the observations were independent of the sequence of dextran infusion. At three hours post-trauma, all animals were transcardially perfused with 250mL phosphate buffered saline followed by 500mL of freshly 124 Chapter 4 Secondary Injury hydrolyzed cold 4% paraformaldehyde. Spinal cords were post-fixed overnight in 4% paraformaldehyde and cryoprotected in graded sucrose (12, 18, 24% in phosphate buffer) before being frozen in isopentane cooled with dry ice and stored at -80\u00C2\u00B0C. Thirty-four spinal cords were cryosectioned at 20pm parasagittally while three cords (one per injury mechanism) were sectioned in the horizontal plane from dorsal to ventral. Sections from each spinal cord were systematically distributed over eight slides (16 to 18 sections per slide) yielding eight replicates for each cord. The remaining two animals served as controls for dextran incubation in the absence of vertebral column exposure. These animals were anesthetized and infused with lOkDa dextran conjugated to fluorescein or cascade blue (Molecular Probes). Animals were maintained under deep anesthesia and perfused at four hours after infusion. Tissue processing was identical to the injury and sham surgical control groups. 4.2.2 Immunohistochemistry Fluorescein-dextran and cascade blue-dextran were immediately visible under epifluorescent microscopy, however, to improve photostability, slides from all animals were also immunostained with goat anti-fluorescein and rabbit anti-cascade blue (Table 4.1). Oxidative stress in neuronal somata was analysed by immunostaining (n = 37) for 3-nitrotyrosine (3NT), which is generated by the reactive oxygen species peroxynitrite (Deng et al., 2007; Xiong et al., 2007), and early indications of apoptosis were investigated by immunostaining (n = 22) for cytochrome c (Springer et al., 1999; Vanderluit et al., 2003). Immunohistochemistry for non-phosphorylated (n = 37) and phosphorylated (n = 12) neurofilament epitopes was used to detect axonal degeneration. In addition, accumulation of (3-Amyloid Precursor Protein (PAPP, n = 37) demonstrated axonal transport dysfunction (L i et al., 1995; Cornish et al., 2000; Stone et al., 2004). Anti-Ibal (Imai et al., 1996; Ito et al., 1998) revealed activated microglial morphology (n = 37) while the extent of reactive astrocytes was assessed by staining for glial fibrilliary acidic protein ( G F A P , n = 21). 125 Chapter 4 Secondary Injury Table 4.1: Immunohistochemical markers for secondary injury Animals Sampled1 Analysis Antibody Type Target Vendor Dilution CT DL DT SH Membrane A-11095 goat polyclonal fluorescein-dextran Molecular Probes 1:200 10 10 10 7 Permeability A-5760 rabbit polyclonal cascade blue-dextran Molecular Probes 1:200 10 10 10 7 Cell Bodies in 1A6 mouse monoclonal nitrotyrosine Upstate 1:100 10 10 10 7 Gray Matter2 6H2.B4 mouse monoclonal cytochrome c Pharmingen 1:500 5 5 5 7 Axons in White Matter2 SMI 32 mouse monoclonal non-phosphorylated neurofilament Sternberger Monoclonals 1:1000 10 10 10 7 SMI 31 mouse monoclonal phosphorylated neurofilament Sternberger Monoclonals 1:1000 3 3 3 3 CT6953 rabbit polyclonal P-Amyloid Precursor Protein Invitrogen 1:500 10 10 10 7 Microglial Activation 01-1974 rabbit polyclonal Ibal Wako 1:1000 10 10 10 7 Reactive Astrocytes2 G-A-5 mouse monoclonal Glial Fibrillary Acidic Protein Sigma 1:400 6 6 6 3 1 Cryosections from each spinal cord were systematically distributed over eight slides to produce eight replicates of the cord from each animal (16 to 18 sections per slide). CT = Contusion, DL = Dislocation, DT = Distraction, SH = Sham. 2 Where there was no conflict in the primary antibodies, sections were also multilabelled with anti-fluorescein and anti-cascade blue. 3 P-Amyloid Precursor Protein that was visualized in brightfield whereas immunofluorescence was used for all other analyses (refer to methods). For immunofluorescence, sections were washed 3 X 5 minutes in 0.01M phosphate buffered saline (PBS). Sections were blocked for 30 minutes in normal donkey serum before being incubated for 3 hours at room temperature in primary antibodies (Table 4.1) diluted in 0 . 0 I M PBS with 0.1% Triton X-100. Sections were then washed 3><5 minutes in 0.01M PBS and incubated for 1 hour in the appropriate donkey secondary antibody. Alexa488 donkey anti-goat (Molecular Probes) was used as the secondary antibody for anti-fluorescein while A M C A donkey anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, P A ) was used to visualize anti-cascade blue. Cy3-conjugated donkey secondary antibodies (Jackson ImmunoResearch; cross adsorbed against rat in the case of anti-mouse primaries) were used to detect all other primary antibodies. Sections were washed 3x5 minutes in 0 . 0 I M P B S then mounted with Fluoromount-G (SouthernBiotech, Birmingham, A L ) . 126 Chapter 4 Secondary Injury For brightfield immunohistochemistry (i.e. PAPP), sections were washed 3x5 minutes in P B S then incubated under agitation for 30 minutes in 0.3% hydrogen peroxide in 100%> methanol to quench endogenous peroxidase activity. Sections were washed 3x5 minutes in P B S then blocked for 30 minutes in normal donkey serum before being incubated for 3 hours at room temperature in rabbit anti-PAPP (Table 4.1) diluted in 0 .01M P B S with 0.1% Triton X-100. Sections were then washed 3x5 minutes in 0.01M P B S and incubated for 30 minutes in avidin-biotin complex ( A B C Standard Elite, Vector Laboratories, Burlingame, C A ) . Sections were washed, incubated for 1 hour in biotinylated donkey anti-rabbit (Jackson ImmunoResearch) and then washed again. Sections were visualized using 3,3'-diaminobenzidine ( D A B ) substrate with nickel (Vector Laboratories), then washed in distilled water, dehydrated in graded ethanol (50, 70, 90, 95, 100%), cleared in isopropanol, followed by toluene, then mounted with Entellan ( E M Science, Gibbstown, NJ). 4.2.3 General Image Acquisition & Analysis A l l images were acquired using a Zeiss AxioPlan2 (Carl Zeiss, Thornwood, N Y ) equipped with a motorized scanning stage (Scan 100x100, Marzhauser, Wetzlar-Steindorf, Germany; M A C 5 0 0 0 controller, Ludl , Hawthorne, N Y ) for systematic random sampling of tissue at either 1mm rostro-caudal intervals or image montaging of contiguous segments as specified below. Sampling at 1mm intervals spanned from 4mm caudal to 4mm rostral to the apparent lesion epicentre (i.e. 9 images, -4mm .. . +4mm) with three series captured (i.e. 3 x 9 images) per region o f interest (e.g. ventral gray matter) in each animal. For montaged images, the rostro-caudal length differed between injury markers, however, for all analyses, three montages were captured per tract per animal. Image analysis was conducted with the investigator blind to the mechanism of injury using customized software (Appendix B) written in Matlab (The MafhWorks, Natick, M A ) to semiautomate thresholding and counting with basic analysis tools (Nikolaidis and Pitas, 2001). A l l image analysis was subjected to manual verification and correction for cases where the general algorithms failed. Random placement of quantitative probes such as counting frames was achieved using a random vector of length 0 to 50pm. A s detailed below, one-way, two-way and three-way Analysis of Variance ( A N O V A ) were carried out with Student-Newman-Keuls post hoc tests using an alpha level of 0.05. 127 Chapter 4 Secondary Injury 4.2.4 Changes in Membrane Permeability Intracellular penetration of fluorescein-dextran and cascade blue-dextran was analysed to assess changes in membrane permeability over the post-traumatic period. Nine specimens were excluded from quantitative analysis due to poor visibility of either dextran tracer (2 contusion, 2 dislocation, 3 distraction, 2 sham controls). Systematic random sampling (-4mm .. . +4mm) was used to image the ventral gray matter (20x objective lens) and ventro-medial white matter (40x objective lens) as these regions were previously representative of the primary membrane compromise in these three injury models (refer to chapter 3 Figures 4.6 & 4.7). Cel l bodies were selected using 1 0 0 x 100pm unbiased sampling frames (Howard and Reed, 1998). Four frames were randomly placed entirely within the gray matter in each image and cells were manually counted i f the intensity of either dextran was three times greater than the background. Dextran penetration into axons was quantified using 100 pm line probes (three per image) randomly placed within 250pm of the ventral tissue border (as in chapter 3 ) . Similar to the gray matter, axons crossing the line probe were manually selected i f they exhibited dextran fluorescence above a consistent background threshold. The intensities of fluorescein-dextran (IFL) and cascade blue-dextran (ICB) within the cell body or axon was defined by averaging 3 x 3 image pixels around the selection point within the cytoplasm of the cell bodies or at the intersection between the axon and the line probe. In order to accommodate for variations in the background level of dextran, the intensity of the dextran within cell bodies and axons was normalized to the background level within the same image: IFL = \u00E2\u0080\u0094 and ICB = \u00E2\u0080\u0094 . Changes in membrane ^ FL_ Background ^CB Background permeability were analysed using three-way repeated-measures A N O V A to compare the effects o f injury mechanism, dextran infusion time (pre-injury vs. post-injury with IFI and ICB as appropriate for the sequence used), and rostro-caudal position. 4.2.5 Cell Bodies in the Gray Matter To compare patterns of oxidative stress between injury models, sections stained for 3 N T were systematically imaged (-4mm .. . +4mm) using a 20x objective lens. Cells exhibiting neuronal morphology and positive for 3 N T above a fixed background threshold were counted i f they fell within four randomly placed 1 0 0 x 1 0 0 p m 2 unbiased sampling frames. Two-way repeated-measures A N O V A compared the effects of injury mechanism and rostro-caudal position on the 128 Chapter 4 Secondary Injury number of 3NT-positive cells per square millimeter. Since count data exhibits a Poisson distribution, where the standard deviation is proportional to the mean, square-root transformations were used to ensure equal variances prior to statistical testing (Zar, 1999). Potentially apoptotic neurons are identifiable by the release of cytochrome c from mitrochondria into the cytoplasm (Springer et al., 1999; Sugawara et al., 2002; Vanderluit et al., 2003), however, classification appeared unreliable in our sections. Hence, cytochrome c immunostaining was analysed qualitatively only. 4.2.6 Axons in the White Matter The ventro-medial white matter tract on slides stained for non-phosphorylated neurofilament were systematically imaged with a 40x objective from 4mm caudal to 4mm rostral to the lesion epicentre thereby paralleling the analysis o f axonal membrane compromise, which is thought to initiate the collapse of neurofilament sidearms (Pettus et al., 1994; Jafari et a l , 1998; Okonkwo et al., 1998). A 200pm wide sampling frame, occupying the entire rostro-caudal length of the image, was randomly placed within 250pm of the superficial tissue border. A fixed threshold segmented individually identified axonal profiles for area measurements. Two-way repeated-measures A N O V A compared the effects of mechanism and rostro-caudal position on changes in the percent area of non-phosphorylated neurofilaments. To confirm the apparent loss of neurofilaments, a subset of adjacent sections was also analysed for phosphorylated neurofilament epitopes. Phosphorylated neurofilaments were not quantified because the staining pattern was not amendable to segmentation of individual axons. Accumulation of PAPP was imaged using a 20x objective lens to capture the dorsal ascending columns, dorsal corticospinal tract, lateral white matter and ventro-medial column. Since (3APP generally exhibits isolated regions of punctate staining, contiguous rostro-caudal montages centered at the lesion epicentre were acquired. For the corticospinal tract 4.5mm image montages were used whereas 3.5mm montages were used for the remaining tracts. BAPP staining outside these regions was negligible. To quantify PAPP accumulation, a sampling frame (200ju.mx4.5mm [corticospinal tract] or 200pmx3.5mm [dorsal, lateral, and ventral tracts]) was randomly overlaid on each montaged image and a fixed threshold was used to determine the total area o f P A P P immunostaining. For each animal, three montages from each tract were quantified except for the lateral column where two montages were used. In addition, the rostro-caudal profile 129 Chapter 4 Secondary Injury of P A P P accumulation was analysed by projecting the 200pm width onto the rostro-caudal axis. Nine specimens (2 contusion, 2 dislocation, 2 distraction, 3 shams) were excluded from quantitative analysis because the level o f endogenous peroxidase quenching was not comparable to the level in the remaining slides. For each tract, one-way A N O V A compared PAPP accumulation between injury mechanisms. Square-root transformations were used to ensure homoscedasticity prior to statistical testing. 4.2.7 Reactive Astrocytes Sections stained for G F A P were montaged with a 10x objective from -4.5mm to +4.5mm rostro-caudal to the lesion epicentre. Area sampling frames, 500pmx9mm, were randomly placed entirely within the central region o f the gray matter and a threshold was applied to determine the percent area of G F A P immunostaining. Arcsine transformations were applied to ensure equal variances of percent data (Zar, 1999) followed by one-way A N O V A to compare the area of reactive astrocytes between injury mechanisms. The rostro-caudal profile of G F A P was analysed by projecting the 500pm sampling frame onto the rostro-caudal axis. White matter astrocytes were not quantified because an immunoreactive gradient was observed between the white/gray matter boundary and the glia limitans thus confounding the placement of a random sampling frame. 4.2.8 Microglial A ctivation A new method was developed and validated to quantify microglial activation. Ramified microglia exhibit thin, highly branched processes which thicken and retract during activation (Glenn et al., 1992; Stence et al., 2001). This activation is often characterized qualitatively or by using semi-quantitative methods to grade microglia as resting, activated, or phagocytic. However, the consistency of subjective classification was deemed untenable for the large current dataset (37 animals x 9 rostro-caudal positions x 4 regions x 3 samples). Quantitative methods such as fractal analysis have shown promise though some disparity remains compared to manual classification (Soltys et al., 2001). To validate our method, a set of 154 cropped images of microglia, visualized with anti-Ibal, were manually classified into fully ramified (Figure 4.2A, from sham surgical controls), initial activation indicated by increased immunoreactivity (Figure 4.2B), low activation indicated by thickened processes (Figure 4.2C), medium activation indicated by retracted processes (Figure 4.2D), high activation indicated by few processes (Figure 4.2E), and full activation indicated by a spherical amoeboid morphology (Figure 4.2F). A threshold was used to segment 130 Chapter 4 Secondary Injury microglial areas (outlined in red Figure 4.2A to 4.2F). A s activation progresses, immunoreactive area initially increases, but this increase is eventually offset by the retraction of processes, thereby rendering area an ambiguous measure of the activation state (Figure 4.2G). In the novel method, each microglia's \"skeleton\"\u00E2\u0080\u0094a common quantity in computational image analysis (Nikolaidis and Pitas, 2001)\u00E2\u0080\u0094was determined by thinning the area profiles (bwmorph(Image, 'thin', inj) Matlab command) until only a one-pixel thick skeleton representation of each microglia remained (blue pixels in Figure 4.2A to 4.2F). A ramification index was defined as the ratio of skeleton pixels to area pixels (R = Skeleton/Area). For a highly ramified microglia with thin processes, the majority of area pixels w i l l also be skeleton pixels (Figure 4.2A and 4.2B) resulting in a greater index (Figure 4.2H A - B on x-axis). A s microglial processes thicken, the area pixels wi l l increase, while process retraction wi l l reduce the number of skeleton pixels, resulting in a progressive decline in the ramification index (Figure 4.2H C-F on x-axis). Hence, once initially activated, microglia exhibit a monotonic decrease in the ramification index (Figure 4.2H B-F on x-axis) making the index a useful metric for quantitative comparisons. Slides stained for Ibal were imaged (20x objective) at 1mm intervals from 4mm caudal to 4mm rostral of the lesion in the ventral gray matter, ventro-medial funiculus, lateral funiculus, and dorsal ascending column. A threshold was applied and the ramification index was calculated within a randomly placed sampling frame (250><325pm2). Two-way repeated-measures A N O V A compared the effects of injury mechanism and rostro-caudal position in each gray and white matter region analysed. 131 Chapter 4 Secondary Injury 14 12 10 < 8 # 6 Ramified Increased IR Process Thicken Process Retract Few Processes Amoeboid II 0.35 0.3 x 0.25 -D S Be 0.2 .9 >. re 2 i< fD \u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2 K 0.1 0.05 Ramification _ Skeleton Pixels Index O i t G A B C Ramified Amoebo id H A Ramified \u00E2\u0080\u00A2 D E F Amoebo id Figure 4.2: Novel microglial ramification index Ibal immunohistochemistry demonstrating progressive microglial activation and validation curves for novel ramification index used to quantify morphology. Ramified microglia (A) exhibit low Ibal immunoreactivity (IR) and fine highly branched (ramified) processes. On initial microglial activation, Ibal-IR increases (B), followed by a thickening of processes (C). As activation evolves, processes retract (D) until microglia exhibit few processes (E). Fully activated microglia (F) exhibit a spherical, amoeboid morphology similar to phagocytic macrophages. In A-F, red pixels outline areas above the background threshold, while blue pixels mark the skeleton of the areas above threshold (refer to methods). G: Percent Ibal-IR area of 154 images manually classified from ramified (A) to amoeboid (F) demonstrates ambiguity of area measurements in delineating the state of microglial activation. H: Ramification index of same images in G shows a progressive decline in the ramification index as microglia evolve from initial activation (B) to amoeboid (F) thereby validating the utility of the (continuous) index for quantitative analysis in place of (discrete) manual classification. Scale bar 20um applies to A-F. Scatter of individual data points are shown in H. Whiskers denote SD. 132 Chapter 4 Secondary Injury 4.3 Results The multi-mechanism injury system produced contusion, dislocation, and distraction injuries with a high repeatability (<5% SD) in the controlled displacements and velocities (Table 4.2). The mean resultant contusion force was 1.5 \u00C2\u00B1 0.4 N (SD). In the dislocation model, the peak force was 24.7 \u00C2\u00B1 5.7 N (SD) while the peak distraction force was 37.9 \u00C2\u00B1 5.4 N (SD). Following dislocation and distraction, repositioning of slight misalignment between C4 and C5 was achieved with gentle axial tension on the vertebrae prior to stabilization with stainless steel rods. The integrity of the stabilization was confirmed during tissue harvest. Table 4.2: Mechanical injury parameters in secondary injury study Mechanism n Displacement (mm) Force (N) Velocity (cm/s) Contusiont 10 1.1 \u00C2\u00B10.02 1.5 \u00C2\u00B10.4 99.8 \u00C2\u00B13 .7 Dislocation1 10 2.5 \u00C2\u00B10.12 24.7 \u00C2\u00B15.7 95.6 \u00C2\u00B13 .2 Distraction 10 4.1 \u00C2\u00B10.03 37.9 \u00C2\u00B15 .4 111.2 \u00C2\u00B13.5 Surgery Controls 7 - - -Dextran Controls 2 - - \u00E2\u0080\u0094 . \u00E2\u0080\u0094 \u00E2\u0080\u0094 - - I -not applicable are denoted by a hyphen (-). ^Mechanical parameters for this group are based on 9 of 10 animals due to software write-error for one animal. 4.3.1 Changes in Membrane Permeability In order to detect changes in membrane permeability, two different dextran-conjugated fluorophores were infused, 1.5 hours before and 2 hours after injury, and analysed by histology at 3 hours. Compromise of membrane integrity due to primary or secondary injury is reflected by characteristic changes in dextran penetration into axons and cell bodies previously shown to be predominantly neurons (i.e. NeuN-positive refer to chapter 3 Figure 3.7). In the gray matter of surgical controls, dextran incubation controls, and uninjured regions distal to the contusion epicentre, the pre-injury and post-injury dextran molecules were excluded from some neurons (arrows Figure 4.3A-C). In these controls, many somata, however, exhibited trace intracellular dextran fluorescence that was similar to background levels (arrows Figure 4.3D-F) suggesting a \"mi ld\" level of dextran penetration or uptake in the absence of trauma. In uninjured white matter, both dextran tracers accumulated at the nodes of Ranvier (arrows Figure 4.3G-I, chapter 3 Figure 3.2) and remained outside the intracellular compartment. In contrast to the gray matter, uninjured white matter rarely exhibited a \"mi ld\" level of dextran penetration. 133 C h a p t e r 4 S e c o n d a r y Injury Pre-injury Dextran Post-Injury Dextran Merged Images \u00E2\u0080\u00A20 & -TJ S 3 s 9 E 01 0> -O c \u00E2\u0080\u0094 % E > Pre-lnjury Infused Dextran in Axons B - 2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Post-Injury Infused Dextran in Axons - 2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB - 2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Figure 4.4: Quantitative analysis of dextran penetration into somata and axons Line graphs comparing the intracellular dextran fluorescence (normalized to the background) in individually identified neuronal cell bodies and axons. In the ventral gray matter (highlighted in A inset), the intensity was similar between the dextran infused before injury (A) and the dextran infused 2 hours after injury (B) suggesting a persistence in membrane permeability. In the ventro-medial white matter (highlighted in C inset), the intensity of dextran infused before injury (C) was greater than that infused after injury (D) indicating membrane resealing. Whiskers denote SEM. 136 Chapter 4 Secondary Injury 4.3.2 Cell Bodies in the Gray Matter Sham controls exhibited little immunostaining for the oxidative stress marker 3NT (Figure 4.5A) whereas extensive staining throughout the neurophil near the lesion epicentre was observed in spinal cords injured by contusion (Figure 4.5B) and dislocation (Figure 4.5C). At the distraction epicentre, 3NT staining appeared localized to sparse hemorrhagic areas (Figure 4.5D). The fluorescent intensity of 3NT-positive cells was generally near background levels (arrowheads Figure 4.5B-D). Contusion -\u00C2\u00BB- Dislocation Distraction * \u00E2\u0080\u00A2 Sham << Caudal - (mm) - Rostral \u00C2\u00BB Figure 4.5: Photomicrographs and quantitative counts of 3-NT immunostaining Representative photomicrographs of immunohistochemistry for the oxidative stress marker 3-nitrotyrosine (3NT) and line graphs showing quantitative distribution of 3NT-positive cells. Spinal cords from sham animals exhibited little 3NT immunostaining (A). Similar patterns of 3NT staining were observed in the gray matter of spinal cords injured by contusion (B) and dislocation (C). Less 3NT immunostaining was detected following distraction injury (D). Near the lesion epicentre, cell bodies permeable to dextran infused before (arrowheads E) and after injury (arrowheads F) were often positive for 3NT (arrowheads G & H). Other dextran-positive cells (arrow E & F), however, were negative for 3NT (arrow G & H) indicating only a weak association between membrane compromise and oxidative stress at this time-point. A significant interaction (p=0.023) between the injury mechanism and rostro-caudal distribution of 3NT-positive cells (I) was found with peak counts in the dislocation model biased rostrally. Scale bars 150um (A-D) and 50um (E-H). Whiskers denote SEM. 137 Chapter 4 Secondary Injury Near the lesion, neurons positive for 3NT {arrowheads Figure 4.5G & H) often exhibited persistent membrane permeability to dextran (arrowheads Figure 4.5E & F), however, dextran-positive neurons were also observed to be negative, or only weakly positive, for 3NT (arrow Figure 4.5E-H). The overall number of 3NT-positive cells was similar between injury groups (p=0.072) though a significant interaction (p=0.007, Figure 4.51) was detected between the injury mechanism and the rostro-caudal distribution of cells. S N K post hoc analysis showed a rostral bias in the number of 3NT-positive cells with more 3NT-positive cells detected at 2mm rostral to the dislocation lesion compared to similar regions in the contusion (p=0.053) and distraction (p=0.012) injuries. There were subtle differences in the pattern of cytochrome c immunostaining between injury groups. Cytochrome c is normally localized within mitochondria (Gulyas et al., 2006) and its release into the cytosol is an early marker of apoptosis as demonstrated by immunohistochemistry (Springer et al., 1999; Sugawara et al., 2002; Vanderluit et al., 2003) and western blot (Springer et al., 1999; Teng et al., 2004). In uninjured animals, the mitochondria within the cytoplasm of neuronal somata exhibited a high intensity of cytochrome c immunofluorescence (arrows Figure 4.6A) against a backdrop of scattered cytochrome c-positive mitochondria in the neurophil (Figure 4.6A). A small population in the lateral penumbra of the contusion lesion exhibited a profound loss of cytochrome c immunostaining (arrowheads Figure 4.6B) indicative of complete cytochrome c release from mitochondria. Attenuation of cytochrome c in neuronal cell bodies was also found in the rostral and caudal penumbra of the dislocation epicentre (arrowhead Figure 4.6C). Distraction injury produced cytochrome c immunostaining patterns similar to that observed in spinal cords from sham surgical controls (arrows Figure 4.6D). Cells displaying a loss of cytochrome c were consistently positive for both dextran tracers (arrowheads Figure 4.6E-H), although dextran-positive neuronal somata were also observed to maintain cytochrome c immunoreactivity (arrow Figure 4.6I-L), suggesting persistent membrane compromise was a necessary, but not sufficient condition to abolish cytochrome c immunostaining. This subpopulation with attenuated immunostaining for cytochrome c was consistently observed following contusion and dislocation, however, the sparseness of these cells, and their narrow localization within the penumbrae of the lesion epicentres, precluded systematic random sampling. Hence, quantitative analysis was not conducted. 138 Chapter 4 Secondary Injury Sham A \u00E2\u0080\u0094 Pre '-injury Dex 1 T E 1 Contusion Dislocation Cyt-c Distraction Cyi A T T J c \u00E2\u0080\u0094 D \u00E2\u0080\u0094 Merged \u00E2\u0080\u00A2 A T T Post-injury Dex Merged Figure 4.6: Cytochrome c immunostaining Photomicrographs showing cytochrome c immunohistochemistry and its association with membrane compromise near the lesion epicentre. Spinal cords from sham surgical controls exhibited granular cytochrome c immunostaining localized to the neuronal cytoplasm (arrows A) with less intense granular staining of mitochondria in the surrounding tissue. In the lateral penumbra of the contusion epicentre, a reduction in cytochrome c was observed in neurons (arrowheads B) and the surrounding hemorrhagic tissue. In the rostro-caudal penumbrae of the dislocation lesion, a loss of cytochrome c immuoreactivity was observed in a small population of cells (arrowhead C) while an increase in immunostaining was observed in surrounding non-hemorrhagic tissue. Cytochrome c immunostaining following distraction injury was similar to that observed in sham animals (arrows D). Cytochrome c-negative neurons in the contusion and dislocation lesion penumbra were consistently positive for dextran (arrowheads E-H), though many dextran-positive neurons were also cytochrome c-positive (arrow I-L). Scale bars 50um. 4.3.3 Axons in the White Matter The compaction of neurofilaments is characteristic of early axonal damage following axolemmal compromise (Pettus et al., 1994) and likely results from the dephosphorylation and collapse of sidearms (Maxwell et al., 1997; Okonkwo et a l , 1998). This dephosphorylation exposes the neurofilament core to earlier proteolytic degradation than its phosphorylated counterparts (Pant, 1988; Schumacher et a l , 1999). SMI32 binds to non-phosphorylated neurofilament (npNF) epitopes in heavy and medium neurofilaments (Sternberger and Sternberger, 1983) revealing thick axons such as those in the ventral white matter (Figure 4.7A), whereas SMI31 detects phosphorylated epitopes (pNF, Figure 4.7B). At the contusion epicentre, non-phosphorylated neurofilament immunostaining declined (Figure 4.7C) while phosphorylated 139 Chapter 4 Secondary Injury neurofilaments were still readily detectable on the adjacent section (Figure 4.7D). In contrast, the dislocation injury mechanism produced an extensive loss of immunostaining for both non-phosphorylated (Figure 4.7E) and phosphorylated (Figure 4.7F) epitopes signifying irreversible white matter damage. Ventral axons at the distraction epicentre appeared predominantly intact and stained robustly for both neurofilament epitopes (Figure 4.7G & H). y The differences in immunoreactive areas between contusion and dislocation were more apparent at the high magnification (40x objective) used for quantification. A t the contusion epicentre, ventral peripheral axons appeared predominantly intact (arrows Figure 4.71). In contrast, at the dislocation epicentre, axonal fragmentation of ventral axons was frequently observed (arrowheads Figure 4.7J) and subpial areas of complete axonal loss were also evident (asterisk Figure 4.7J). Quantitative analysis (Figure 4.7K) showed a significant effect of mechanism (p=0.024), rostro-caudal position (pO.OOl) and interaction o f these two parameters (p<0.001). A t the lesion epicentre, dislocation injuries resulted in a greater loss of non-phosphorylated neurofilament area compared to both contusion (p=0.004) and distraction (p<0.001). The area of non-phosphorylated neurofilaments at the contusion site was not significantly less than the area at the sham lesion (p=0.167) and reflects that many axons at the contusion epicentre were still detectable above the background threshold, while areas of axonal loss were partially offset by axonal swelling. The elevation in the mean area of non-phosphorylated neurofilaments following distraction (green profile Figure 4.7K), though not significant (p>0.127) compared to sham controls, may be indicative of subtle neurofilament dephosphorylation. 140 Chapter 4 Secondary Injury -\u00E2\u0080\u00A2\u00E2\u0080\u00A2 Contusion Dislocation Distraction 1 A. Sham << Caudal 0 \u00E2\u0080\u00A2 (mm) \u00E2\u0080\u00A2 Rostral >> Figure 4.7: Neurofilament degradation in the ventro-medial white matter Representative photomicrographs and quantitative line graphs show the dislocation injury mechanism accelerates the degeneration of neurofilaments in the ventro-medial white matter. Robust non-phosphorylated (npNF) and phosphorylated (pNF) neurofilament immunostaining was observed in spinal cords from sham surgical controls (A & B). A reduction in immunostaining for non-phosphorylated neurofilaments was observed at the contusion epicentre (C) though staining for phosphorylated neurofilament epitopes revealed many axons were still intact (D). Extensive loss of ventro-medial neurofilaments - both non-phosphorylated (E) and phosphorylated (F) - was observed in the dislocation model. The majority of ventral axons appeared intact in the distraction injury model (G & H). High magnification images at the lesion epicentres reveal peripheral ventral axons were generally spared following contusion (arrows I). In contrast, dislocation injury resulted in more axonal fragmentation (arrowheads J) and regions completely devoid of peripheral axons (asterisk J). Quantitative measurements of the percent area of non-phosphorylated neurofilament immunoreactivity showed the greatest loss in neurofilaments at the dislocation epicentre (p<0.004 vs. contusion and distraction). A reduction in non-phosphorylated neurofilament area was detected at the contusion epicentre though most axons exhibited immuoreactivity above the background threshold at high magnification. Scale bars lOOum (A-H) and 50um (I & J). Whiskers denote SEM. We next assessed the accumulation of (3APP which is indicative of disrupted fast axonal transport and reveals white matter injury following neurotrauma in humans (Cornish et al., 2000; Smith et al., 2003) and animals (L i et al., 1995; Gomes-Leal et al., 2005). In the lateral white matter, following contusion (Figure 4.8A & D) and distraction injuries (Figure 4.8C & F), diffuse punctate staining of PAPP was observed. In contrast, dislocation produced widespread staining of 141 Chapter 4 Secondary Injury PAPP accumulation in ascending and descending fibers of the lateral funiculi (Figure 4.8B & E) near primary axotomies from the direct shearing between C4 and C5 vertebrae (arrowheads Figure 4.8B). Quantitatively in the distraction model, the diffuse level of PAPP accumulation in all white matter tracts rendered these areas negligible relative to the more extensive accumulations observed following contusion and dislocation injuries (Figure 4.8G & H , p<0.001 for all comparisons relative to distraction). In contrast to the diffuse staining observed in distraction, contusion and dislocation injury mechanisms produced similar levels of PAPP accumulation in the dorsal column (p=0.647), dorsal corticospinal tract (p=0.145), and ventro-medial column (p=0.206). However, as observed qualitatively, dislocation injuries resulted in quantitatively much greater PAPP immunostaining in the lateral column than either contusion (p=0.001) or distraction (p<0.001) injuries. Although PAPP accumulation levels were often similar between the contusion and dislocation groups - indicating a comparable number of axons injured - the rostro-caudal position of the P A P P accumulation front in the corticospinal tract was shifted rostrally following dislocation indicating a more extensive damage of this tract by a dislocation injury mechanism (Figure 4.81). 142 Chapter 4 Secondary Injury c o u D \u00E2\u0080\u00A2 _ F -APP Accumulation in White MatterTracts ,4 APP Immunoreactive Profile in Dorsal Corticospinal Tract Contusion Dislocation Distraction Sham -1 0 \u00C2\u00AB Caudal (mm) Rostral>> Figure 4.8: pAPP accumulation in white matter Representative photomicrographs, bar graphs and rostro-caudal profile of PAPP immunostaining. In the lateral funiculi of spinal cords injured by contusion (A & D), dislocation (B & E) and distraction (C & F), PAPP accumulation was most extensive following dislocation injury. Characteristic axotomy from the dislocation mechanism's shear forces was evident in the lateral column (arrowheads B). Quantitative analysis of PAPP accumulation in the dorsal corticospinal tract (G), dorsal column, lateral column, and ventral column (H) showed relatively diffuse injury following distraction. In the white matter, contusion and dislocation injuries produced similar areas of pAPP immunostaining except in the lateral column where dislocation resulted in significantly greater PAPP accumulation (p=0.001). A rostral bias in the boundary of descending PAPP accumulation was observed in spinal cords injured by dislocation (I). The profile in I was averaged over all animals analysed excluding 9 slides due to differences in the background level of endogeneous peroxidase quenching (refer to methods). Scale bars 250um (A-C) and 50pm (D-F). Whiskers denote SEM. 143 Chapter 4 Secondary Injury 4.3.4 Reactive Astrocytes Both contusion and dislocation injury mechanisms resulted in extensive astrogliosis near the boundary of the lesion epicentre. G F A P immunostaining in spinal cords from sham surgical controls revealed well defined astrocytes that exhibited dim immunofluorescence (Figure 4.9A). The intensity of G F A P immuoreactivity was increased near the spinal cord lesion following contusion (Figure 4.9B & E), dislocation (Figure 4.9C & G) and distraction (Figure 4.9D) injuries. In the contusion injury model, astrogliosis appeared predominantly contained within 2mm of the lesion epicentre (compare Figure 4.9E versus 4.9F). In contrast, following dislocation, astrogliosis was evident near the lesion boundary (Figure 4.9G) but also in regions at 3 to 4mm distal to the epicentre (Figure 4.9H). In spite of this qualitative observation, quantitative analysis of G F A P immunoreactive areas showed no significant difference in percent G F A P area between contusion and dislocation (Figure 4.91, p=0.992). A n analysis of the rostro-caudal distribution of G F A P (Figure 4.9J) suggests the average density of G F A P after dislocation and contusion was indeed similar, though it was more confined to the lesion following contusion, and extended more rostrally following dislocation. 144 Chapter 4 Secondary Injury Sham A E \u00E2\u0080\u0094 Contusion E \u00E2\u0080\u00A2 F \u00E2\u0080\u00A2 B \u00E2\u0080\u0094 F _ Dislocation H \u00E2\u0080\u00A2 u c \u00E2\u0080\u0094 G _ Distraction D \u00E2\u0080\u0094 H \u00E2\u0080\u0094 GFAP Immunoreactive Profile Figure 4.9: Distribution of reactive astrocytes Low and high magnification photomicrographs, quantitative bar graphs and rostro-caudal profile of GFAP immunostaining. Spinal cords from sham surgical controls exhibited low levels of GFAP (A) while an increase in reactive astrocytes was observed following contusion (B), dislocation (C), and distraction (D) spinal cord injuries. The increase in astrogliosis was localized to the lesion epicentre following contusion (E vs. F) while following dislocation, reactive astrocytes were observed both near the lesion epicentre and rostrally (G vs. H). Quantitative analysis in the gray matter showed no significant differences in immunoreactive areas between the injury mechanisms (I), however, the rostro-caudal profile of immunostaining suggests a broader distribution of reactive astrocytes following dislocation injury (J). Percent areas in I were based on 500umx9mm sampling frames. Identical exposure and background thresholds were used for all images. These levels were selected to prevent saturation of the exposure and threshold areas in images from injured animals resulting in very low GFAP levels in sham surgical controls (A and I). Scale bars 500um (A-D) and 50um (E-H). Whiskers denote SEM. 145 Chapter 4 Secondary Injury 4.3.5 Microglial A ctivation Microglial activation is another hallmark of central nervous system trauma and is characterized by a morphological transition from a resting ramified to an activated amoeboid shape (Figure 4.2 and methods section). Each injury mechanism resulted in distinct rostro-caudal patterns of microglial activation. Microglia in sham surgical control animals exhibited fine highly ramified processes characteristic of resting microglia although some evidence of low activation was observed at the sham lesion presumably due to surgical manipulation of the vertebral column (Figure 4.1 OA). Microglia at the contusion (Figure 4.1 OB) and dislocation (Figure 4.IOC) gray matter epicentres were predominantly amoeboid in morphology indicative of full activation. In contrast, following distraction injury, few microglia were fully activated except those found near regions exhibiting tissue necrosis (Figure 4.10D). A t 4mm rostral to the lesion, microglia in sham (Figure 4.10E), contusion (Figure 4.1 OF) and distraction (Figure 4.1 OH) groups exhibited morphologies indicative of resting microglia or microglia in a low activation state. In contrast, highly activated microglia with few processes were still observed at 4mm rostral to the dislocation epicentre (Figure 4.10G). Quantitatively, in the gray matter, contusion and dislocation resulted in similar levels of microglial activation at the lesion epicentre (p = 0.210) which were significantly greater than those detected at the distraction lesion (p<0.001, Figure 101 0mm). Microglial activation in the dislocation model remained elevated along the length of the spinal cord from -4mm caudal (p=0.017 compared to sham controls) to +4mm rostral (p=0.003 compared to sham controls) of the lesion epicentre. Microglia in animals injured by vertebral distraction tended to have a greater ramification index compared to shams at 2mm (p<0.001), 3mm (p<0.004), and 4mm (p=0.052); indicative of the increased immunoreactivity of microglia in the earliest phase of activation (Figure 4.101, cross-reference with Figure 4.2B & 2H). Within the white matter, microglial activation patterns in animals injured by dislocation differed between tracts. Following dislocation, microglial activation was greater rostrally in the lateral (Figure 4.10J) and dorsal (Figure 4.1 OK) columns but symmetric about the lesion epicentre in the ventro-medial tract (Figure 4.10L). In contrast, contusion injuries produced symmetric patterns of microglial activation with the highest activation in the dorsal (Figure 4.1 OK) and ventral columns (Figure 4.10L). In the distraction model, the microglia in the white matter were quantitatively similar to those in sham surgical control specimens. 146 Chapter 4 Secondary Injury .-- v v U - - : A , ^ _ B V. \u00E2\u0080\u0094 ! \u00C2\u00BB H \u00C2\u00AB\u00E2\u0080\u00A2 -v 'S , ' ***** *\u00C2\u00AB E v _ \"v- #r . N A* 3 F \"'\u00E2\u0080\u00A2 H \u00E2\u0080\u00A2 _ * \u00E2\u0080\u00A2 r \u00E2\u0080\u00A2 j . \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 * \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 . . * \u00C2\u00BB \u00E2\u0080\u00A2 - \u00C2\u00BB # . 0.25 r 11 \u00C2\u00B0-2 c 3 \u00C2\u00A3 2 I < o.-K ; Gray Matter j ,$ Lateral Column -\u00E2\u0080\u00A2. Contusion Dislocation \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Distraction Sham Dorsal Column 0.25 0.1 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Ventro-Medial Column << Caudal - (mm) - Rostral >> L 5 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Figure 4.10: Microglial activation Representative photomicrographs of Ibal immunostaining and morphometry of microglia in the gray and white matter show asymmetry of microglial activation following dislocation injury. Microglia in sham surgical controls were highly ramified and exhibited low levels of Ibal immunostaining at the lesion and rostrally (A & E). Microglia at the contusion epicentre were amoeboid (B) while rostrally, only low levels of activation were detected (F). Microglia were highly activated at both the dislocation epicentre (C) and rostrally (G). At the distraction epicentre, microglia exhibited process thickening characteristic of low levels of activation (D) while rostrally, most microglial exhibited increased Ibal immunoreactivity but remained highly ramified (H). Analysis of the ramification index (Figure 2) in the gray matter (I) showed greater rostro-caudal microglial activation following dislocation injury. Microglial morphology in the lateral (J), dorsal (K), and ventral (L) funiculi showed a dorsal to ventral asymmetry in activation following dislocation injury. Scale bars 50um (A-H). Insets I-L highlight region quantified. Whiskers denote SEM. 147 Chapter 4 Secondary Injury 4.4 Discussion This study compared the initiation o f secondary pathology following distinct clinically relevant mechanisms of SCI. A n early post-traumatic time-point was examined because it is within this acute phase that neuroprotective strategies have demonstrated promising efficacy, and thus it is here, where an understanding of residual secondary effects from differences in primary damage (chapter 3) might eventually improve future clinical treatment paradigms. Relative to spinal cord contusion, the dislocation injury accelerated neurofilament degeneration, produced a wider axonal regeneration gap, and extended the rostro-caudal zone of microglial and astrocyte activation. In contrast, although distraction injury has been previously shown to produce widespread primary cellular membrane compromise (chapter 3), in this study, modest secondary pathology was detected that might be attributable to the combination of membrane resealing and this model's modest primary hemorrhage that is central to the initiation of degeneration (Balentine, 1978; Guha and Tator, 1988). A study limitation was that the short survival period prevented the analysis of post-traumatic behaviour. The profound functional deficits from cervical injuries (Pearse et al., 2005; Gensel et al., 2006) ethically necessitate the demonstration of meaningful differences between these injury mechanisms before proceeding with a survival study. The degeneration in the lateral funiculi following dislocation injury could precipitate deficits in digit dexterity due to damage of the rubrospinal tracts (Schrimsher and Reier, 1993) or hindlimb impairments due to loss of ventro-lateral reticulospinal fibers (Loy et al., 2002; Schucht et al., 2002). Clinically, spinal shock confounds the reliability of functional scores taken at the time of hospital admission (Tator, 2006), while later, initial differences may be masked by irreversible degeneration of the spinal cord. Hence, the focus was on contrasting the early secondary cascade that precedes the period of reliable functional assessments. In the present study dextran tracers were used but this technique has some limitations in detecting membrane compromise. Fluorescence from extracellular dextran blurs the intracellular signal and reduces the reliability of detecting dextran within fine structures (chapter 3). A wider range of graded dextran penetration was observed at three hours post-trauma compared to a primary injury time-point (chapter 3) and is likely due to a combination of protracted endogenous uptake mechanisms (Vercelli et al., 2000) and membranes exhibiting variable stages of progressive 148 Chapter 4 Secondary Injury degeneration or recovery. Approximately 40% of cells have been observed to reseal to Texas Red-conjugated dextran by 2 hours following diffuse traumatic brain injury (Farkas et al., 2006). In the current study, the limited resealing detected in the gray matter reflects the greater severity of the injuries produced. The limited resealing observed in the gray matter is unlikely to be an artifact of retrograde transport of the second dextran tracer back to the cell body because there was little penetration of the second dextran into axons and the duration of incubation of the second dextran species was only 1 hour prior to euthanasia. Differences between the studies due to the dextran species utilized (Nance and Burns, 1990) are unlikely since the results were consistent with preliminary trials employing tetramethylrhodamine-dextran (Choo et al., 2005) and confirmed by reversing the order of dextran infusion. In addition, the use of two dextran-conjugated fluorophores limited our ability to use multiple labels for detecting pathologies such as oligodendroglial apoptosis; though the literature indicates this population is more readily detectable at one week after the period analysed here (Shuman et al., 1997). In the three injury models, dextran penetration across compromised cell membranes previously demarcated distinct primary injury fields but these boundaries have been obscured by the evolution in membrane permeability. Contusion was previously shown to damage cell membranes in a symmetric pattern at the lesion epicentre while the injury fields following dislocation and distraction were asymmetric - extending rostrally (chapter 3). These primary differences were less evident following the secondary evolution of membrane integrity (Figure 4.4B & D). The pre-injury dextran in the white matter, however, still reflected the primary injury asymmetry (Figure 4.4C) suggesting membrane resealing retains the dextran that entered during mechanical injury (Figure 4.4A). Although transient, the loss of membrane integrity is associated with delayed cell death (Geddes et al., 2003; Geddes-Klein et al., 2006) and axonal degradation (Banik et al., 1982; Pettus et al., 1994; Jafari et al., 1998), while membrane repair strategies have shown therapeutic benefits (Borgens and Shi, 2000; Serbest et al., 2006). The rostro-caudal patterns of secondary neuropathology appeared related to the characteristics of primary injury. In the dislocation model, the distribution of activated microglia, and to a lesser extent reactive astrocytes, parallels the extended rostral primary membrane injury previously observed in chapter 3 for this model. This rostral bias in glial activation may suggest mechanically induced membrane compromise allows the release of adenosine triphosphate 149 Chapter 4 Secondary Injury (Ahmed et al., 2000) into the extracellular space which in turn activates glia via purinergic receptors (Rathbone et al., 1999; Inoue, 2002; Davalos et al., 2005). Microglial activation also exhibited a dorsal to ventral asymmetry. This phenomena may highlight the susceptibility to injury of dorsal funiculi that has also been reported in contusion models (Bresnahan et al., 1991). Moreover, signs of primary damage in the lateral funiculi following dislocation injury (chapter 3) were evident in the rapid accumulation of PAPP at this lateral epicentre. Parallels between primary and early secondary damage following distraction injury were difficult to draw. This seems consistent with the delayed time-course o f neuropathology reported in other animal models o f distraction (Myklebust et al., 1988; Maiman et al., 1989; Dabney et al., 2004), as well as in vitro models that have demonstrated the robustness of neurons to stretch injury (Smith et al., 1999; Lusardi et al., 2004; Chung et al., 2005) and their vulnerability to delayed apoptosis (Pike et al., 2000; Arundine et al., 2004). In the current study, although similar primary injury severities were modelled, distinct secondary axonal injury populations were identifiable. Neurofilament degradation and P A P P accumulation are well characterized hallmarks of axonal pathology (Banik et al., 1982; Iizuka et al., 1987; L i et al., 1995). The loss of axolemmal integrity is thought to precipitate the collapse of neurofilament sidearms (Jafari et al., 1998), possibly by proteolytic cleavage (Pettus et al., 1994) or dephosphorylation (Okonkwo et al., 1998), rendering these cytoskeletal proteins more susceptible to calpain degradation than their phosphorylated counterparts (Pant, 1988; Schumacher et al., 1999). Non-phosphorylated neurofilaments were not lost following distraction injury; an observation similar to diffuse axonal injury models where an increase in neurofilament dephosphorylation has been reported (Chen et al., 1999; Saatman et al., 2003). In contrast, both contusion and dislocation injuries resulted in reduced immunostaining for non-phosphorylated neurofilament, with degeneration in the dislocation model extending to the phosphorylated epitopes that degrade slower (Pant, 1988). Thus, treatments such as calpain inhibition (Banik et al., 1998; Schumacher et al., 2000), may have a broader time-window in contusion and distraction injuries. In the gray matter, contusion and dislocation injury mechanisms shared similarities as well as differences that could indicate which processes initiate degeneration regionally. The oxidative stress marker 3NT has been shown in both traumatic brain and spinal cord injuries to increase by 150 Chapter 4 Secondary Injury one hour post-trauma (Deng et al., 2007; Xiong et a l , 2007). Overall, the similar counts of 3NT-positive cells following contusion and dislocation suggest an equal priority for anti-oxidant or anti-lipid peroxidation strategies in these two mechanisms with a lower priority for this degenerative pathway in distraction injuries. However, the broader microglial activation in the gray matter o f spinal cords injured by dislocation suggests this glial response may lead the degeneration in the rostral and caudal fields (Inoue, 2002; Schwartz, 2003; Block and Hong, 2005; Bye et al., 2006) with other pathologies trailing soon after. In addition, the release of cytochrome c from mitochondria has been established to precede caspase-3 mediated apoptosis (Springer et al., 1999). The few cytochrome c-negative neurons observed in the lateral and rostro-caudal penumbrae of the contusion and dislocation lesions respectively may presage the leading edge of apoptosis that could be amendable to targeted pharmacotherapies (Teng et al., 2004). This study contributes some perspective on the clinical translation of the myriad therapies directed at mitigating secondary damage. Clinically, vertebral fracture-dislocation have been associated with more severe functional deficits (Marar, 1974; Tator, 1983). The contusion and dislocation injury severities used in this study were previously shown to produce nearly identical levels of intramedullary hemorrhage as well as comparable levels of membrane compromise at the lesion epicentre (chapter 3). The accelerated axonal degeneration and widespread resident inflammatory response of microglia suggest a narrower neuroprotective time-window following dislocation injuries, and may partially explain the limited efficacy of therapies tested in the heterogeneous human population (Pointillart et al., 2000; Geisler et al., 2001; Tadie et al., 2003). Moreover, the rostral asymmetry in the dislocation model may explain the increased clinical severity of fracture-dislocations since the loss of each cervical level is highly detrimental to neurological function (Hedel and Curt, 2006). In a clinical series of 186 cervical SCI's (Harrop et al., 2001), ascension of the lesion was observed in 12 (6%) patients, of which 9 appeared to have suffered a vertebral dislocation injury with concomitant flexion-distraction reported in 6 patients. This study also suggests that repair and regenerative strategies, specifically targeted to contusion injuries, may have limited efficacy when applied to other injuries. Although in this study astrogliosis was far from complete, our early observations might suggest that dislocation generates a wider astrocytic barrier to regeneration. In the dorsal corticospinal tract, the rostral shift in the (3APP accumulation front following dislocation delineates a longer regeneration 151 Chapter 4 Secondary Injury distance. When combined with the extensive damage detected in the lateral funiculi, the differences in white matter deterioration suggest repair strategies may favour contusion and distraction injuries, whereas for dislocations, substantial axonal regeneration, however elusive, may be required for improving function. 152 Chapter 4 Secondary Injury 4.5 References Ahmed S M , Rzigalinski B A , Willoughby K A , Sitterding H A , Ell is E F (2000) Stretch-induced injury alters mitochondrial membrane potential and cellular A T P in cultured astrocytes and neurons. JNeurochem 74:1951-1960. Arbogast K B , Margulies SS (1998) Material characterization of the brainstem from oscillatory shear tests. J Biomech 31:801-807. 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In these new animal models, the biomechanical injury mechanism dictated the pattern of primary damage within the spinal cord which in turn influenced the initial characteristics of secondary pathology. Contusion injuries were highly focused at the lesion epicentre whereas primary damage following dislocation and distraction injuries was distributed over several vertebral levels. Secondary degeneration following dislocation was particularly rapid perhaps indicating a narrower therapeutic time-window for this injury mechanism. In contrast, the extent of secondary pathology following distraction injuries was less pronounced than in either spinal cord contusion or dislocation. These mechanism-specific patterns of injury emphasize that novel therapies should be tested in a broader range of clinically relevant injury mechanisms prior to human trials. The work presented in this thesis demonstrates how an understanding of the biomechanical injury mechanism may contribute to the development of future clinical treatment paradigms. 5.2 Modelling Considerations The three SCI models developed in this thesis are unique from the animal models already in widespread use. Cervical dislocation and distraction in the rat spine are entirely new experimental paradigms while cervical contusion models have only been developed recently (Pearse et al., 2005; Baussart et al., 2006; Gensel et al., 2006). The SCI animal models that are widely used control variables such as the injury force or injury displacement while maintaining a transverse contusion or compression mechanism of injury. In this thesis, the biomechanical mechanism of injury was explicitly varied in order to characterize its effect on primary damage and the subsequent initiation of secondary neuropathology in the spinal cord. 159 Chapter 5 Discussion & Conclusion The injuries in all three novel models were delivered at speeds of approximately lOOcm/s. This high speed is on the order of that believed to occur in human SCI (Panjabi et al., 1995; Nightingale et al., 1996; Wilcox et al., 2002). In spinal cord contusion injuries, the impact velocity has been previously shown to control the extent of blood-spinal cord barrier disruption (Maikos and Shreiber, 2007), intramedullary hemorrhage (Anderson, 1985), and electrophysiological function (Kearney et al., 1988). Several in vitro and ex vivo studies have also emphasized the need to model trauma at high rates (Cargill and Thibault, 1996; Geddes et al., 2003; LaPlaca et al., 2005; Shi and Whitebone, 2006). Pneumatic devices can produce contusion injuries at impact speeds of several meters per second (Anderson, 1982; Kearney et al., 1988) though these devices are not widely used today. In the most common contusion models, impact speeds can range from 13cm/s (Scheff et al., 2003) to approximately lOOcm/s (Maikos and Shreiber, 2007), while other injury models such as the aneurysm clip compression (Rivlin and Tator, 1978) and forceps compression (Gruner et al., 1996) do not control the injury speed. Thoracolumbar dislocation has been modelled at injury velocities between 5cm/s and 15cm/s (Fiford et al., 2004) while the first computer-controlled distraction model utilized Harrington rods and laminar hooks to displace vertebrae at speeds of 0.5cm/s to lcm/s (Dabney et al., 2004). In the cervical spine, I observed the lamina to fracture when applied displacements exceeded speeds of a few centimeters per second ( A . M . Choo et al., unpublished observations). In this thesis, injuries at lOOcm/s were achievable due to the novel vertebral clamping strategy developed which held the vertebrae laterally beneath the transverse processes. In addition to the reduced vascular damage (Anderson, 1985; Maikos and Shreiber, 2007) discussed above, slower impact speeds appear to produce less primary axotomy thereby enhancing the relative significance of demyelination in this type of lesion (W. Tetzlaff, personal communication). Some injuries in humans likely occur at lower velocities and hence, it is important that injury devices are capable of modelling both high and low speed trauma. Cervical contusion models have been recently reported that use the weight-drop to produce unilateral (off-centre) contusions (Gensel et al., 2006) and controlled displacements to produce contusions centred at C5 (Pearse et al., 2005). The contusions reported in this thesis utilized novel vertebral clamping devices that were developed to continuously support the vertebrae beneath the transverse processes. This new design eliminates the vertebral slippage that is a well known 160 Chapter 5 Discussion & Conclusion criteria for excluding animals from an experiment following contusion injuries (Stokes et a l , 1992; Jakeman et al., 2000; Scheff et al., 2003). These cervical vertebral clamps have also been utilized with other injury devices such as the Ohio State University impactor where cervical hemi-contusion injuries with impact forces of 1.6 \u00C2\u00B1 0.2N (SD) have been found to produce highly repeatable behavioural deficits (Plunet et al., unpublished observations). In addition, this new clamping strategy has also been successfully adapted to the thoracic spine (Sjovold et al., 2005). In this thesis, the variability of the contusion forces was on the order of 25% which is somewhat higher than the 5-20% reported by others who have modelled thoracic contusion injuries (Bresnahan et al., 1987; Somerson and Stokes, 1987; Bresnahan et al., 1991; Behrmann et al., 1992). This increase in variability may stem from the infusion of dextran tracers into the cerebrospinal fluid (CSF). In thoracic spinal cord contusions, sectioning of the dura in order to release CSF prior to impact results in a - 5 0 % reduction in the impact force (n = 1, A . M . Choo et al., unpublished observations). Hence, the infusion o f dextran into the C S F may introduce some variability into the pressure within the subarachnoid space. In the cervical contusion injuries modelled, the 0.3N contact force used to define the initial position of the impactor was observed to be immediately fully reversible thereby suggesting dextran infusion did not disrupt CSF dynamics. Given that the focus of the study was to compare differences between injury mechanisms, an increase in the within-group variability was deemed an acceptable compromise in order to gain insight into the distribution of primary membrane compromise which was afforded by the infusion of dextran-conjugated fluorophores. A l l animal models, including the novel set developed in this thesis, possess strengths that are ideally suited for distinct objectives. Although, spinal cord transections are believed to be rare (Norenberg et al., 2004), these models are important for the clear demonstration of axonal regeneration (Kwon et al., 2002). Similarly, slow contusion or compression injuries might produce a more focal lesion that involves less variability in cellular damage and thus could enhance the detection o f therapeutic benefits. The novel models developed in this thesis are surgically more invasive than thoracic contusion injury paradigms and require more extensive post-traumatic care because the lesions are in the cervical region. Consequently, these new models may be inefficient for screening of pharmacotherapies. However, the models add a new dimension\u00E2\u0080\u0094the biomechanical injury mechanism\u00E2\u0080\u0094to the range of injury paradigms available for research. 161 Chapter 5 Discussion & Conclusion Typically, animal models are designed to possess mild, moderate, and severe injury grades (Rivlin and Tator, 1978; Behrmann et al., 1992; Jakeman et al., 2000; Scheff et al., 2003). Therapeutic strategies are often honed at moderate injury severities because in mild injuries, control animals recover as well as treated animals whereas in severe injuries, neither treated nor control animals recover. Additional injury mechanisms may provide the flexibility to produce different damage patterns while maintaining an overall level of moderate functional deficits thereby augmenting the repertoire o f models for assessing the robustness of novel therapies prior to clinical trials. 5.3 Biomechanics of Primary Injury Patterns Primary mechanical injury is generally deemed to be irreversible and hence studies have typically focused on mitigating secondary pathomechanisms as well as enhancing repair and regeneration strategies. Primary injury in spinal cord contusions are characterized by central hemorrhagic necrosis (Balentine, 1978a; Rawe et al., 1978) to the highly vascularized gray matter with prominent sparing of the surrounding white matter tracts (Blight, 1983; Bresnahan et al., 1991). The primary injury pattern in other clinically relevant injuries had not been addressed to determine the role o f the biomechanical injury mechanism in the pathophysiology of SCI. In traumatic SCI, the primary mechanical injury occurs on the order of milliseconds thereby making it difficult to capture the dynamic strain distribution within the spinal cord. Mechanical trauma has been shown to induce breaches in the cellular plasma membrane which can be detected by the intracellular penetration of macro molecules (Pettus et a l , 1994; Shi and Pryor, 2002). In vitro neuronal stretch models demonstrated that lOkDa dextrans were able to differentiate moderate injury severities (Geddes et al., 2003). Hence, it was hypothesized that the spatial distribution of primary injury could be captured by analyzing the patterns o f intracellular dextran penetration immediately after trauma before secondary degenerative events had progressed. 5.3.1 Contusion Following contusion, the distribution of primary damage was similar to that widely reported in thoracic contusion models with primary hemorrhage focused in the central gray matter (Balentine, 1978a; Rawe et a l , 1978). Membrane compromise in both the gray and white matter was localized within 1 to 2mm from the lesion epicentre (Figures 3.6 and 3.7). The biomechanical basis for this pattern has been discussed by Blight and Decrescito (Blight and Decrescito, 1986; 162 Chapter 5 Discussion & Conclusion Blight, 1988) who used a gel filled cylinder with ink tracks to show that the maximum rostro-caudal displacement of tissue occurred centrally (Figure 5.1 A ) . Blight & Decrescito india ink tracks \u00C2\u00A9 \u00C2\u00A9 \u00C2\u00AE -' ' (7) tensile strains or shear strains at the edge of the impactor can produce dorsal axotomy (j) compressive stresses damage vasculature & displace fluid (3) tensile (Poisson) strains produce tissue failure (4) opposite shear strains produce symmetry at epicentre D Figure 5.1: Central damage pattern in contusion injuries A. Deformation pattern of a gelatin model of the spinal cord under compression, adapted from Blight and Decrescito, Neuroscience 19(1) 1986, who used india ink tracks to show that gel displacement during compression was greatest at the centre of the gelatin filled tube. B. Analysis of the deformation pattern shows distinct biomechanical strain regions. Axons on the surface (zone 1) experience tensile strains, though shear strains may also be present at the contact point between the impactor's edge and the spinal cord. The injury is symmetric about the line of impact with compressive stresses (zone 2) resulting in substantial rostro-caudal and lateral tensile strains (zone 3) since neural tissue is often considered incompressible. Away from the lesion epicentre, shear strains dominate (zone 4). C. Photomicrograph of parasagittal section stained with H&E from an animal perfused immediately following contusion injury shows the pattern of primary hemorrhage. D. Photomicrograph of parasagittal section stained with anti-GFAP to reveal reactive astrocytes at 3 hours following contusion injury shows central pattern of primary damage and surrounding reactive astrocytes. Arrows in C and D highlight discontinuity in damage between gray and white matter. Scale bars 500pm in C and D. (Illustration in A \u00C2\u00A9 IBRO, 1986, adapted by permission) Blight and Descrescito proposed that this central tissue displacement accounted for the sparing of subpial axons and the progressive loss of central white matter axons which is observed in the chronic SCI lesion (Blight and Decrescito, 1986; Blight, 1988). In another study, compression of isolated white matter strips resulted in greater membrane compromise at the centre of the white matter strips compared to the superficial fibres (Shi and Pryor, 2002). More recently, finite element analysis of the mechanics of contusion has indicated a similar pattern of von Mises 163 Chapter 5 Discussion & Conclusion strains (average of three-dimensional strains) focused centrally within the spinal cord (Greaves et al., 2004). The finite element analysis also showed stress concentrations at the superficial surface of the spinal cord at the point of contact with a cylindrical impactor (Greaves et al., 2004). Somewhat counterintuitively, the superficial axons at the site of contact with the impactor are usually spared except in cases of deeper impact displacement into the spinal cord (Blight, 1988). Impactors with a beveled edge are often used to reduce shear stress concentrations at the contact point between the edge of the impactor and the spinal cord (Stokes et al., 1992). In this thesis, a spherical head impactor was selected to further minimize these edge effects. Further analysis of the displacement pattern of the gel filled tube shows there are distinct biomechanical injury zones during contusion (Figure 5.IB). Subpial axons in the dorsal column are subjected to tensile strains, though the local strain distribution at the point o f contact w i l l depend on the geometry of the impactor (zone 1 in Figure 5.IB). Neural tissue is generally considered to be incompressible (King et al., 1995) and hence, dorso-ventral compressive stresses (zone 2 in Figure 5.IB) wi l l result in substantial tensile Poisson strains (zone 3 in Figure 5.IB) that stretch tissue rostro-caudally and laterally. There is some symmetry along the line of impact though the true strain field in the transverse plane wi l l be complicated by geometric factors such as the shape of the dorsal and ventral gray horns. The tensile strains (zone 3 in Figure 5. IB) along the line of impact have lead some to the hypothesize that white matter is predominantly injured in tension (Fiford et al., 2004; Henderson et al., 2005). Although tension certainly produces axonal injury (Smith et al., 1999; W o l f et al., 2001), shear strains (zone 4 in Figure 5. IB) along the axis o f axons are likely also important in white matter injury. Away from the centre of impact, shear strains may be responsible for damage to myelin laminae which have been reported to exhibit early ultrastructural disruption, sometimes in the presence of an intact axolemma (Balentine, 1978b; Anthes et al., 1995). In this thesis, dextran-conjugated fluorophores often accumulated at nodes of Ranvier thereby highlighting them. A s reported in chapter 3, however, the zones flanking the contusion epicentre (e.g. zone 4 in Figure 5.IB) were often devoid of dextran positive axons as well as visible nodes of Ranvier. This observation may suggest that in the penumbra of the contusion lesion epicentre, longitudinal shear strains disrupt the organization of myelin at the nodes of Ranvier without causing the axolemma compromise that leads to intracellular dextran penetration. 164 Chapter 5 Discussion & Conclusion This analysis of the central displacement pattern during contusion can also apply to the gray matter. Primary hemorrhage was localized to the compressive region (Figure 5.1C) while secondary astrocyte activation extended into the lesion penumbra (Figure 5.ID). Interestingly, a discontinuity in tissue damage was often encountered at the gray and white matter tissue boundary (arrows Figure 5.1C & D) in contrast to the continuous pattern of gel deformation discussed above (Figure 5.1 A ) . This phenomenon may reflect the greater susceptibility of gray matter to mechanical damage compared to white matter. Several lines of evidence predict injury during contusion wi l l initiate in the central gray matter due to its inherent susceptibility to primary damage. In vitro evidence suggests gray matter fails at lower tensile stress levels than white matter (Ichihara et al., 2001), though the relative strength of these two tissue components remains uncertain (Ozawa et al., 2001). The gray matter is more highly vascularized than the white matter rendering it more susceptible to hemorrhagic necrosis when the impact speed is high enough (Anderson, 1985). In addition, the blood-spinal cord barrier exhibits a lower mechanical injury threshold in the gray matter compared to the white matter (Maikos and Shreiber, 2007). In this thesis, neuronal somata in the vicinity o f the surgical exposure of sham control animals exhibited some susceptibility to primary membrane compromise (Figure 3.7A) in contrast to white matter axons (Figure 3.6). In addition, the extent of plasma membrane resealing appears to be slower in neuronal cell bodies than axons (Figure 4.4). In summary, contusion impacts produce a central zone of compressive strains and tensile Poisson strains encapsulated by a region of shear strains. These strains produce hemorrhagic necrosis at the contusion epicentre which is flanked by sublethal zones of neurons and axons that exhibited primary membrane compromise. Shear strains in the lesion penumbra may be responsible for primary disruption of myelin structure at the nodes of Ranvier as well as activation of secondary effects such as reactive astrocytes. In addition to the pattern of biomechanical strains, the primary injury is also influenced by the apparent susceptibility of gray matter to injury. The net result is a focal region consisting of both necrotic and salvagable populations of neurons, axons, and glia. 5.3.2 Dislocation The dislocation injury resulted in primary hemorrhagic necrosis within the gray matter similar to that observed in contusion, but spinal cords injured by a dislocation mechanism also 165 Chapter 5 Discussion & Conclusion exhibited primary injury patterns that were unique from the central cavitation of the contusion model. Primary axotomy in the lateral funiculi was a distinctive feature o f the dislocation model that was not observed in either of the other two injury mechanisms. During dislocation, the lateral white matter column is sheared between the vertebral margins of C4 and C5 (Figure 5.2A). Given the high prevalence of dislocation injuries in the human population, this feature of the dislocation injury mechanism may suggest some axotomy occurs during clinical fracture-dislocations which is not evident with post-traumatic imaging. Damage to the lateral aspect of the spinal cord has also been reported in a model of thoracolumbar dislocation where the vertebrae are dislocated laterally (Fiford et a l , 2004). However, the biomechanics of this thoracolumbar model differs from the cervical fracture-dislocation developed in this thesis. In the thoracolumbar model, the lateral dislocation was distributed over T12, T13, and L I . Further analysis of this model has shown this distributed dislocation results in two injury epicentres at the junction of T12-T13 and T13-L1 (Clarke et al., 2006). The region between T12 and L I appear to be under tensile strain that produces diffuse hemorrhage in the lateral gray matter (Fiford et al., 2004) in contrast to the lateral white matter axotomy observed following the C4/5 dislocation injuries modelled in this thesis. In addition, dislocation between T12 and L I produced tissue failure in the T13 region (Clarke et al., 2006) along an axis that indicates tissue rupture occurs in tension (arrows Figure 5.2B). 166 Chapter 5 Discussion & Conclusion reference marks . T12 Spinal Cord . Tensile Tissue Failure B Distributed Thoracolumbar Dislocation Model Spinal Cord C5 Shear, C4 D ventral Unconstrained Displacement Vertebral Body I Contact Forces l t t t t t t t f f T 1 i i Lamina Unconstrained Displcement -< Tissue Failure r \u00E2\u0080\u00A2 I I Figure 5.2: Injury patterns following fracture-dislocation A. Illustration of C4/5 vertebral dislocation that results in shearing of the lateral white matter funiculi (arrows). B. Illustration of distributed thoracolumbar dislocation (T12-T13-L1) where tissue fails in tension (arrows) producing an oblique pattern of tissue rupture. C. Photomicrograph of parasagittal section stained with H&E from an animal perfused immediately following C4/5 dislocation injury shows central hemorrhage with some evidence of primary tissue failure in an oblique plane opposite to that expected of tensile failure (arrowheads). D. Photomicrograph of parasagittal section stained with anti-GFAP to reveal reactive astrocytes at 3 hours following dislocation injury shows oblique pattern of tissue failure that is also opposite to the hypothetical principal tensile plane (arrowheads). E. The strain distribution within the spinal cord is complex, but the pattern of injury may be explained by the unconstrained movement of the free surfaces and shear failure between the narrow margins of C4 and C5. F. Hypothetical tissue failure pattern if the spinal cord is fully constrained (i.e. via greater cranial flexion that increases spinal cord axial tension). Illustration in B is adapted from data in Clarke et al. J Neurotrauma 23, 2006. Circles and squares are reference marks for tracking deformation. Scale bars 500|im in C and D. 167 Chapter 5 Discussion & Conclusion In the C4/5 dislocation modelled in this thesis, the gray matter usually\u00E2\u0080\u0094though not always\u00E2\u0080\u0094failed along an oblique plane that might suggest a shear mode of tissue failure rather than a tensile mode (arrowheads Figure 5.2C & D). The internal strains within the spinal cord are complex and vary rostro-caudally as well as laterally. The cervical spinal cord (~2><3mm) is not fully constrained within the vertebral canal (~3><4.5mm) and this may allow unconstrained displacement of the free surfaces (i.e. surfaces without vertebral contact) during dislocation that results in the oblique failure pattern often observed (Figure 5.2E). In this thesis, the angle of the rat's cranium within the stereotaxic frame was standardized such that the dorsal surface of the skull was vertical, thereby placing the head in flexion and removing cervical lordosis. A n increase in flexion angle should increase the axial tension in the spinal cord (Breig, 1970) which may constrain the spinal cord and alter the angle of tissue failure (Figure 5.2F). These differences in failure patterns demonstrate the need to understand contrasting injury patterns even within seemingly similar injury mechanisms. In this thesis, the intracellular penetration of dextran-conjugated fluorophores was used to map the distribution of primary mechanical damage within the spinal cord. This technique gave an indication o f the traumatic strain distributions, but the technique does not yield information regarding the local strain components (i.e. local tension, compression, and shear) relative to the orientation of the cells and their processes. The local strain field sensed by individual neurons was recently analysed in vitro (Cullen and LaPlaca, 2006). Neurons cultured in a three-dimensional matrix exhibited greater cell death following shear strain trauma compared to neurons cultured in a planar configuration. A n analysis of the orientation of the neurite processes extending from the neuronal cell body showed that the neurites of neurons cultured in three-dimensions sampled a broader range of shear, tensile, and compressive strains. Consequently, neurons cultured in three-dimensions were more likely to experience a broader range of traumatic strains thereby increasing the likelihood of neuronal injury. The cervical dislocation modelled in this thesis also produced a distinctive rostral bias in the membrane compromise particularly in the ventral white matter (Figure 5.3A). This pattern of membrane compromise suggests a region of tensile strain rostral to the C4/5 epicentre (label 2 Figure 5.3B). A complementary caudal bias was not detected in the dorsal white matter. This is likely due to the reduced sensitivity of detecting fine caliber dextran-positive axons. A s discussed 168 Chapter 5 Discussion & Conclusion in chapter 3, dextran fluorescence above and below fine processes and smaller cell bodies reduces the sensitivity of discriminating the intracellular level of dextran penetration relative to the background. Contusion Dislocation \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 ' Distraction 1 * Sham Lamina A-2 0 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Figure 5.3: Ventro-medial axolemma compromise following dislocation injury A. Rostro-caudal pattern of primary axolemma compromise evidenced by the intracellular penetration of lOkDa fluorescein-dextran in ventro-medial axons from animals euthanized immediately following dislocation injury (repeated from chapter 3). B. Expected distribution of contact loads (arrowheads) delivered to the spinal cord during fracture-dislocation. Maximum number of dextran-positive axons are located near the lesion epicentre (/). Tensile forces within the ventral aspect of the spinal cord result in membrane compromise rostral to the lesion epicentre (2). Inset in A highlights region of tissue sampled. Illustration in B is not to scale and does not include the dura and cerebrospinal fluid. (Graphics in A from: Choo AM, et al. 2007. Contusion, dislocation, and distraction: primary hemorrhage and membrane permeability in distinct mechanisms of spinal cord injury. J Neurosurg Spine 6:261. \u00C2\u00A9 Journal of Neurosurgery, 2007, adapted by permission.) Hence, fracture-dislocation produces a distinct primary injury pattern compared to the focal central damage typically reported in contusion models. The dislocation lesion epicentre shares similarities with both contusion and partial transection models. The rostral membrane compromise, however, is a unique feature of this injury model and demonstrates this injury mechanism produces a broader population of injured cells within the spinal cord. 5.3.3 Distraction In contrast to the contusion and dislocation injury models, distraction injury did not exhibit a focal hemorrhagic lesion epicentre and the extent of membrane compromise was similar at C4/5 and the rostral spinal cord suggesting a more diffuse and uniform injury pattern. The coupling between the vertebral column and the spinal cord remains unresolved. Others have suggested that distractive forces are transmitted to the spinal cord through the denticulate ligaments (Tani et al., 1987). In the dorsal column, membrane compromise was detected in axons between 2mm caudal 169 Chapter 5 Discussion & Conclusion and 2mm rostral to the lesion epicentre (label 1 Figure 5.4A). Some axolemma compromise was also detected between 2 and 4mm rostral to the lesion. This periodic pattern of primary dextran penetration into dorsal axons might suggest that the translating vertebrae apply traction forces through the nerve roots (label 1 & 2 Figure 5.4B). The greater membrane compromise in the ventral column suggests traction forces are mainly transferred from the flexed vertebrae and the ventral aspect of the spinal cord (arrowheads Figure 5.4B). 70 r 60 50 | 40 20 10 -\u00C2\u00AB\u00E2\u0080\u00A2 Contusion -m- Dislocation \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Distraction Sham C4 / Vertebral Body Nerve Roots - 4 - 2 o 2 \u00C2\u00AB Caudal - (mm) - Rostral \u00C2\u00BB Endplate Fracture Figure 5.4: Dorsal axolemma compromise following distraction injury A. Rostro-caudal pattern of primary axolemma compromise evidenced by the intracellular penetration of lOkDa fluorescein-dextran in dorsal axons from animals euthanized immediately following distraction injury (repeated from chapter 3). B. Expected distribution of contact loads (arrowheads) delivered to the spinal cord during flexion-distraction. Maximum number of dextran-positive axons are located near the lesion epicentre (/). The mechanism of coupling between the spinal cord and vertebral column remains uncertain. Tensile forces (arrows show hypothetical distribution) may be transferred to the dorsal column via the nerve roots (2) that are spaced at approximately 2mm intervals. Inset in A highlights tissue region sampled. Illustration in B is not to scale and does not include the dura and cerebrospinal fluid. (Graphics in A from: Choo AM, et al. 2007. Contusion, dislocation, and distraction: primary hemorrhage and membrane permeability in distinct mechanisms of spinal cord injury. J Neurosurg Spine 6:261. \u00C2\u00A9 Journal of Neurosurgery, 2007, adapted by permission.) Maiman et al. used radio-opaque markers to estimate the extent of vertebral column and spinal cord coupling during distraction of cat spines (Maiman et al., 1989a). When the spinal column was placed under 147N (15kg) of distraction, the coupling ratio between the column and cord ranged from 0.38 at C2/3 to 0.62 at C6/7. The difference in coupling ratios may suggest the presence of distributed coupling mechanisms such as spinal nerve roots but could also stem from differences in the cross-sectional area of the spinal cord. The study was not able to isolate the source o f vertebral column-spinal cord force transfer. The coupling between the spinal cord and vertebral column might be estimated by tracking the deformation of superficial vasculature within the spinal cord which is visible through the translucent dura. Glare from changes in reflected 170 C h a p t e r 5 D i s c u s s i o n & C o n c l u s i o n lighting as the cord deforms, camera vibration, and slow camera acquisition speeds are some challenges that need to be overcome in order to realize this method ( A . M . Choo et a l , unpublished observations). Distraction is a frequently observed component of spinal injuries such as flexion-distraction and extension-distraction, and hence, a great deal remains to be learned about the characteristics of this important component of SCI. In the absence of overt hemorrhage, it is possible that spinal cords injured by a distractive injury mechanism may under go a protracted time-course of pathology that results in delayed pathologic features (Maiman et al., 1989b; Dabney et al., 2004). 5.4 Association of Primary and Secondary Injury The injury mechanisms modelled produced different patterns of primary damage, but also altered the characteristics of early secondary pathology. This relationship between primary and secondary injury suggests that a consideration of the injury mechanism may aid in the targeting of post-traumatic neuroprotective as well as repair and regeneration strategies. The interaction between primary and secondary damage mechanisms have been advocated before (Blight, 1988; LaPlaca and Thibault, 1997) and evidence continues to accumulate which shows how mechanical injury alters the course of secondary molecular events (Deridder et al., 2005; Geddes-Klein et al., 2006). 5.4.1 Contusion The secondary pathologies observed following contusion were localized to the vicinity of the lesion epicentre where primary damage was focused. The inflammatory response of activated microglia was localized to \u00C2\u00B12mm (blue circles Figure 5.5B) which corresponds to the central primary hemorrhagic necrosis and distribution of primary membrane compromise of neuronal somata (blue circles Figure 5.5A). Likewise, the rostro-caudal distribution of reactive astrocytes was also predominantly restricted to this \u00C2\u00B12mm injury field. The presence of cytochrome c negative neurons in the lateral penumbra of the lesion, but not in the rostro-caudal penumbra, might stem from a greater primary strain field in the lateral zone because it is rigidly confined by the boundaries of the spinal canal compared to the unconfmed gray matter displacement rostro-caudally along the axis of the spinal cord. In the white matter, axonal degeneration was most prominent near the boundary of hemorrhagic gray matter while subpial axonal fibers that exhibited extensive primary membrane 171 Chapter 5 Discussion & Conclusion compromise were still largely intact at the 3 hour time-point analysed. This observation suggests that primary hemorrhagic necrosis accelerates degeneration of axonal fibers adjacent to the gray matter while subpial fibers are able to repair breaches in the axolemma. The most prominent accumulation of PAPP was found in the dorsal and ventral funiculi (Figure 4.8G & H) along the direct line of the contusion impact and further demonstrates the role of the primary injury in dictating the pattern of secondary pathology. Primary Secondary Membrane Compromise Microglial Activation Figure 5.5: Parallel patterns of primary and secondary injury Distribution of primary mechanical injury was detected by the intracellular penetration of fluorescein-dextran (A & C, repeated from chapter 3) in animals euthanized immediately following injury. Indicators of secondary pathology, such as microglial activation (B & C, repeated from chapter 4), appear to parallel primary injury thereby demonstrating that secondary injury was not yet an independent cascade at 3 hours following SCI. Measurement of primary and secondary injury markers demonstrated mechanism-specific patterns that may aid in the targeting of therapeutic strategies. A. Percent of dextran-positive neuronal somata immediately following injury. B. Ramification index of microglial morphology in the gray matter at 3 hours post-trauma. A lower index represents increased microglial activation. C. Number of dextran-positive axons in the dorsal column immediately following injury. D. Ramification index of microglial morphology in the dorsal column at 3 hours post-trauma. (Graphics in A & C from: Choo AM, et al. 2007. Contusion, dislocation, and distraction: primary hemorrhage and membrane permeability in distinct mechanisms of spinal cord injury. J Neurosurg Spine 6:261. \u00C2\u00A9 Journal of Neurosurgery, 2007, adapted by permission.) 172 Chapter 5 Discussion & Conclusion 5.4.2 Dislocation The secondary degeneration in the dislocation model also closely paralleled the primary mechanical damage observed in this injury mechanism. In the gray matter, the rostro-caudal extent of microglial activation (red squares Figure 5.5B) strongly coincided with the central hemorrhage and rostro-caudal distribution of primary membrane compromise in the gray matter (red squares Figure 5.5A). A distinct rostral bias was observed in the activation of microglia in the dorsal column (red squares Figure 5.5D). This feature may be related to the slight rostral bias in membrane compromise in dorsal axons (red squares Figure 5.5C) but could also stem from hemorrhagic necrosis in the dorsal gray horns that are likely subjected to a contusive impact with the C4 lamina. A s with the microglia, reactive astrocytes also extended rostrally thereby paralleling the asymmetric distribution of primary injury in the dislocation injury mechanism. In contrast to contusion, cytochrome c negative neurons were not located in the lateral penumbra of the lesion but instead were localized to the rostral and caudal boundaries of the lesion epicentre. In the lateral penumbra of the lesion, cytochrome c-negative neurons were not observed due to the extensive necrosis in this region. The pattern of secondary white matter damage following dislocation further reduced the population of available axons that might be salvaged with neuroprotection. In the lateral funiculi, primary axotomy lead to P A P P accumulation in transected axons, but the surrounding spared white matter also exhibited extensive axonal transport dysfunction resulting from the high shear strains in this \"lateral epicentre\" following vertebral fracture-dislocation. A l l white matter tracts exhibited extensive PAPP accumulation following dislocation though it is not clear why less P A P P accumulation was detected in the dorsal ascending columns (Figure 4.8H). Although the boundary conditions likely differ between the dorsal and ventral columns, a similar disparity in PAPP accumulation was observed in the contusion model and suggests this may be a physiological difference between these axonal populations. In the ventro-medial white matter, axonal degeneration was observed to extend outward from the hemorrhagic gray matter, as well as inward from the subpial rim. This suggests that in the dislocation injury mechanism, primary gray matter hemorrhage and shear strains of ventral axons may combine to narrow the therapeutic time-window available for this population. This may emphasize that urgent treatment priority should be given to axonal cytoskeletal protective strategies such as calpain inhibition (Stys and Jiang, 2002; Zhang et al., 2003; Thompson et al., 2006). 173 Chapter 5 Discussion & Conclusion 5.4.3 Distraction Although distraction injury exhibited extensive rostro-caudal membrane compromise in both the gray and white matter, the overall extent of secondary pathology was much less than that observed in both the contusion and dislocation injury models. The patterns of secondary degeneration following contusion and dislocation emphasized that vascular damage exacerbates processes such as microglial activation and axonal degeneration. Little overt mechanical damage to vascular structures was detected in the distraction model and this appears to be an important factor in the early progression of secondary damage. Vascular damage was widely studied in the past (Balentine, 1978a; Rawe et al., 1978; Anderson, 1985; Khan et al., 1985; Fehlings et al., 1989; Guha et al., 1989; Noble and Wrathall, 1989) but has received less attention recently as the field has gravitated towards the precision afforded by molecular approaches. It has been suggested that it would be beneficial to further investigate the role of hemorrhagic factors in SCI research (Norenberg et al., 2004). Following distraction, some evidence of increased neurofilament dephosphorylation was detected though complete cytoskeletal degeneration was not observed suggesting a high tolerance of axons to mechanical injury when hemorrhagic factors are absent. This tolerance to trauma could be a consequence of both axolemma resealing (Figure 4.4D) and the lack of overt primary intramedullary hemorrhage (Figure 3.4). Recovery of axons to stretch injuries has been widely observed in both ex vivo (Shi et al., 2000; Shi and Pryor, 2000, 2002) and in vitro (Galbraith et al., 1993; Smith et al., 1999) injury preparations where hemorrhage is absent. Indeed, axons o f cultured dorsal root ganglion can be elongated up to 8mm/day without any obvious pathology (Pfister et al., 2004). Neuronal somata also exhibit membrane resealing characteristics (Prado and LaPlaca, 2004) though at sub-lethal levels, they may be more vulnerable to delayed secondary insults (Arundine et al., 2004; Geddes-Klein et a l , 2006). 5.4.4 Summary of Primary and Secondary Injury B y analysing an early post-traumatic time-point, it was possible to identify early secondary injury features that relate to the mechanism of SCI. The patterns of primary mechanical tissue damage immediately restrict the effectiveness of neuroprotective strategies. From a secondary injury perspective, the primary mechanism of injury alters the rostro-caudal distribution of salvageable populations. Following contusion, it is thought that secondary factors spread outward 174 >ter 5 Discussion & Conclusion to destroy otherwise healthy tissue. In dislocation and distraction, however, the sublethal injury in the rostral and caudal zones may already prime these cells for delayed apoptosis (Arundine et a l , 2004) or heighten their sensitivity to secondary insults (Geddes et al., 2003). Thus, the cells in these sublethal zones are distinct from necrotic populations as well as naive uninjured populations. The balance between different cellular populations could, hypothetically, be crucial in the calculation to determine which treatment combination wi l l preserve the maximum number of neurons, axons, and glia. A s the lesion grows over time, it may be increasingly difficult to detect functional differences between injury mechanisms. Degeneration of the lateral column following contusion would likely result in similar deficits as primary axotomy following fracture-dislocation. Indeed, the pattern of membrane recovery in the white matter at 3 hours already masks the primary injury distribution. However, these effects should not diminish the significance of the injury mechanism. For example, though the lateral columns may eventually deteriorate in both the contusion and dislocation injury mechanisms, the rostro-caudal extent of the injury is still critical. Greater rostro-caudal injury following dislocation may translate to greater axonal die-back requiring a longer distance for axonal regeneration. Alternatively, sublethal biomechanical injuries in the rostro-caudal zone may increase the extent of oligodendroglial apoptosis and the resultant dysmyelination or demyelination of spared fibers. In addition, in the cervical spine, a rostral degeneration of gray matter has profound implications for the neurons in this region which control forelimb and respiratory function. A n SCI treatment needs to save a diverse population of motoneurons, sensory neurons, interneurons, large axons, small axons, myelinated axons, unmyelinated axons, and supporting glia, amidst a cascade of interrelated and sometimes competing events. Calcium entry precipitates secondary degeneration (Young, 1992), yet, calcium entry is also needed for membrane resealing (Shi et al., 2000; Prado and LaPlaca, 2004). Oxygen is essential to maintain respiration, yet reoxygenation produces more free radicals (Stys, 2004). Inflammation risks bystander damage, but debris clearance is an essential phase of repair. The mechanism of injury alters the initial distribution of these populations and so may serve as a rationale for identifying treatment strategies. 175 Chapter 5 Discussion & Conclusion 5.5 Clinical Relevance It is understood and accepted that experimental animal models can only reflect an idealized typical human SCI (Tator, 2006). Human clinical trails are therefore necessary in order to prove the efficacy of novel therapies. In recent years, the rodent-to-human paradigm for testing pharmacotherapies has increased in prevalence compared to the traditional progression of testing therapies in higher mammals (Tator, 2006). The work presented in this thesis demonstrates that it is technically feasible to examine other types of clinically relevant injury mechanisms, and more importantly, that these injury mechanisms alter the characteristics of the SCI lesion. Hence, pre-clinical therapies designed in a contusion or compression injury paradigm, may not directly apply to the broader range of injury mechanisms encountered clinically. Pre-clinical therapies have had limited success in clinical trials and thus it is imperative that any novel strategy prove its robustness in alternate clinically relevant injury paradigms before making the leap from rodents, to higher mammals, to humans. The results in this thesis further highlight the importance o f the early post-traumatic period as a critical time-window for intervention. The results showed that vertebral fracture-dislocation consistently produced primary axotomy in the lateral funiculi and white matter pathology that was further exacerbated by rapid axonal cytoskeletal degeneration at the shear epicentre. This pattern of white matter damage may explain the greater deficits often associated with vertebral dislocations (Kiwerski, 1991) while the rostro-caudal injury distribution may hypothetically contribute to some instances of ascension of the SCI lesion (Harrop et al., 2001). The mechanism of SCI may be inferred from the type of vertebral column injury that is visible on radiographs and magnetic resonance images. From the primary and secondary injury patterns found in this thesis, the injury mechanism may give clinicians an indication of the distribution of neuropathology within the cord and eventually aid in the development of therapies targeted to salvage these cellular populations that are established in a mechanism-specific manner. 176 Chapter 5 Discussion & Conclusion 5.6 Limitations This thesis focused on the primary mechanical injury and the secondary events that were initiated soon after trauma. A s time elapses after injury, secondary events contribute to the characteristics of the lesion thereby confounding the ability to specifically attribute differences to either the initial mechanical insult or secondary factors. It is important to distinguish between these two components as some characteristics of the primary insult, such as direct axotomy, immediately constrain the amount of tissue that can be spared by therapeutic intervention. Hence, it was reasoned that primary mechanical injury should first be characterized and then its relation to early secondary injury could be delineated at later time-points. The feasible time-window to exploit differences between injury mechanisms could be determined by systematically lengthening the survival time. This rationale was supported by both experimental (Rosenberg and Wrathall, 2001; Gris et al., 2004) and clinical (Bracken et al., 1990; Bracken et al., 1997) observations that earlier treatment was often more effective at preserving post-traumatic function. The short survival time analyzed in this thesis introduced several limitations. Post-traumatic behavioural function was not assessed. Cervical SCI are more severe than thoracic injuries and thus ethically necessitated that relevant differences be first established at short survival times. Cervical SCI models have some advantage over thoracic models. Approximately 55% of SCIs occur in the cervical region in humans (Sekhon and Fehlings, 2001). In addition, among quadriplegics, restoration of upper limb function\u00E2\u0080\u0094rather than walking\u00E2\u0080\u0094has been identified as the most important treatment priority (Anderson, 2004). Cervical injury paradigms are more sensitive for testing both gray and white matter neuroprotective strategies than thoracic lesions which are largely insensitive to gray matter sparing (Onifer et al., 2005). Also, the mobility of the cervical region facilitated modelling SCIs at clinically relevant injury velocities and this was an important consideration given the clinical rationale behind comparing contusion, dislocation, and distraction injury mechanisms. Modelling injuries such as vertebral dislocation in the thoracic region are not without some drawbacks. A deeper surgical exposure is necessary in order to hold the vertebral body in this region since the transverse processes may not have sufficient strength to support a high-speed fracture-dislocation (~38N chapter 2 table 1). Given this invasiveness, longer-term survival in thoracic dislocation injuries has also been difficult to achieve (Clarke et al., 2006). 177 Chapter 5 Discussion & Conclusion This thesis aimed to compare clinically relevant SCI mechanisms using a rat model, though there are clearly differences between rats and humans. In the rat, the dorsal corticospinal tract located in the deep dorsal column was found to be extensively injured in both the contusion and dislocation models. In humans, however, the main corticospinal tract is located in the dorso-lateral funiculus\u00E2\u0080\u0094a region which was pooled with the lateral column in this thesis. There are also some differences in the cellular response between rats and humans. In rats, the infiltration of neutrophils during the early inflammatory response peaks at 1 day whereas in humans, the number of neutrophils peaks between 1 and 3 days post-trauma (Fleming et al., 2006). In addition, humans exhibit less demyelination than has been observed in rat models (Norenberg et al., 2004). In spite of these differences, rats, along with mice, are widely used as they are currently the most practical in vivo models of experimentally modelling SCI. This thesis did not assess pre-clinical therapies. The greater rostro-caudal microglial response observed in the dislocation model could suggest a greater therapeutic benefit from anti-inflammatory treatment such as minocycline (Stirling et al., 2004; Teng et al., 2004) which is currently under clinical investigation (Baptiste and Fehlings, 2006). In addition, calpain inhibitors have previously been shown to spare white matter and improve behavioural function following contusion (Schumacher et al., 2000) and this treatment strategy may have a greater efficacy in a dislocation injury paradigm. The investigation of pre-clinical interventions must be deferred until long-term survival has been established. The three injury mechanisms analysed\u00E2\u0080\u0094contusion, dislocation, and distraction\u00E2\u0080\u0094are themselves still only representative of a portion of SCI mechanisms encountered in humans. Human SCI occur in different combinations such as flexion-distraction with facet dislocation or extension-distraction. In addition, torsion also occurs in human SCI resulting in unilateral facet dislocation (Magerl et al., 1994). Hence, the mechanisms modelled here were chosen as stereotypical vertebral fracture patterns that cover a range o f clinically relevant injuries to the spinal cord though there are many other injury mechanisms that merit investigation. 5.7 Recommendations The injury models should be further developed in order to examine additional biomechanical aspects. In the contusion model, the correlation between the impact force and the hemorrhage volume over a relatively narrow range of displacements might suggest vascular 178 Chapter 5 Discussion & Conclusion perfusion pressure, which is not typically regulated in experimental models, might contribute to the undesirable variability observed in contusion models (Scheff et al., 2003; Ghasemlou et al., 2005; Kloos et al., 2005). In addition, both the dislocation and distraction models could benefit from additional pressure sensors (Chavko et al., 2007) to provide force feedback from within the spinal canal. In the distraction model, the coupling between the vertebral column and spinal cord remains an important quantity that should be determined in order to better understand the mechanics of distractive force transfer to the cord. In addition, this thesis did not vary the extent of axial tension on the spinal cord prior to injury. Traction on the spinal cord has been shown in a small series of animals to increase the cord's susceptibility to compression injury (Fujita and Yamamoto, 1989). Hence, the extent of cranial flexion, or extension, may change the pattern of spinal cord damage as hypothesized in the discussion on failure patterns following fracture-dislocation (Figure 5.2). Although the recommended approach for the near future would be to further develop the existing models, the multi-mechanism injury system was designed to be a flexible device that can be adapted to model injury mechanisms beyond contusion, dislocation, and distraction. The devices developed have already been adapted to produce oblique hemi-contusions (Choo and Tetzlaff, unpublished observations), cervical hemi-contusions (Plunet, L i u , and Tetzlaff, unpublished observations), thoracic contusions with residual compression (Sjovold et al., 2005), as well as thoracolumbar fracture-dislocation (Clarke et al., 2006). Hyper-extension is also a common injury mechanism associated with falls (Pickett et al., 2006) that was not addressed in this thesis that should likely be investigated. From a neurobiology perspective, a survival model, particularly for fracture-dislocation would be an important next step given the high prevalence of this injury mechanism in humans. Some important questions would be to determine i f the rostro-caudal increase in primary membrane compromise eventually translates to greater oligodendroglial apoptosis, axonal die-back, demyelination, and a broader chronic glial scar. Also important might be to determine i f the axonal cytoskeletal degradation near the lesion epicentre might be amendable to treatment by early calpain inhibition (Banik et al., 1998; Schumacher et al., 2000). Clinically, a retrospective review of patients stratified by injury mechanism might be used to compare the pattern of functional deficits with the distribution of primary and secondary damage 179 Chapter 5 Discussion & Conclusion observed in this thesis. A reanalysis of the results of the National Acute Spinal Cord Injury Studies might yield interesting results regarding the efficacy of methylprednisolone in mechanism-specific patient populations. 5.8 Contributions In the broader scope, this thesis attempts to bridge the relation between the biomechanics of injury and the neurobiology of SCI through animal models that bear closer resemblance to the injuries encountered clinically in humans. The significance of the work can only be assessed as the knowledge matures over time. It is hoped that this work contributes some small perspective on the broad range of SCI in humans and stimulates thoughts about alternative approaches to solving this devastating injury. Some specific contributions include: 1. The development of a novel multi-mechanism injury system. The device can be used to model a broad range of injuries in addition to those analysed in this thesis. 2. The development o f three clinically relevant cervical SCI models. This is the first development of vertebral clamps for delivering distinct high-speed injuries to the rat vertebral column. This is the first development of dislocation and distraction injuries in the rat cervical spine. 3. The characterization of primary injury fields in contusion, dislocation, and distraction injuries. This established a comparative reference map of how traumatic injury is distributed in these injury mechanisms. 4. The characterization of similarities and differences in early secondary injury resulting from differences in the mechanism of spinal cord injury. 5. The development of a novel method to quantitatively analyse the morphology of microglia during activation. Although the method is a minor aspect of this thesis, it may be of general interest in other areas of neuroinflammation. 180 Chapter 5 Discussion & Conclusion 5.9 Conclusion In humans, spinal cord injury can occur by a range of injury mechanisms that have not been previously compared experimentally. Three new animal models were concurrently developed in this thesis to compare three clinically relevant injury mechanisms\u00E2\u0080\u0094contusion, dislocation, and distraction. The results showed that the mechanism of injury dictates the pattern of primary mechanical damage within the spinal cord which in turn alters the initial characteristics of secondary neuropathology. The primary and secondary neuropathology in human SCI is more diverse than the central cavitating lesion produced by the commonly used contusion and compression injury paradigms and hence pre-clinical therapies should be tested in a range of injury mechanisms before human trials. 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J Neurotrauma 20:59-67. 186 Appendix A S C I T E S T S Y S T E M 187 Appendix A SCI Test System 5.11 Test System Assembly Base z-coupl ing Tower Armature z-Axis xy-Table Surgical Table Actuator Complete System Figure A . l : Test system assembly sequence 188 Appendix A SCI Test System 5.12 Test System Drawings does not Include radius Material: Aluminum Tolerance: +/- 0,001\" Figure A.2: Accelerometer housing Flange f o r b a l l - n o s e sp r i ng p lunger F lange f o r b a l l - c a t c h e s Mate r ia l ! 304 o r 316 S t a i n l e s s S t e e l T o l e r a n c e s : nominal + / - 0.005' n o n - c r i t i c a l dins Figure A.3: Rotary detent 189 Appendix A SCI Test System 5.13 Sensors 20 15 ^ 10 Z i 5 c 3 0 01 CC 1 - 5 TO c _J - 1 0 - 1 5 - 20 Calibration Model 31: 22.5N 1 1 -20 - 1 5 - 1 0 - 5 0 5 Load Applied (N) 10 15 20 Applied (N) -19.3387 -14.6707 -9.8851 -4.7856 4.7856 9.8851 14.6707 19.3387 Readout (N) -19.353 -14.694 -9.909 -4.795 4.801 9.921 14.715 19.415 RMS Error = 0.0366 N Percent Error = 0.1627% FS Slope = 1.0024 y-intercept = 0.0126 Max Non-linearity = 0.0201 N Percent Max Non-linearity = 0.0894% FS Figure A.4: Load cell calibration model 31: 22.5N Calibration Model 31:225N 100 s-s 50 -o \u00E2\u0080\u0094 TO 01 \u00E2\u0080\u0094 o -100 -100 Applied (N) -90.7213 -69.2055 -45.0027 -24.2028 -19.3387 -14.6707 -9.5713 -4.7856 4.7856 9.5713 14.6707 19.3387 24.3303 42.9139 67.2442 87.2988 100 Load Applied (N) Readout(N) -90.51 -69.08 -44.83 -24.13 -19.31 -14.65 -9.53 -4.76 4.75 9.52 14.62 19.29 24.26 42.85 Percent Error = 0.0393% FS Slope = 0.9988 y-intercept = 0.0304 Max Non-linearity = 0.1338N Percent Max Non-linearity = 0.0595% FS RMS Error = 0.0885N 67.33 87.33 Figure A.5: Load cell calibration model 31: 225N 190 Appendix A SCI Test System Figure A . 6 : Load cell calibration model 208C02: 444N 191 Appendix B I M A G E ANALYSIS 192 Appendix B Image Analysis 5.14 Image Analysis Tools Figure B . l : StereolTools interface and screen images A. StereolTools interface built in Matlab. Upper window is browser for selecting the image specimen, tract, series, and rostro-caudal position. Automated processing option runs software code to analyse the particular image series (e.g. detect neurons, detect axons, classify neurons, colocalize etc.). Blind mode codes the specimen number based on a cipher file that is opened after the completion of the analysis. Lower window shows quantification controls for laying down probes and counting objects. B. Representative screen shot showing gray matter immunostained for fluorescein-dextran, cascade-blue dextran, and 3-nitrotyrosine. C. Representative example of hemorrhage quantification using point probes. A high point density is necessary to achieve accurate area measurements, though accurate volume estimates can also be achieved with a low density of point probes and a greater number of sections. Simple automated algorithms such as colour segmentation and morphological feature detection accelerates quantification and is most efficient when combined with manual verification. 193 Appendix C A N O V A T A B L E S 194 Appendix C ANOVA Tables Table C. l : ANOVA summary - Acute dextran penetration into axons Sum of Degrees of Region Effect Squares Freedom Mean Square F P Dorsal Column Mechanism1 181.399 2 90.699 7.309 0.003 Error 297.821 24 12.409 Position 930.603 10 93:060 45.954 <0.001 Mechanism x Position 221.739 20 11.087 5.475 <0.001 Error 486.020 240 2.025 Lateral Column Mechanism1 6.239 2 3.119 0.542 0.589 Error 138.045 24 5.752 Position 581.903 10 58.190 35.192 O.001 Mechanism x Position 240.671 20 12.034 7.278 <0.001 Error 396.848 240 1.654 Ventro-Medial Mechanism1 207.496 2 103.748 5.955 0.008 Error 418.103 24 17.421 Position2 989.799 9 109.978 53.095 <0.001 Mechanism x Position 342.007 18 19.000 9.173 <0.001 Error 447.410 216 2.071 Ventro-Lateral Mechanism1 147.421 2 73.710 4.281 0.026 Error 413.222 24 17.218 Position2 956.825 9 106.314 54.014 <0.001 Mechanism x Position 388.397 18 21.578 10.963 O.001 Error 425.147 216 1.968 Square root transforms were applied to data prior to statistical testin g because counts (axons/mm) exhibit a Poisson distribution. ' Sham surgical controls were not included in the ANOVA because dextran penetration was not detected in the white matter. 2 The position at 5mm caudal was excluded as there was no variance on which to perform an analysis. Table C .2: ANOVA summary - Acute dextran penetration into neuronal somata Sum of Degrees of Region Effect Squares Freedom Mean Square F P Ventral Gray Matter Mechanism 6.653 3 2.217 11.324 <0.001 Error 6.071 31 0.196 Position 12.249 10 1.224 31.341 O.001 Mechanism x Position 9.737 30 0.324 8.305 <0.001 Error 12.116 310 0.039 Arcsine transforms were applied to data prior to statistical testing because proportional data (% of cells) exhibit a binomial distribution. 195 Appendix C ANOVA Tables Sum of Degrees of Region Effect Squares Freedom Mean Square F P Ventral Gray Matter Mechanism 28.121 3 9.374 2.29 0.104 Error 98.273 24 4.095 Dextran 2.255 1 2.255 6.08 0.021 Dextran x Mechanism 2.682 3 0.894 2.41 0.092 Error 8.897 24 0.371 Position 62.560 8 7.820 9.74 O.001 Position x Mechanism 42.226 24 1.759 2.19 0.002 Error 154.137 192 0.803 Dextran x Position 0.316 8 0.040 0.429 0.903 Dextran x Position x 1.555 24 0.065 0.703 0.845 Mechanism Error 17.693 192 0.092 Ventro-Medial White Matter Mechanism 35.495 3 11.832 1.056 0.386 Error 268.951 24 11.206 Dextran 46.909 1 46.909 18.217 O.001 Dextran x Mechanism 48.777 3 16.259 6.314 0.003 Error 61.800 24 2.575 Position 16.527 8 2.066 1.390 0.203 Position x Mechanism 72.815 24 3.034 2.041 0.004 Error 285.445 192 1.487 Dextran * Position 31.705 8 3.963 10.194 <0.001 Dextran x Position x 13.754 24 0.573 1.474 0.080 Mechanism Error 74.644 192 0.389 Dextran denotes comparison between tracers that were infused before and after injury. 196 Appendix C ANOVA Tables Table C.4: ANOVA summary - 3NT-positive cells Sum of Degrees of Region Effect Squares Freedom Mean Square F P Ventral Gray Matter Mechanism 178.203 3 59.401 2.562 0.072 Error 765.195 33 23.188 Position 133.800 8 16.725 4.114 <0.001 Mechanism x Position 187.901 24 7.829 1.926 ' 0.007 Error 1073.400 264 4.066 Square root transforms were used because count data (cells/mm ) exhibit a Poisson distribution. Table C .5: ANOVA summary - Neurofilament degradation in ventro-medial white matter Sum of Degrees of Region Effect Squares Freedom Mean Square F P Ventro-Medial White Matter Mechanism 207.991 3 69.330 3.563 0.024 Error 642.148 33 19.459 Position 45.042 8 5.630 4.949 <0.001 Mechanism x Position 65.582 24 2.733 2.402 <0.001 Error 300.342 264 1.138 Table C.6: ANOVA summary - PAPP accumulation in axons Sum of Degrees of Region Effect Squares Freedom Mean Square F P Corticospinal Tract Mechanism1 534810 2 267405 34.938 O.001 Error 160730 21 7654 Dorsal Column Mechanism1 31561.3 2 15780 17.114 <0.001 Error 19363.9 21 922.1 Lateral Column Mechanism1 78156.1 2 39078.0 10.359 O.001 Error 79217.6 21 3772.3 \" Ventral Column Mechanism1 94527.2 2 47263.6 14.908 <0.001 Error 66577.7 21 3170.4 Square root transforms were used to ensure homoscedasticity prior to statistical testing. 1 Sham surgical controls were not included in the ANOVA because sections from these animals did not exhibit any positive staining for PAPP. Table C .7: ANOVA summary - Reactive astrocytes Sum of Degrees of Region Effect Squares Freedom Mean Square F P Gray Matter Mechanism 0.035 3 0.012 3.5 0.038 Error 0.056 17 0.003 Arcsine transforms were used to ensure homoscedasticity of proportional data (% area) prior to statistical testing. 197 Appendix C ANOVA Tables Table C . 8 : A N O V A summary - Microglial activation Sum of Degrees of Region Effect Squares Freedom Mean Square F P Ventral Gray Matter Mechanism 0.310 3 0.103 34.864 <0.001 Error 0.098 33 0.003 Position 0.114 8 0.014 43.759 <0.001 Mechanism x Position 0.076 24 0.003 9.759 O.001 Error 0.086 264 0.0003 Lateral Column Mechanism 0.081 3 0.027 9.436 O.001 Error 0.094 33 0.003 Position 0.011 8 0.001 5.475 O.001 Mechanism x Position 0.020 24 0.0008 3.356 O.001 Error 0.065 264 0.0002 Dorsal Column Mechanism 0.253 3 0.084 30.124 <0.001 Error 0.092 33 0.003 Position 0.029 8 0.004 9.911 O.001 Mechanism x Position 0.048 24 0.002 5.573 <0.001 Error 0.095 264 0.0004 Ventral Column Mechanism 0.038 3 0.013 4.207 0.013 Error 0.098 33 0.003 Position 0.025 8 0.003 17.369 <0.001 Mechanism x Position 0.019 24 0.0008 4.318 <0.001 Error 0.047 264 0.0002 198 Appendix D E T H I C S B O A R D C E R T I F I C A T E S OF A P P R O V A L 199 Appendix D Ethics Board Certificates lygCI T H E UNIVERSITY O F BRITISH C O L U M B I A ANIMAL CARE CERTIFICATE Application Number: A04-0] 86 Investigator or Course Director: TonxR. Oxland Department: Orthopaedics Animals: Rats Sprague-Dawley 180 Start Date: January 1.2003 Approval Date: March 6,2007 Funding Sources: Funding Agency: B C Neurotrauma Funding Title: Spinal cord injury mechanisms Funding Agency: Natural Science Engineering Research Council Funding Title: Injury mechanisms o f the spinal cord Funding Agency: Rick Hansen M a n In Motion Foundation Funding Title: Spinal cord injury mechanisms Funding Agency: Canadian Institutes of Health Research Funding Title: The effect o f distinct modes o f spinal column failure on spinal cord injury Funding Agency: B C Neurotrauma Funding Title: Spinal Cord Injury Mechanisms Unfunded title: N / A The Animal Care Committee has examined and approved the use o f animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the C C A C and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office o f Research Services and Administration 102, 6190 Agronomy Road, Vancouver, B C V 6 T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 202 "@en . "Thesis/Dissertation"@en . "10.14288/1.0080720"@en . "eng"@en . "Mechanical Engineering"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Clinically relevant mechanisms of spinal cord injury : contusion, dislocation, and distraction"@en . "Text"@en . "http://hdl.handle.net/2429/31276"@en .