published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

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Access to and subsequent control of spinal cord function are critical considerations for design of optimal therapeutic strategies for SCI patients. Electrical stimulation of the spinal cord is capable of activating behaviorally-relevant populations of neurons for recovery of function, and is therefore an attractive target for potential devices. A promising method for accessing these spinal circuits is through their axons, which are organized as longitudinal columns of white matter funiculi along the cord exterior. For this thesis, I hypothesized that these funiculi can be selectively recruited via electrodes appropriately placed on the surface of the spinal cord, for functional activation of relevant motor circuitry in a chronically-transected spinal cord. My tandem design goal was to fabricate and implement a conformable multi-electrode array (MEA) that would enable this selective stimulation. To accomplish this design goal, I participated in the design, fabrication, and electromechanical testing of a conformable MEA for surface stimulation of spinal tracts. I then assessed the fundamental capability of this MEA technology to stimulate white matter tracts in a precise, controlled, and functionally-relevant manner. This was accomplished via in vitro experiments that explored the ability of this MEA to locally activate axons via single- and dual-site surface stimulation. The results from these evaluation studies suggest that spinal-cord surface stimulation with this novel MEA technology can provide discrete, minimally-damaging activation of spinal systems via their white matter tracts. To test my hypothesis that surface stimulation can be used to recruit distinct populations in the spinal cord, I performed studies that stimulated lateral funiculi in both chronically-transected and intact in vitro spinal cords. Results from these studies reveal that selective surface stimulation of white matter tracts in the ventrolateral funiculus (VLF) elicit motor outputs not elicited in intact cords. In addition, I was able to demonstrate that the spinal systems activated by this surface stimulation involve synaptic components and are responsive to spatial, temporal, and pharmacologic facilitation. Corresponding labeling of the axonal tracts projecting through the T12 VLF indicate that, after chronic transection, the remaining spinal neurons whose axons travel through the VLF include those with cell bodies in both the intermediate region and dorsal horn. These electrophysiological results show that surface-stimulating technologies used to control motor function after injury should include focal activation of interneuronal systems with axons in the ventrolateral funiculus. As a whole, these studies provide essential starting points for further use of conformable MEAs to effectively activate and control spinal cord function from the surface of the spinal cord.

Doctor of Philosophy / The general aim of this current work was to examine spinal cord injury (SCI), and in particular to examine the pathology of injury as it relates to changes in sensory transmission. Due to the limited possibilities for experimentation in humans, a range of animal models of SCI have been developed and are reviewed here. The weight drop SCI model is the most similar to the clinical presentation of SCI in humans and has been widely used in the rat. It was selected for the series of experiments reported in this thesis. Many of the functional deficits produced by SCI result from a cascade of biochemical events set into motion by the injury. Included amongst these is the activation of the enzyme nitric oxide synthase which produces the gaseous neuromodulator, nitric oxide (NO). NO is amongst the most widely distributed and widely utilised molecule in virtually all living organisms, and it is an important signalling molecule in the nervous system. One of the major functions performed by NO appears to relate to sensory transmission, and thus alterations in sensory transmission observed as a result of SCI may involve alterations to NO synthesis. One of the principal aims of this thesis was to examine the effect of SCI on the NO producing cells of the spinal cord and to consider what any changes in NO synthesis may suggest in regards to sensation. NO producing cells were examined using NADPH diaphorase (NADPH-d) histochemistry. As the symptoms of SCI such as motor loss and changes in sensory processing are functional changes, it was also useful to examine changes in neuronal function as a result of SCI. Widespread neuronal function was examined via immunohistochemical detection of the gene product of the immediate early gene, c-fos. It is not known how extensive the biochemical changes resulting from SCI may be, thus another of the aims of the present thesis was to examine the effects of SCI on NO synthesis not only at the level of injury, but also distant to the injury. Findings of the present thesis indicated that traumatic SCI resulted in a decrease in the number of NADPH-d positive cells from the superficial dorsal horn (SDH) of the spinal cord, while the number of these cells are increased in the ventral horn. These changes were restricted to spinal segments adjacent to the injury. Fos expression was also altered by injury and was found to decrease. The most profound changes were found to occur in lamina III, although the other laminae also demonstrated similar changes. Changes in fos expression however were notably more widespread than those for NADPH-d and were not restricted to the level of the injury, occurring at all levels of the spinal cord examined. It was interpreted that alterations in NO synthesis appear to be modulated by the local injury-induced environment while fos expression may be altered by widespread changes to the global level of activity within the central nervous system. Having observed that the number of NADPH-d positive cells of the SDH is reduced following injury, it was of interest to determine whether these cells were in fact killed, or whether they were still present but with reduced NADPH-d activity. Cell counts suggested that the NADPH-d positive cells, which were likely to represent a population of inhibitory interneurons, were not killed following injury, but rather are disrupted such that their normal biochemistry is altered. Since these cells were likely to be inhibitory and were located in laminae involved in sensory transmission, the question arose how disruption of these cells may relate to the neuropathic pain observed to develop following SCI. Thus both NADPH-d and fos expression were again examined, but this time in conjunction with the sensory function of the rats. Sensory thresholds to pain-like behaviour were determined prior to and after injury using Von Frey filaments. Rats that demonstrated a decrease in sensory threshold of at least two Von Frey filament gradations (>70%) were classed as allodynic, while those with a less than a 70% decrease in threshold were classed as non-allodynic. A subpopulation of each of the groups of rats (uninjured, non-allodynic and allodynic) underwent a somatic stimulation paradigm. It was found that stimulation resulted in an increase in the number of NO producing cells but only in the allodynic group of animals. Since this group of animals by definition would perceive this stimulation as noxious, it is likely that the noxious nature of the stimulation resulted in the increased number of NO producing cells observed. This effect occurred only in segments adjacent to the injury. When fos expression was examined in the uninjured animals it was noted that somatic stimulation resulted in a decrease in fos expression, almost exclusively in lamina III. Following injury, there was no change in fos expression in lamina III observed. Instead the only change observed was an increase in fos expression in the deep dorsal horn (DDH, lamina IV and V). This occurred most profoundly in the allodynic group. These results suggested that SCI may lead to misprocessing of sensory signals such that non-noxious somatic stimuli are processed in the DDH rather than lamina III following SCI. It is proposed here that this change in laminae processing may be responsible for the perception of pain towards a non-noxious stimulus, and that the reported injury-induced loss of NO producing inhibitory interneurons in the SDH may be responsible for this alteration in sensory processing following SCI. Sensation is also processed by a number of supraspinal structures and a number of these have been implicated in the development of neuropathic pain states. The effects of SCI on neuronal activity as well as NO synthesis were examined in the periaqueductal grey region of the mid brain (PAG). SCI was shown to result in reduced neuronal activity in the PAG. This reduction in activity did not follow the somatotopy of the lateral column of the PAG (lPAG). It was suggested the reduced activity may not be solely caused by reduced spinal input as a result of SCI. Reduced neuronal activity in the PAG may indicate reduced PAG function, which includes descending modulation of spinal sensory transmission. Injury was not found to alter NADPH-d expression in the PAG. The effect of traumatic lumbar SCI on the parietal (sensorimotor) cortex of the rat was also examined, as loss of inputs following SCI have been shown to result in a profound reorganisation of the cortex. Results indicated that SCI results in a virtual cessation of neuronal activity in areas 1 and 2 of the parietal cortex, likely as a result of lost afferent drive. Theories of cortical plasticity suggest that while the primary inputs via the lumbar spinal cord may be lost following SCI, other less dominants input will remain and become more dominant. It has been proposed previously that cortical reorganisation involves a rapid reorganisation of the entire sensory system. It was interpreted that a similar process may explain the system-wide reduction in neuronal activity observed in the present series of studies.

Background: Spinal Cord Injuries (SCIs) are traumatic, life-changing injuries that can affect every aspect of an individual's life and can lead to death if not treated timeously and appropriately. Respiratory complications occur frequently after the SCI and are the leading cause of mortality and morbidity. Respiratory complications are predictable based on the neurological level of impairment of the spinal cord lesion; the higher the neurological injury, the more severe the respiratory complication. Changes in pulmonary function, poor cough, hypersecretion, immobility and bronchospasm all contribute to the development of respiratory complications. If the patient is unable to protect his/her airway or if respiratory failure occurs, mechanical ventilation is often required. Many patients require prolonged ventilation and subsequently need to go for tracheostomies. The critical care nurse plays an important role in the early identification of complications and can, therefore, act to limit and prevent these complications, which may be a direct result from the injury or treatment modality such as mechanical ventilation. Respiratory management has been promoted in preventing and treating respiratory complications and is associated with better prognosis in the SCI patient. Design and method: The research study aims to explore and describe existing literature and to make recommendations for the respiratory management of a mechanically ventilated spinal cord injured patient in a critical care unit (CCU). A systematic review was undertaken with clear inclusion and exclusion criteria. Ethical principles were maintained throughout the study. The quality of the study was ensured by critically appraising data that was utilized in the systematic review. It is envisaged that the results from this systematic review will improve the respiratory management of the SCI patient and prevent any variations in practice. Results: Were presented under the following themes: priorities of care for the SCI patient in the acute phase, during the critical care phase and preventative care. Conclusion: The SCI patient regardless of the neurological level or completeness of injury should be admitted to the CCU for intensive ventilatory, cardiopulmonary support and hemodynamic monitoring in order to detect and prevent respiratory complications. The use of larger tidal volumes is associated with improved comfort and less dyspnea however if a patient has acute lung injury or ARDS the use of low tidal volumes 6ml/kg is recommended. Prevention and early identification of respiratory complications is associated with improved outcomes for the SCI patient.

This thesis assessed the suitability of unilateral, electrolytic lesions as a model of spinal cord damage and repair in the adult rat. This type of lesion resulted acutely in localized damage in the upper motor neuron at the L2-L3 level of the spinal cord. Minimal acute damage to ascending sensory pathways was indicated by preserved somatosensory evoked potentials elicited by stimulation of the tibial nerve. Immediately after lesion generation one of several substrates was injected into the lesion cavity. These substrates were saline buffer, liquid collagen solution, foetal spinal cord cells from 14 day old rat embryos, and a mixture of collagen and E 14 foetal spinal cord cells. The 4 groups were compared for functional recovery over 3 months using the inclined plane test and a Tarlov movement scale. After sacrifice, the tibialis anterior muscles were dissected and weighed to assess atrophy due to lower motor neuron injury. After removing and embedding the spinal cords in paraffin, transverse and longitudinal sections were taken for cytoarchitectural investigation. Cresyl violet was used to indicate Nissl substance, Luxol fast blue stained for myelin and anti - glial fibrillary acidic protein (GFAP) antibody revealed the expression of GFAP in the cord sections. Chronic electrolytic lesions were characterized by the highly variable degree of cavitation, demyelination and macrophage infiltration that was present. There was no significant performance deficit on the inclined plane test in any of the lesioned groups when compared to unoperated animals. The tibialis muscles from all groups were of normal weight, indicating that the lower motor neurons were not significantly damaged by the lesions used. There was, however, a marked decrease in the number of GFAP reactive astrocytes in the lesioned animals when compared to unlesioned controls (P < 0.01, Wilcoxon test). Moreover, this reduction of GFAP - like immunoreactivity was not prevented by implants of foetal neurons, collagen or foetal neurons suspended in collagen. Possible explanations for the reduced GFAP - like immunoreactivity seen in all electrolytically lesioned cords are discussed. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

An in vitro model of Rapid Acceleration Impact (RAI) Injury was used to study the effects of multiple impact (220 g/impact, 3-5 sec intervals) trauma on cultures of murine CNS cells. Investigations with spinal cord cultures showed that 1) multiple impacts delivered tangential to the plane of cell growth caused neuronal death (12% after 3 impacts to 46% after 10 impacts); 2) multiple impacts delivered normal to the plane of cell growth were much less effective (8% dead after 10 impacts); 3) most neuronal death occurred within 15 minutes after injury 4) morphological changes observed included increased nuclear prominence and somal swelling; and 5) pretreatment with ketamine (0.1mM) reduced cell death from 51 to 14% and reduced somal swelling. Identical studies performed on cortical cultures revealed minimal differences between the two tissues in their response to multiple tangential impacts.

This study aimed to determine health promotion needs of physically disabled youth with spinal cord injury. The study specifically explored health-related behaviours with reference to participation in physical activity and substance usage, factors that influenced these behaviours and major issues that needed to be targeted in health promotion.

研究目的：脊髓損傷是中樞神經系統的嚴重創傷，致殘率高。脊髓損傷後的再生修復一直是當前醫學的難題。迄今為止，脊髓損傷依然缺乏一種有效地治療方法。既往研究證明，胰島素樣生長因子-1對鼠和兔脊髓損傷有保護作用，為了進一步把這些發現應用到臨床方面，我們採用與人類生理更相近的豬只作為實驗動物，構建與臨床相似的脊髓損傷動物模型，并以此為基礎，系統性研究胰島素樣生長因子-1的脊髓保護作用，評估該治療的功效。 / 研究方法：以運動誘發電位為指導，通過直接壓迫和牽拉造成脊髓損傷。18頭猪只隨機分為3組：胰島素樣生長因子-1治療組、生長激素治療組及生理鹽水對照組。脊髓損傷后1小時、24小時及48小時經鞘內注射給藥。于術後第1天、第3天及第21天收集腦脊液檢測胰島素樣生長因子-1和生長激素濃度。連續21天使用修正的 Tarlov 評分標準對動物的運動功能進行評估。第21天處死動物並取材，檢測脊髓中NeuN, GFAP, caspase-3 的活性，并通過TUNEL染色觀察細胞凋亡情況，比較各組之間有無差別。 / 研究結果：通過這種方法建立的脊髓損傷動物模型穩定可靠，各組之間無明顯差異。鞘內給藥24小時及48小時后，腦脊液中胰島素樣生長因子-1和生長激素濃度明顯升高，術後21天檢測，其濃度恢復至基礎值。胰島素樣生長因子-1治療組的運動功能的恢復優於其它各組。與生理鹽水對照組比較，胰島素樣生長因子-1治療組可以明顯提高脊髓損傷后神經元的存活數量，抑制星形膠質細胞增生，減少細胞凋亡。而生長激素治療組僅抑制星形膠質細胞增生，其它方面與生理鹽水對照組無明顯差別。 / 結論：胰島素樣生長因子-1通過提高神經元存活數量，抑制星形膠質細胞增生，以及減少細胞凋亡促進脊髓損傷的恢復。 / Objective: Spinal cord injury is a devastating condition that leads to long-term disabilities. Currently, there is no effective treatment that minimizes spinal cord damage or enhances neurological recovery. Recent studies in rats or rabbits suggested that neurologic recovery after spinal cord injury could be improved with the administration of neurotropic hormones, such as insulin-like growth factor-1 (IGF-1). In order to apply such bench-side discovery to clinical practice, we conducted a study in a higher animal model, akin to human physiology, to evaluate the effectiveness of intrathecal injections of IGF-1to improve neurological recovery in a porcine model of acute traumatic spinal cord injury. / Methods: Traumatic spinal cord injury model was produced by controlled compression and distraction of the exposed T12 segment of the spinal cord. Eighteen pigs were randomly assigned to receive intrathecal injections of either IGF-1, growth hormone or saline at 1, 24 and 48 hours after spinal cord injury. Locomotor function was assessed daily using the validated modified Tarlov’s scale for 21 days. Spinal cord segments were then harvested and the survival of neurons, reactive astrogliosis and apoptosis were determined using neuronal-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. / Results: Intrathecal injections of IGF-1 and growth hormone significantly increase the concentrations of the neurotropic hormones in the cerebrospinal fluid after injury (p < 0.01). These concentrations returned to baseline by 21 days after drug delivery. Motor deficits on the first day after injury were comparable between animals in the treatment and control groups. By the end of the third week, neurologic recovery was better in animals receiving IGF-1 treatment (p < 0.05). Immunohistological and western blot studies of the injured segments of spinal cord showed that treatment with both IGF-1 and growth hormone prevented reactive astrogliosis (p < 0.05) while only IGF-1 improved the survival of mature neurons (p < 0.05). IGF-1 also inhibited apoptosis after spinal cord injury (p < 0.05). / Conclusions: In our clinically relevant model of traumatic spinal cord injury in pigs, intrathecal injection of IGF-1 demonstrated beneficial effects on neurological and histological recovery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Qinzhou. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-122). / Abstract also in Chinese. / Declaration of origination --- p.I / Abstract --- p.II / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.XII / List of Figures --- p.XIII / Abbreviations --- p.XVIII / Chapter Part 1 --- Spinal Cord Injury: A Review --- p.1 / Chapter Chapter 1-1 --- Acute Spinal Cord Injury: Epidemiology, Socioeconomic Impact --- p.2 / Chapter 1.1.1 --- Epidemiology of Spinal Cord Injury --- p.2 / Chapter 1.1.2 --- Socioeconomic Impact of Acute Spinal Cord Injury --- p.5 / Chapter Chapter 1-2 --- Mechanisms of Spinal Cord Injury --- p.6 / Chapter Chapter 1-3 --- Putative Treatments for Spinal Cord Injury --- p.8 / Chapter 1.3.1 --- Methylprednisolone --- p.8 / Chapter 1.3.2 --- Stem Cell Therapy --- p.11 / Chapter 1.3.3 --- Riluzole --- p.11 / Chapter 1.3.4 --- Other Pharmacological Therapies for Spinal Cord Injury --- p.12 / Chapter Chapter 1-4 --- Insulin-like Growth Factor-1 for the Treatment of Spinal Cord Injury --- p.13 / Chapter Chapter 1-5 --- Summary --- p.17 / Chapter Part 2 --- Insulin-like Growth Factor-1 and Growth Hormone for Spinal Cord Injury --- p.18 / Chapter Chapter 2-1 --- Hypothesis and Objectives --- p.19 / Chapter Chapter 2-2 --- Establishment of Animal Models for Acute Spinal Cord Injury --- p.22 / Chapter 2.2.1 --- Introduction --- p.22 / Chapter 2.2.2 --- Experimental Animals --- p.22 / Chapter 2.2.3 --- Anesthesia --- p.23 / Chapter 2.2.4 --- Transcranial Electrical Motor Evoked Potential --- p.26 / Chapter 2.2.5 --- Surgery --- p.28 / Chapter 2.2.6 --- Statistics --- p.34 / Chapter 2.2.7 --- Results --- p.34 / Chapter 2.2.8 --- Discussion --- p.38 / Chapter Chapter 2-3 --- Optimal Stimulation Protocols for Transcranial Electrical Motor Evoked Potential. --- p.42 / Chapter 2.3.1 --- Introduction --- p.42 / Chapter 2.3.2 --- Methods --- p.42 / Chapter 2.3.2.1 --- Experimental Animals and Anesthesia --- p.42 / Chapter 2.3.2.2 --- Transcranial Electrical Motor Evoked Potential Recording --- p.44 / Chapter 2.3.2.3 --- Stimulation Protocol --- p.44 / Chapter 2.3.3 --- Analyses --- p.44 / Chapter 2.3.4 --- Results --- p.45 / Chapter 2.3.5 --- Discussion --- p.52 / Chapter Chapter 2-4 --- Evaluation of the Efficacy of Insulin-like Growth Factor-1 and Growth Hormone in a Porcine Model --- p.54 / Chapter 2.4.1 --- Introduction --- p.54 / Chapter 2.4.2 --- Materials and Methods --- p.54 / Chapter 2.4.2.1 --- Study Design --- p.54 / Chapter 2.4.2.2 --- Intrathecal Injection and Collection of Cerebrospinal Fluid --- p.58 / Chapter 2.4.2.3 --- Measurements --- p.58 / Chapter 2.4.2.3.1 --- Clinical Evaluation --- p.58 / Chapter 2.4.2.3.2 --- Biochemical Assessments --- p.58 / Chapter 2.4.2.3.3 --- Spinal Cord Section, Histological and Immunochemical Staining --- p.63 / Chapter 2.4.2.3.4 --- Western Blot --- p.69 / Chapter 2.4.3 --- Statistical Analysis and Sample Size Calculation --- p.72 / Chapter 2.4.3.1 --- General Analysis --- p.72 / Chapter 2.4.3.2 --- Sample Size --- p.72 / Chapter 2.4.4 --- Results --- p.73 / Chapter 2.4.4.1 --- Changes of TceMEP --- p.73 / Chapter 2.4.4.2 --- Motor Deficit after Spinal Cord Injury at Baseline --- p.75 / Chapter 2.4.4.3 --- Insulin-like Growth Factor-1 and Growth Hormone in Cerebrospinal Fluid --- p.77 / Chapter 2.4.4.4 --- Clinical Assessment --- p.80 / Chapter 2.4.4.5 --- Demyelination, Neuron Survival and Astrocyte Reaction --- p.85 / Chapter 2.4.4.6 --- Apoptosis --- p.89 / Chapter 2.4.5 --- Discussion --- p.93 / Chapter 2.4.5.1 --- Principal Findings --- p.93 / Chapter 2.4.5.2 --- Insulin-like Growth Factor-1 and Neuroprotection after Spinal Cord Injury --- p.93 / Chapter 2.4.5.3 --- Growth Hormone and Neuroprotection after Spinal Cord Injury --- p.95 / Chapter 2.4.5.4 --- Strengths and Limitations of Our Study --- p.96 / Chapter 2.4.5.5 --- Summary --- p.97 / Chapter Part 3 --- Summary and Future Directions --- p.99 / Chapter Chapter 3-1 --- Summary --- p.100 / Chapter Chapter 3-2 --- Future Directions --- p.103 / Chapter Part 4 --- References and appendixes --- p.104 / References --- p.105 / Appendixes --- p.123

This study aimed to determine health promotion needs of physically disabled youth with spinal cord injury. The study specifically explored health-related behaviours with reference to participation in physical activity and substance usage, factors that influenced these behaviours and major issues that needed to be targeted in health promotion.