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No abstract available / Dissertation (MArch (Prof))--University of Pretoria, 2007. / Architecture / unrestricted

The Vascular system of the human spinal cord at birth has been investigated, and a report is submitted on the detailed pattern of the arterial and arteriolar vessels in 27 cadavers. In 21 instances, the anterior median spinal arterial trunk and in 9 instances the postero-lateral spinal arterial trunks have been recorded. Selection of cadavers has been on the basis of the absence of obvious congenital deformity or disease of the spine and spinal cord. Particular attention has been paid to the feeder arteries which contribute to the main longitudinal channels, both anteriorly and posteriorly. The number of feeder vessels, their ;.., sources of origin .and their approximate size~ have been determined. In 21 specimens the anterior vessels have been recorded and charted in detail. (Plates I - VII). In 9 specimens the posterior vessels have been recorded and the detailed charts of 3 of the latter are presented. (Plate X, p.46). Material and Methods Newborn cadavers, obtained on average 3-5 days after death from various causes such as prematurity, cerebral trauma, pneumonia and pulmonary hyaline membrane have been used. In each case the body was warmed to room temperature, before the introduction into the right femoral artery of a plastic tubular cannula of appropriate gauge. Dilute Ammonia, 2-3 ml., was introduced to promote the flow of the injection material and the specimens were injected with latex under pressures which varied from 5 lbs per square inch to 20 lbs per square inch. The manometric readings of intra-arterial pressure were between 300 mms. Hg and 1200 mms. Hg, and the optimal pressure of delivery was 15 lbs per square inch. Both the arterial and the venous sides of the circulation were well-filled by the injection material, which in all instances was administered via the femoral artery. Rupture of vessel walls with leakage of injection material was not excessive. The number of cadavers rejected on account of inadequate filling or excessive leakage was less than 10 out of approximately 50 cadavers. The injection material consisted of a prevulcanised latex, "Revultex", coloured red with an appropriate dye, and stained through fine muslin before use to ensure freedom from solid particles which could cause obstruction of vessels of fine calibre. The injection material was allowed to penetrate the vascular tree for periods varying from 5 to 15 minutes and the cadaver was then embalmed, injecting the body cavities and infiltrating the limbs as far as possible with a standard embalming fluid consisting of: White Industrial Spirits, 45%; Glycerine, 35%; Formaldethyde, 15%; Thymol, q.s. The cadaver was preserved in a 50% solution of the embalming fluid, and dissections were commenced not less than 14 - 21 days after preparation. Use was made in all instances of the binocular surgical microscope, and magnifications of 10 or of 16 were found to be optimal. Photographic reproductions were made in colour, and the dissected specimens have been preserved. The findings in respect of the anterior arterial system of supply of the cord have been consistent with those of a number of workers, in particular Woollam and Millen (1958) of Cambridge. Additional findings have been reported in respect of the smaller arteries (arteries of the 4. 4th order) including the perforating arteries of the median sulcus. The value of a computerised "average" for the vascular pattern of the cord is disputed. In the opinion of the author, it is likely to be misleading to the clinician and to the surgeon. Reliance upon an average picture in an individual case is liable to be harmful; in the present series of 21 specimens, the average picture as offered by Suh and Alexander (1939) was, in some respects only, applicable to 1 case. The presence of arterio-arterial anastomoses as a common feature in the spine is reported, and the possible significance discussed. Reference is made to a "critical narrow zone" which the author has determined by the radiological examination of 50 vertebral columns in people of all ages. When the region of the "narrow critical zone" is related to the regional blood supply of the spinal cord, then a significant factor in the post-operative development of paraplegia in scoliosis cases has been found to emerge. Vascular factors have been sought in the etiology of idiopathic scoliosis, but not found.

Modulation of synaptic transmission in the spinal cord dorsal horn plays an important role in development and maintenance of pathological pain states. The indisputable part of this modulation is conducted via activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and the cannabinoid receptor 1 (CB1), expressed on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Under physiological conditions, activation of TRPV1 receptors is pronociceptive while CB1 receptor activation leads to attenuation of nociceptive signalling. However, both receptors share also one endogenous agonist anandamide (AEA) that may be produced from N-arachidonoyl phosphatidylethanolamine (20:4-NAPE). Main objective of this thesis focuses on the effect of 20:4-NAPE on nociceptive synaptic transmission in spinal cord slices under naïve and inflammatory conditions and consequent on the possible interaction of TRPV1 and CB1 receptors. First, 20:4-NAPE application induced significant release of anandamide from spinal cord slices under in vitro conditions. Next, patch- clamp recordings of excitatory postsynaptic currents (mEPSC and sEPSC) from superficial dorsal horn (DH) neurons in acute spinal cord slices were used. 20:4-NAPE application under the physiological...

Neurotrauma is a general term describing injury to the central nervous system (CNS); which comprises of the brain and spinal cord. The damage resulting from neurotrauma includes primary injury, which occurs from different sources such as compressed air hitting the brain (bTBI) or an object/bone contusing the spinal cord, resulting in a spinal cord injury (SCI). These various means of primary brain and spinal cord injury are further complicated by the many possible combinations of severity levels and frequencies. However, primary injuries are regarded in many cases as unavoidable with the immediate nerve damage being largely irreversible. Despite all this, primary injuries of the CNS are related by common biochemical pathways in secondary injury. Secondary injury is the cause of declining outcomes after neurotrauma and poor recovery. Secondary injury begins immediately after primary injury and can continue to trigger death of neurons for years later. Given this contribution to poor recovery and its slow progression, secondary injury provides an excellent window of opportunity for therapeutic intervention. A major factor and key link in secondary injury and its perpetuation is reactive aldehyde formation, such as acrolein, from lipid peroxidation. The common formation of acrolein in neurotrauma is attributed to the unique structure of the CNS: with neurons containing a high lipid content from myelin and heavy metabolic activity they are vulnerable to acrolein formation. Thus, acrolein in secondary injury is a point of pathogenic convergence between the various forms of neurotrauma, and may play a role as well in the development of neurotrauma linked disorders and related neurodegeneration. The overall goal of this thesis is to therefore develop better strategies for acrolein removal. We explore here endogenous clearance strategies and targeted drug delivery in SCI, investigate detailed cellular structure changes in bTBI, and acrolein formation and removal in Parkinson’s disease. These findings of pathology, and effectiveness of new or existing acrolein removal strategies, will allow us to better employ treatments in future studies.

The present study was undertaken to evaluate the effects of ultra low concentrations (10-9 or 10-14 M) of morphine on neurite elongation in cultured neurons dissociated from rat spinal cords and cerebral cortex. In fetal serum (FS) or fetal serum-free supplemented with cAMP media, the length of longest neurite was significantly increased by 10-9 or 10-14 M morphine. For example, 10-14 M morphine increased neurite length by 24±0.5% and 27±0.3% in spinal cord neurons, and 18±0.2% and 17±0.6% in cortical neurons. Morphine (10-6 M) had no significant effect on neurite length of spinal and cortical neurons. The relative frequency distribution of neurite length revealed 61±2.7% of spinal neurons and 48±2.6% of cortical neurons are responsive to ultra low concentrations of morphine. In the responsive populations, morphine (10 -14 M) enhanced the neurite outgrowth in spinal neurons by 58±0.9% and 48±1.2% and in cortical neurons by 31±0.6% and 28±0.9% in FS and cAMP-supplemented media, respectively. Pretreatment with naloxone did not prevent the morphine effect. The result shows that morphine at ultra low concentrations enhances neurite outgrowth of spinal and cortical neurons via a naloxone-independent mechanism.

Part I. Development of an isoxylitone analog as an antiepileptic drug candidate. Delphinium denudatum is a medicinal plant traditionally used to treat a variety of conditions in Central Asia. Its interesting anticonvulsant effects were determined to be a property of the compound isoxylitone. Prior work from our group in collaboration with the Poulter group from Western University investigated this compound and generated a large number of isoxylitone analogs in order to optimize its antiepileptic activity. This led to the discovery of the prodrug 13 and the active form 15 shown below, which emerged as the most potent. In this work, the library of analogs was further expanded with 22 new compounds with several which matched the activity of 13 and 15, such as compounds 22 and 37, which led to valuable new insights on the activity of these analogs, and suggested other possible future improvements. In addition, efforts were continued regarding developing compound 15 as a clinical trial candidate. Optimization of the synthesis was performed to drastically reduce costs and waste of chemicals, as well as accelerating the duration of the synthesis. The purification of the final product was also greatly facilitated by the direct synthesis of 15, compared to the prior process of first preparing 13 and hydrolyzing the ester. Efforts were exerted to gather additional knowledge on the characteristics of the compound, with structural and conformational analysis via X-ray crystallography and NOE NMR as well as accelerated stability studies to test the viability of 15 in long-term storage under various conditions. All the information gathered throughout this work supported 15 and its sodium salt as excellent clinical trial candidates as treatments for epilepsy. Part II. Synthesis of SOX9 inhibitors as promoters of recovery from spinal cord injury. According to the World Health Organization, 250 to 500 thousand people develop a spinal cord injury each year with a large portion resulting in tetraplegia. A common misconception is that this is permanent because the damaged nerves cannot be repaired. In fact, nerves can and do regrow after being damaged, but cannot do so after spinal cord injuries due to formation of scar tissues which physically and chemically prevents the healing. The Brown group at Western University identified the SOX9 transcription factor as an important promoter of the formation of this scar and showed that SOX9 inhibitors could improve recovery and mobility in mice affected by spinal cord injuries. In collaboration with their group, previous work in our lab performed and SAR study on the lead compounds ZO2(1) and STL26 (2), shown below. The different sections of the molecule have been designated units A to D, to simplify discussion. Initial work by our group established an efficient method to prepare a library of analogs of the lead compounds. A number of compounds were prepared, which primarily investigated small amines as unit A and phenols with small aliphatic substituents as unit D. The initial SAR data confirmed the validity of STL26 as lead compound, as most alterations to the structure were detrimental to the SOX9 inhibitory activity. The objective of this work was to build on these preliminary SAR results, and expand the library of analogs. Larger substituents were introduced in unit A and D and showed that any group larger or smaller than diethylamide in unit A was detrimental to the activity, but that there seemed to be ample space to increase the size of the unit D isopropyl group. Analogs investigating unit B showed that adding substituents at most of the positions was detrimental, as well as changing the relative positions of unit A and B to be ortho or para to each other. However, the C4 on ring B seemed to be very tolerant to various electron donating or withdrawing functional groups. During this SAR study, a recurring theme was the awful solubility of the compounds in water, which heavily complicated their administration to mice during the bioassays. While none of the analogs tested proved superior to 2, the knowledge accrued during this work painted a clear path forward on which areas of the structure could be safely altered to improve solubility without negative impacts on SOX9 inhibition. Some additional efforts were put into obtaining an accurate three-dimensional structure of an active STL26 (2) analog, and information on the primary conformation in solution. Achieving these goals required the use of NOE NMR experiments and X-ray crystallography. One conformation was discovered to be strongly favoured as a result of an intramolecular hydrogen bond even in protic solvents. Subsequently, a small number of additional analogs were prepared containing modifications that would strongly favor or hinder the preferred conformation, in order to better understand its role in the inhibitory activity. The presence of this hydrogen bond appeared to be key to the activity of the compounds.

Spinal cord injury (SCI) is a physically and mentally devastating condition for which there is no curative treatment. It involves primary trauma from the impact and secondary damage in the form of biochemical cascades that threaten the integrity of functional tissue. Therapeutic interventions can only prevent secondary damages, given the irreversibility of the primary laceration. Experimental therapies for SCI can aim to promote neuronal growth and/or regeneration, promote neuroplasticity in surviving neurons and networks, and enhance neuroprotection, or the survival of spared neurons. Surgical decompression and hypothermia are neuroprotective strategies that usually precede rehabilitational strategies in SCI. Hyperbaric oxygen (HBO) treatment constitutes another promising therapy that can increase the amount of oxygen dissolved in the blood, and therefore, the amount delivered to tissues. Both pre-clinical and clinical studies have illustrated that HBO therapy can enhance motor recovery and exert neurological improvements after SCI. A plethora of pre-clinical studies have elucidated several aspects of its function in SCI; HBO seems to suppress apoptosis, edema, and inflammation, as well as mitigate oxidizing conditions. It can also promote angiogenesis, enhance nerve conduction, and inhibit neural degeneration. The limited number of clinical studies and the heterogeneity of protocols allow for fewer conclusions on the roles of HBO in human SCI: motor benefits are hinted in several clinical trials, while neuroprotective effects include increases in blood oxygen, and suppression of inflammatory responses. However, the number and variety of pre-clinical studies suggest that HBO can exert additional neuroprotective benefits in human SCI, which remain to be explored in the future.

The failure of axonal regeneration is due to myriad reasons both cell intrinsic and extrinsic. In this thesis, I sought to investigate an intrinsic reason for regeneration failure in the CNS. Specifically, I investigated the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for two weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Interestingly, transection of axons within the sciatic nerve resulted in mitochondrial fission but did not result in the fragmentation of mitochondria. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDivi-1) blocked fragmentation. Additionally, it was determined that there is increased mitophagy in CST axons following spinal cord injury based on increased colocalization of mitochondria and LC3. In vitro models revealed that mitochondrial calcium uptake is necessary for injury induced mitochondrial fission, as inhibiting mitochondrial calcium uptake using RU360, a mitochondrial calcium uniporter inhibitor, prevented injury induced fission. This phenomenon was also observed in vivo. These studies indicate that following injury, both in vivo and in vitro, axonal mitochondria undergo increased fission, which may result in an ATP deficit that contributes to the lack of regeneration seen in CNS neurons. / Biomedical Sciences

An increased prevalence and earlier onset of cardiovascular disease (CVD) occurs in persons with spinal cord injury (SCI); the higher risk may be explained by novel CVD risk factors of aerobic capacity and peripheral vascular structure and function. Physical inactivity likely contributes to the basis of increased CVD risk after SCI, however evidence on the effectiveness of exercise programs in attenuating CVD risk in SCI is insufficient. The present thesis evaluated novel CVD risk factors in a cohort of individuals with chronic SCI, and examined the effects of a single bout of exercise and exercise training on CVD risk. The first study demonstrated dramatic decreases in body composition, aerobic capacity, and sublesional endothelial function via flow-mediated dilation (FMD) in adults with chronic SCI vs. able-bodied (AB) controls. The second, third, and fourth studies assessed the role of shear rate (SR) patterns on FMD. Elevated retrograde SR had a detrimental effect on brachial and superficial-femoral-artery (SFA) FMD in both SCI and AB, but elevated anterograde SR had a favorable effect on SFA FMD in AB only. The fifth study demonstrated that sublesional vasculature does not respond to a 4-month combination aerobic and resistance-training program using the recently released physical activity guidelines for adults with SCI (PAG). The results of this thesis highlight the multilayered regulation of sublesional vasculature, and that it may respond differently to a single bout of exercise and exercise training when compared to an AB population. This information is crucial when designing strategies to combat impaired vascular structure and function after SCI. The results from this thesis also indicate the potential for the PAG to improve aspects of anthropometrics, body composition, and carotid vascular health in adults with SCI. Further investigations are necessary to delineate the effects of SCI itself, and of exercise, on CVD risk in this population. / Dissertation / Doctor of Science (PhD)