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)

Eight functionally identified group Ia muscle afferents from triceps surae or plantaris muscles were labeled intraaxonally with horseradish peroxidase (HRP) in seven adult cats. Subsequently, HRP was injected into two to six homonymous or heteronymous α-motoneurons per animal (total = 22), each identified by motor unit type and located near the site of afferent injection. The complete trajectories of labeled afferents were reconstructed, and putative synaptic contacts on HRP-labeled motoneurons were identified at high magnification. Dendritic paths from each contact were also mapped and measured. A total of 24 contact systems (the combination of a group Ia afferent and a postsynaptic motoneuron) were reconstructed, of which 17 were homonymous, and seven were heteronymous. Overall, homonymous contact systems had an average of 9.6 boutons, whereas heteronymous contact systems had an average of 5.9 boutons. The average number of boutons found on type S motoneurons in homonymous contact systems was smaller (6.4, range 3-17) than in systems involving types FF or FR motoneurons (FF: 10.4, range 4-18; FR: 11.3, range 4-32). Neither of these differences were statistically significant. In contrast to earlier reports, a majority (15/24) of contact systems included more than one collateral from the same Ia afferent. The complexity (number of branch points) in the arborization pathway leading to each contact (overall mean 8.4 ± 3.3) was virtually identical in all contact systems, irrespective of the type of the postsynaptic motoneuron. The three- dimensional distribution of group Ia contacts was not coextensive with the radially organized dendrites of motoneurons: Dendrites oriented in the ventromedial to dorsolateral axis had the fewest (8%) contacts, whereas rostrocaudal dendrites had the most (63%) contacts. Nevertheless, contacts were widely distributed on the motoneuron surface, with few on and near the soma (≤200 μm radial distance from the soma) or on the most distal parts of the tree (≤1,000 μm). The boutons in individual contact systems also showed wide spatial and estimated electrotonic distributions; only 3/24 systems had all contact located within a restricted spatial/electrotonic region. The relations between these anatomical results and existing electrophysiological data on group Ia synaptic potentials are discussed.

The studies reported in this dissertation constitute the first evidence that methylprednisolone (MP) has been shown to ameliorate specific components of secondary trauma including ischemia, lactic acidosis, and lipid peroxidation.

Thesis (Ph.D. (Health Sciences)) -- University of Limpopo, 2021 / Rehabilitation programmes for PLWSCI aim to give them as much independence as possible and perfect integration at a later stage. Proper rehabilitation involves effective community integration of a person with the SCI. Rehabilitation and community integration are key to return the person with an SCI to play his/her role in the community as an effective, independent, and important person. Globally, PLWSCI are facing numerous barriers and limitations, especially in rural areas, where rehabilitation resources are limited. There is a dearth of rehabilitation centres in the Limpopo Province, which might impact negatively on the rehabilitation and community integration outcomes for PLWSCI in this region. The absence of rehabilitation centres in this province point to the need for the development of extra skills for the professionals who work in the rehabilitation team in this Province. Aim and Objectives The overall aim of this study was to develop a rehabilitation programme to enhance community reintegration for PLWSCI in rural areas of the Limpopo Province, South Africa. Methodology This study used a convergent parallel mixed method design in phase 1. A mixed methodology approach consists of a set of designs and procedures in which both quantitative and qualitative data are collected, analysed, and mixed in a single study. Quantitative data were generated and collected regarding the rehabilitation and community integration challenges of PLWSCI using the Spinal Cord Injury Community Reintegration Measure (SCICRM) tool. Qualitative data were collected through focus group discussions (FGDs). In phase 2 of the study, the Delphi study design was utilised and conducted as a forecasting method based on the results of phase 1. vi Results Quantitative results show that 37% of the respondents were fully reintegrated. Moderate reintegration was above average (54.4%), while 5.7% were minimally integrated, and 3.9% failed to reintegrate into their communities. The rehabilitation teams’ home visits were further rated as “never happened” (51%) and “rarely happened” (14.3%). Patients who received a home visit from their rehabilitation team were more likely to fully reintegrate into their community, in this case, 75%. Of those who did not receive any home visits, only 39% managed to fully reintegrate into their communities. The qualitative findings, which is based on information-rich interviews with participants, indicated an urgent need for the implementation of a rehabilitation programme to enhance a successful and better community reintegration for PLWSCI. The qualitative results also highlighted the importance of home visits and following up on the patients after they have returned to their communities. The qualitative findings were presented according to six themes that were also divided into several sub-themes. In phase 2 of this study, experts agreed that the rehabilitation programme to enhance community integration for people with SCIs in the Limpopo Province should include the identification of the rehabilitation team, distribution of powers between the team members, documentation and referral letters, mobility, assistive devices, sub-acute rehabilitation tasks, home programmes, and the outcome of the successful rehabilitation. Conclusion Rehabilitation is the bridge between the SCI and successful community integration. A comprehensive rehabilitation programme implemented by a multidisciplinary team should enhance the successful reintegration of PLWSCI.

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 debilitating condition that disrupts the communication between the brain and the spinal cord. Several studies have sought to determine how to revive dormant spinal circuits caudal to the lesion to restore movements in paralyzed patients. So far, recovery levels in human patients have been modest at best. In contrast, animal models of SCI exhibit more recovery of lost function. Recovery of lost function could arise from structural changes in spinal circuits following spinal cord injury. Previous work from our lab has identified dI3 interneurons as a spinal neuron population central to the recovery of locomotor function in spinalized mice. We seek to determine the changes in the circuitry of dI3 interneurons and motoneurons following SCI in adult mice. After a complete transection of the spinal cord at T9-T11 level in transgenic Isl1:YFP mice and subsequent treadmill training at various time points of recovery following surgery, we examined changes in three key circuits involving dI3 interneurons and motoneurons: 1) Sensory inputs from proprioceptive and cutaneous afferents, 2) GABAergic inputs onto sensory afferents (GABApre), 3) Central excitatory glutamatergic synapses from spinal neurons onto dI3 INs and motoneurons. Furthermore, we examined the possible role of treadmill training on changes in synaptic connectivity to dI3 interneurons and motoneurons. Our data suggests that sensory inputs from the periphery labelled by VGLUT1⁺ to dI3 interneurons decrease transiently or only at later stages after injury, whereas levels of VGLUT1⁺ remain the same for motoneurons after injury. Levels of central excitatory inputs labelled by VGLUT2⁺ to dI3 INs and MNs may show transient increases but fall below levels seen in sham-operated mice after a period of time. Levels of GABApre boutons onto the VGLUT1⁺ sensory afferents that project onto to dI3 INs and MNs can rise shortly after SCI, but those increases do not persist. However, levels of these GABApre boutons onto VGLUT1⁺ inputs never fell below levels observed in sham-operated mice. For some synaptic inputs studied, levels were higher in spinal cord-injured animals that received treadmill training, but these increases were observed only at some time points. Changes in spinal circuitry could be maladaptive. For example, spasticity is a common consequence of SCI, disrupting motor function and resulting in significant discomfort. Spasticity may arise from maladaptive changes in spinal circuits. Current models of hindlimb spasticity are lacking, hindering the study of mechanisms or treatments of spasticity. Therefore, we have generated a novel mouse model of SCI-related spasticity that utilizes optogenetics to activate a subset of cutaneous VGLUT2⁺ sensory afferents to produce reliable incidences of hindlimb spasticity. To examine the efficacy of this optogenetic spasticity model, a T9-T10 complete transection injury was performed in Isl1-Vglut2ᒼᵃᵗᒼʰ mice, followed by the implantation of EMG electrodes into the left and right gastrocnemius and tibialis anterior muscles. Beginning at 9 days post-injury, EMG recordings were performed during episodic optogenetic stimulation. During each recording session, an optic fiber coupled to a 470nm wavelength LED was used to deliver light pulses to the palmar surface of each hindpaw. The results of these recordings demonstrated significant increases in the amplitude of EMG responses to the light stimulus from 2 weeks post-injury to 5 weeks post-injury, indicating hyperreflexia. Interestingly, this hyperreflexia was significantly greater in the female cohort in comparison to the males. Incidences of prolonged involuntary muscle contraction and clonus were also detected through EMG and visual observation during the testing period, supporting the presence of spasticity. Overall, the results in my thesis suggest remodelling of spinal circuits involving spinal interneurons that have previously been implicated in the recovery of locomotor function after spinal cord injury in mice. In addition, we have developed an optogenetic mouse model that appears to reliably elicit spasticity in SCI mice and may be valuable for the study of SCI-related limb spasticity mechanisms due to the maladaptive changes within the spinal cord.