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Certain organophosphorus (OP) compounds produce a delayed neuropathy in man and susceptible animal species after early inhibition and aging of the enzyme, neurotoxic esterase (neuropathy target esterase, or NTE). In this study, the human neuroblastoma cell line, SY-5Y, was examined for its potential to serve as a nonanimal model for the study of the early effects of neuropathy-inducing OPs. For these investigations, the time course of inhibition and aging of NTE after toxicant treatment in neuroblastoma cells was compared to that in brain tissue from the adult chicken, which is the recognized animal model for organophosphorus ester-induced delayed neuropathy (OPIDN). Concentrations of toxicants to be used for treating neuroblastoma cells were determined after observing viability of treated differentiated SY-SY cells incubated for 24 hours with a range of concentrations of an OP (mipafox) that induces neuropathy, an OP (paraoxon) that does not induce neuropathy, a carbamate (aldicarb), a neurotoxicant (β, β’-iminodipropionitrile, or IDPN) that acts by a different mechanism than the OPs, and a cholinergic agonist (carbachol). Treatment concentrations were chosen that caused less than 30% loss of viability over the 24 hour period. The time course and extent of detrimental effects of mipafox, which induces OPIDN in hens, and the carbamate, aldicarb, on NTE were similar in the SY-5Y cells to those observed in homogenized chicken brain tissue after the same treatments. Mipafox produced rapid inhibition and aging of NTE, with maximal effects occurring within 10 minutes of exposure. Aldicarb inhibited NTE but did not age the enzyme. Instead, spontaneous reactivation was observed both in SY-5Y cells and in brain tissue. None of the other negative control compounds (paraoxon, IDPN, carbachol) affected NTE activity in either SY-5Y cells or chicken brain tissue. To determine if the neuroblastoma cells could be used to study early events that could lead to modification of OPIDN, NTE inhibition and aging were determined in the differentiated SY-5Y cell line after mipafox was removed. Removal of mipafox from the cell culture medium at 5 minutes after exposure, or earlier, resulted in essentially no NTE inhibition or aging. NTE inhibition and aging were also determined after treatment of the SY-5Y cells with the neuropathy-inducing OP, mipafox, and representatives of 2 classes of compounds (carbamates and calcium channel blockers) previously demonstrated to modify OPIDN in hens. The modifiers (aldicarb and verapamil) were used as a 5 minute pre-treatment, simultaneous treatment, and a 2 minute post-treatment. Significant prevention of most of mipafox-induced NTE inhibition and aging was observed. Effects on NTE inhibition and aging in differentiated SY-5Y cells after each mipafox-aldicarb combination and mipafox-verapamil combination of treatments were similar to those in chicken brain homogenate. These results indicated that both aldicarb and verapamil protected NTE against the early biochemical effects of mipafox that are thought to initiate OPIDN in vivo. The temporal relationship of NTE inhibition and aging to other detrimental effects on neuroblastoma cells was assessed by the capability of mipafox to cause changes in free intracellular calcium ion concentration, measured using fluorescent calcium probes, and by its capability to alter cell morphology as assessed by phase contrast microscopy. NTE inhibition and aging preceded these changes. Capability to inhibit activity of the neural enzyme, acetylcholinesterase, was also determined. The results of these studies indicate that the SY-5Y model system shows promise for use in the determination of initial mechanisms contributing to the development of organophosphorus-induced delayed neuropathy. / Ph. D.

>Magister Scientiae - MSc / Background: Patients who suffer from diabetic peripheral neuropathy in the leg experience a greater risk of developing gait deviations due to a decrease in strength of the lower extremities, especially the tibialis anterior and triceps surea muscle groups. Aim: The aim of the study was to determine the effect of an exercise training programme on blood pressure, fasting blood glucose, muscle strength, range of motion, balance and gait pattern deviations in patients with diabetic neuropathies. Methods: A total of fourteen participants, who had been diagnosed with diabetic peripheral neuropathy or nocturnal allodynia in either one or both extremities, were asked to participate in this study. Participants were purposively selected from two private Podiatry practices based on their signs and symptoms of diabetic neuropathy, age, gender and doctor’s clearance to participate in any form of physical activity. Dependent variables included isometric strength of the muscles surrounding the hip, knee and ankle, the range of motion of the ankle in plantarflexion and dorsiflexion using goniometry, an assessment of balance using the stork stand test, and a gait pattern analysis, using the modified Tinetti Gait pattern Assessment Scale. Study design: The study was a single-blinded, pre-test and post-test experimental study design using a quantitative approach. Intervention: The researcher (a registered biokineticist) developed a scientifically-based exercise intervention programme to specifically target the entire kinetic chain, and to reduce fall risks, improve quality of life and to assist in developing a standard protocol for patients with DPN. The intervention programme consisted of a combination of ankle, hip and knee rehabilitation, including gait pattern specific rehabilitation. The intervention took place 2-3 times a week for 45 minutes per session and was divided in four categories: Range of motion exercises, strengthening exercises, balance and proprioception and gait pattern training exercises. Results: The Mann-Whitney and Wilcoxon Sign Rank Tests were used to evaluate the differences in dependent variables from pre- to post-intervention. The level of significance was set at p<0.05. An increase in range of motion only in the left ankle dorsiflexion were observed and an increase in balance time for the left leg were observed in the intervention group after a 10-week follow up assessment. Clinical significance was observed in the intervention group, post-intervention, with a decrease in systolic (-9.09%) and diastolic blood pressure (-13.89%) and a decrease in blood glucose levels (-17.89%), however, an increase in these variables was observed in the control group post-intervention. An increase in plantarflexion, 8% (left) and 8% (right) and dorsiflexion 5.26% (left) and an 11.11% (right) increase in range of motion for both left and right ankles, and balance time for both legs, 200% (left) and 159% (right) was observed in the intervention group post-intervention. Although the muscular strength variables showed a mix of an increase and decrease in strength post-intervention in the intervention group, however a clinically significant decreased amount was observed in the control group post-intervention for the majority of muscular strength variables. Conclusions: Although not many findings of this study are statistically significant, clinical significance were observed with most of the variables of this study. The findings of this study can assist future researchers in the development of exercise interventions for patients who suffers from DPN.

Hyperglycaemia caused by insulin deficiency is believed to play a major role in the de-velopment of neuropathy in diabetic patients. The clinical and pathological features of diabetic neuropathy vary considerably, although sensory and autonomic dysfunctions are the most common characteristics. Normalisation of the blood glucose level by ef-fective insulin treatment decreases the incidence of diabetic neuropathy in patients. However, intensive insulin therapy may result in more frequent hypoglycaemic epi-sodes than are provoked by less ambitious diabetes control. Neuropathy might also be induced by severe hypoglycaemia in diabetes or insulinoma. Accordingly, it seems that the diversity in clinical symptoms of diabetic neuropathy may be due to the combined effects of hyperglycaemia and hypoglycaemia. Based on that assumption, the general aim of this project was to study the relationship between severe hypoglycaemia and pe-ripheral neuropathy in diabetic rats. To understand how the development of neuropathy is related to glycaemic control, we needed to be aware of the glucose dynamics in the animal model that we used. The aim was to ascertain whether the diabetic rats were similar to type 1 diabetic patients with regard to such dynamics. To achieve that goal, we used a MiniMed continuous glucose monitoring system (CGMS®) to measure sub-cutaneous glucose in freely moving rats over a period of 72 hours. The glucose monitor worked well, and it showed that the insulin-treated diabetic BB/Wor rats with a hyper-glycaemic insulin regimen have a glycaemic status similar to that of type 1 diabetic patients with poor glycaemic control. The diabetic rats with a hypoglycaemic regimen generally had low blood glucose levels. Prolonged hypoglycaemia led to axonal de- and regeneration of large myelinated fibres in vagus nerve destined to the laryngeal muscle. Axonal de- and regeneration was also observed in the gastrocnemius and sural nerves, although the frequency of degeneration was much lower in the sural nerve. Small myelinated and unmyelinated nerve fibres were normal in these nerves. These results suggest that hypoglycaemia preferentially damages muscle-related nerve fibres. In contrast, in the diabetic rats exposed to pro-longed hyperglycaemia, only the sural nerve exhibited decreased myelinated fibre diameter in the absence of obvious axonal degeneration. In situ glucose measurements by microdialysis showed that the glucose concentrations in blood and subcutaneous tissue were similar in healthy, diabetic hyperglycaemic, and diabetic hypoglycaemic rats. In the healthy and hyperglycaemic animals, the lowest glucose level was found in the peripheral nerve. Moreover, in controls, the glucose level was lower in muscle than in blood. In hypoglycaemic rats, there were no signifi-cant differences in glucose concentrations between different tissues.

<p>Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an <i>in vitro</i> cell model. The major findings were as follows:</p><p>i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. </p><p>ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. </p><p>iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection.</p><p>iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 </p><p>v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.</p>

Diabetes, which is characterized by elevated plasma glucose, can have a devastating effect on peripheral nerves frequently leading to the clinical symptoms of neuropathy. Diabetic autonomic neuropathy (DAN) results from damage to autonomic nerves, and the most troubling forms of DAN often lead to cardiovascular abnormalities and premature death. Despite the prevalence of DAN and the impact to quality and life expectancy, the precise mechanisms underlying these pathologies are poorly understood. Recently, a new model for the onset of DAN was proposed where hyperglycemia-induced oxidative stress inactivates nicotinic acetylcholine receptors (nAChRs), the main receptor driving autonomic synaptic transmission at sympathetic ganglia. This inactivation leads to the depression of synaptic transmission, and consequently triggers the onset of autonomic neuropathy in diabetic mice. However, the source and pathways contributing to the elevation of reactive oxygen species (ROS) and oxidative stress remained unclear. In recent years it has been shown that the accelerated formation of advanced glycation end products (AGEs) and activation of their receptor (RAGE) in diabetes play a major role in the induction of oxidative stress in sensory nerve damage. Thus we hypothesized that the activation and up-regulation of RAGE during high glucose conditions is a major source of ROS production in sympathetic neurons leading to the inactivation of nAChRs and autonomic malfunction. In this thesis we show for the first time that RAGE is expressed in cultured sympathetic neurons and is also up-regulated during high glucose conditions. Our results further demonstrate that direct RAGE activation by its natural ligands leads to an increase in cytoplasmic ROS which in turn induces the inactivation of nAChRs in sympathetic neurons. We also report that high glucose-induced ROS generation and subsequent inactivation of nAChRs is prevented in sympathetic neurons from RAGE knock-out mice. The results of this dissertation suggest RAGE to be a pivotal source of ROS production leading to the functional deficits observed in sympathetic neurons during high glucose conditions.

La surdité est l'un des déficits sensoriels les plus fréquents dans nos sociétés industrialisées. Parmi les pathologies de l'audition, les surdités de perception ou neurosensorielles sont les plus répandues. Les surdités de perception sont dues à un dysfonctionnement de la cochlée impliquant l'homéostasie ionique, la perte des cellules sensorielles et des neurones ganglionnaires. Alors qu'une altération de l'homéostasie ou que la perte de cellules sensorielles entraine immanquablement la survenue d'une surdité, l'impact de la perte de neurones ganglionnaires est mal connu.L'objet de cette thèse était d'évaluer l'impact des pertes neuronales sur l'audition. Pour ce faire, nous avons développé un outil pharmacologique capable de créer une perte sélective de neurones auditifs primaires, sans endommager les structures pré-synaptiques telles que les cellules sensorielles et la strie vasculaire. Pour ce faire, nous avons appliqué des doses croissantes de ouabaïne sur la membrane de la fenêtre ronde chez la gerbille. Les tests électrophysiologiques (produits de distorsions acoustiques, potentiel endocochléaire et potentiel d'action composite du nerf auditif) ont été réalisés avant et 6 jours après l'application de ouabaïne. A la fin des explorations fonctionnelles, les cochlées étaient prélevées et préparées pour réaliser des évaluations morphologiques en microscopie confocale et en microscopie électronique à transmission.Jusqu'à 80 µM, la ouabaïne n'entrainait aucun changement significatif des produits de distorsions acoustiques ce qui reflétait le bon fonctionnement des cellules ciliées externes, ni du potentiel endocochléaire témoin du fonctionnement normal de la strie vasculaire. En revanche, les mêmes concentrations de ouabaïne provoquaient une diminution dose-dépendante de l'amplitude du potentiel d'action composite du nerf auditif, étroitement associée à une perte de neurones ganglionnaires et de synapses afférentes. Si l'amplitude du potentiel d'action composite du nerf auditif constitue un bon indicateur du nombre et de l'état fonctionnel des neurones ganglionnaires et des synapses afférentes, ce n'est donc pas le cas pour les seuils audiométriques. En effet, ce n'était qu'avec une perte de 75 % des synapses afférentes et supérieure à 55 % des neurones ganglionnaires, qu'une élévation des seuils audiométriques était observée, après une perfusion de 80 µM de ouabaïne. A 100 µM de ouabaïne, l'élévation des seuils auditifs résultait de la perte cumulée des cellules sensorielles et de l'altération de la strie vasculaire, se surajoutant aux dommages neuronaux et synaptiques.L'ensemble de nos résultats montrait que l'application de ouabaïne sur la membrane de la fenêtre ronde chez la gerbille constitue un excellent modèle pour étudier l'impact de la perte sélective des neurones ganglionnaires sur la fonction auditive. Il apparaît aussi nécessaire de développer des outils d'investigation plus précis que le simple audiogramme pour évaluer les pertes neuronales chez l'homme. / Deafness is one of the most frequent sensory deficits in our industrialized societies. Among the auditory pathologies, sensorineural deafness is the most wide-spread. Sensorineural deafness is due to a dysfunction of the cochlea involving the ionic homeostasis, loss of sensory cells and spiral ganglion neurons. While an alteration of the homeostasis or the loss of sensory cells induce inevitably the appearance of deafness, the impact of spiral ganglion neuron loss is unknown.The object of this thesis was to estimate the impact of spiral ganglion neuron losses on the auditory function. We developed a pharmacological tool capable of creating a selective loss of spiral ganglion neurons, without damaging the presynaptic structures such as the sensory cells and the stria vascularis. To do this, we applied increasing doses of ouabain to the round window membrane in the gerbil. Electrophysiological evaluations such as the distortion product otoacoustic emissions, the endocochlear potential and the compound action potentials of the cochlear nerve were recorded before and 6 days after application of ouabain. At the end of the functional evaluations, the cochlea were removed and prepared for morphological evaluations using confocal microscopy and transmission electron microscopy.Our results showed that up to a concentration of 80 µM, ouabain did not induce any significant change of the amplitude of the distortion product otoacoustic emissions, which indicated a normal functional state of the outer hair cells, nor of the endocochlear potential which reflected an intact stria vascularis. On the other hand, the same concentrations of ouabain led to a dose-dependent decrease of the amplitude of the compound action potentials, which was strictly associated with a loss of spiral ganglion neurons and afferent synapses, as assessed by morpho-anatomical analyses. If the amplitude of the compound action potentials constitutes a good indicator of the number and the functional state of the spiral ganglion neurons and the afferent synapses, it is not the case for the audiometric thresholds. Indeed, a loss of 75 % of afferent synapses and more than 55 % loss of the ganglion neurons was necessary before an elevation of the audiometric thresholds was observed in the cochleae perfused with 80 µM ouabain. At 100 µM ouabain, the elevation of the auditory thresholds may result from the accumulated loss of sensory cells, damage to the stria vascularis, in addition to the loss of the spiral ganglion neurons and afferent synapses. All these results indicate that the application of ouabain onto the round window membrane in the gerbil is an excellent model to study the impact of the selective loss of the spiral ganglion neurons on hearing function. More generally, this study points towards the necessity of developing more precise tools, beyond the simple audiogram, for the investigation of auditory neuron loss in humans.

In newborns, unconjugated bilirubin (UCB) is not readily excreted, and when bilirubin levels exceed the serum albumin binding capacity, pathological levels of UCB exist. Hyperbilirubinemia may lead to auditory damage and ultimately cause a hearing disorder called auditory neuropathy/dys-synchrony, characterized by absent or abnormal brainstem auditory evoked potentials (BAEPs) with evidence of normal inner ear function assessed by either otoacoustic emissions or cochlear microphonic responses. Phototherapy and double volume exchange transfusion are used as treatment methods for neonatal hyperbilirubinemia. Spontaneously jaundiced Gunn rat pups given sulfadimethoxine to displace bilirubin from serum albumin develop bilirubin encephalopathy and have abnormal BAEPs comparable to human neonates. BAEPs are a noninvasive electrophysiological measure of neural function of the auditory system. High levels of calcineurin activity are believed to be involved in the mechanism of this bilirubin induced auditory neuropathy. FK506, a calcineurin inhibitor, was administered 3 hours prior to sulfa in concentrations of 0.1mg/kg, 1.0mg/kg, and 10.0mg/kg body weight. Due to the observation that all animals had abnormal BAEPs after treatment with FK506 and sulfa, it can be concluded that none of the treatment doses protected against bilirubin induced auditory impairment.

Key words: Peripheral neuropathy, HIV/AIDS, Progressive resisted exercise, Performance oriented mobility, Quality of life. Background: Distal symmetrical poly-neuropathy (DSP) has emerged as one of the major neurological complication associated with HIV/AIDS and antiretroviral therapy. People with DSP commonly have problems with pain, mobility, altered gait and balance all which affect their quality of life. While therapeutic strengthening exercise has been reported to attenuate these impairments in other co-morbid conditions like diabetes mellitus and in HIV/AIDS, there is no evidence available on the effects of exercise on DSP in people living with HIV/AIDS (PLWHA).The purpose of this study was to determine the effects of progressive resisted exercises (PRE) on performance oriented mobility and health related quality of life in (PLWHA) related DSP. Objectives of this study were to determine the effects of PRE on gait, balance and pain levels and establish if there is a relationship between performance-oriented mobility and health-related quality of life in PLWHA related DSP. Methods: In order to fulfil the objectives, an assessor-blinded randomized controlled trial was conducted over two studies, with a combined sample of 160 participants sourced from two family care clinics at two central hospitals and ten anti-retroviral therapy dispensing municipal clinics in Harare, Zimbabwe. While the experimental group with 80 participants had an intervention program of PRE sessions of one hour for the lower limbs, done twice per week over 12 weeks, the control group of 80 participants was given advice to walk unsupervised at home. Loss to follow up in this study was at 60% (n=97) and the data was analysed using an intention to treat analysis approach. Results: Participants of an average age of 42.2 years (SD=8.5) constituted of 70.6% (n=113) female participants. Combination antiretroviral therapy containing stavudine, was used by 59% (n=94) of the participants and 59% (n=94) of the participants had moderate to severe neuropathy. Proximal muscles exhibited weakness (hamstring muscles strength = 3.43 kg force (SD=1.5)) when compared to leg muscles (gastrocnemius muscles strength = 12.8 kg force (SD=2.0)). Gait and balance scores did not show differences in effect between the intervention and the control group (95%CI 0.00-0.02, p = 0.8). Similarly there were no differences of effect for muscle strength (95%CI 0.00-0.08, p=0.13-0.8) and pain (95%CI 0.0-0.06, p>0.13). However the effect on quality of life changes were significantly different between the two groups (95%CI 0.00-0.12 p= 0.04). Quality of life was positively associated with gait, odds ratio 1.01 (95%CI 1.00 – 1.04), moderately associated with balance odds ratio 0.68, (95%CI 0.52 – 0.93) negatively associated with pain odds ratio 0.98 (95%CI 0.97 – 0.99). Conclusion: This research study established that progressive resisted exercises have positive effects on the health related quality of life in PLWHA related DSP. However this study did not show a difference of the effects of progressive resisted exercises on performance oriented mobility in PLWHA related DSP when compared to advice to exercise at home. The study findings may not be generalized to all individuals living with HIV/AIDS who have DSP as the participants were from a particular demographic setting. This project may be continued at the participating family care clinics as a roll on of the perceived benefits of exercise for people with HIV related DSP.

Thesis. 1977. Ph.D.--Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Vita. / Bibliography : leaves 175-184. / Ph.D.