Traumatic injuries to the spinal cord and brachial plexus induce a significant inflammatory response in the nervous tissue with progressive degeneration of neurons and glial cells, and cause considerable physical and mental suffering in affected patients. This thesis investigates the effects of the antioxidants N-acetyl-cysteine (NAC) and acetyl-L- carnitine (ALC) on the survival of motoneurons in the brainstem and spinal cord, the expression of pro-apoptotic and pro-inflammatory cell markers, axonal sprouting and glial cell reactions after spinal hemisection in adult rats. In addition, a novel MRI protocol has been developed to analyse the extent of neuronal degeneration in the spinal cord. Rubrospinal neurons and tibial motoneurons were pre-labelled with the fluorescent tracer Fast Blue one week before cervical C3 or lumbar L5 spinal cord hemisection. The intrathecal treatment with the antioxidants NAC (2.4mg/day) or ALC (0.9 mg/day) was initiated immediately after injury using Alzet2002 osmotic mini pumps. Spinal cord injury increased the expression of apoptotic cell markers BAX and caspase 3, induced significant degeneration of rubrospinal neurons and spinal motoneurons with associated decrease in immunoreactivity for microtubule-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker glial fibrillary acidic protein and microglial markers OX42 and ED1 was markedly increased. Treatment with NAC and ALC attenuated levels of BAX, caspase 3, OX42 and ED1 expression after 2 weeks postoperatively. After 4-8 weeks of continuous intratheca ltreatment, NAC and ALC rescued approximately half of the rubrospinal neurons and spinal motoneurons destined to die, promoted axonal sprouting, restored the density of MAP2 and synaptophysin immunoreactivity and reduced the microglial reaction. However, antioxidant therapy did not affect the reactive astrocytes in the trauma zone. The inflammation modulating properties of ALC were also studied using cultures of human microglial cells. ALC increased the microglial production of interleukin IL-6 and BDNF, thereby possibly mediating the anti-inflammatory and pro-regenerative effects shown in vivo. To study degeneration in the spinal cord following pre-ganglionic and post-ganglionic brachial plexus injuries, adult rat models of ventral root avulsion and peripheral nerve injury were used. A novel MRI protocol was employed and the images were compared to morphological changes found in histological preparations. Ventral root avulsion caused degeneration of dendritic branches and axonal terminals in the spinal cord, followed by significant shrinkage of the ventral horn. Extensive astroglial and microglial reactions were detected in the histological preparations. Peripheral nerve injury reduced the density of dendritic branches but did not cause shrinkage of the ventral horn. Quantitative analysis of MRI images demonstrated changes in the ventral horn following ventral root avulsion only, thus validating the developed MRI technique as a possible tool for the differentiation of pre-ganglionic and post-ganglionic nerve injuries.

Purpose: Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment. Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined. Results: SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations. Conclusion: SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013

Purpose: Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment. Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined. Results: SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations. Conclusion: SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013

Orientador: Ana Angélica Henrique Fernandes / Resumo: A obesidade é considerada um dos maiores problemas de saúde pública em muitos países, uma vez que está associada queda da qualidade de vida. Embora existam vários fatores que corroboram com o desenvolvimento para tal fato, os hábitos alimentares seja o fator relevante. Os transtornos metabólicos podem resultar em alterações na funcionalidade do fígado, podendo desenvolver Doença Hepática Gordurosa Não Alcoólica (DHGNA). Como o número de obesos e as co-morbidades associadas ao sobrepeso vêm aumentando abruptamente nas últimas décadas, vários modelos de obesidade experimental têm sido propostos para investigar os distúrbios metabólicos envolvendo suas causas e consequências. O glutamato monossódico é amplamente utilizado na culinária e também por indústrias alimentícias, contudo atua no sistema nervoso central e promove a degeneração de áreas importantes do hipotálamo que leva a distúrbios da saciedade e, consequentemente acúmulo excessivo de gordura abdominal. Com a finalidade de estudar substâncias que apresentem potencial atividade terapêutica no controle dos distúrbios metabólicos, o N-acetil-cisteína possui propriedades antioxidantes e exerce hepatoproteção. Desta forma, o objetivo do presente estudo foi evidenciar a indução da obesidade pelo glutamato monossódico e determinar o efeito do N-acetil-cisteína sobre os parâmetros calorimétricos, metabolismo energético, atividade dos complexos respiratórios e estresse oxidativo no tecido hepático. Foram utilizados 32 ratos Wins... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity is considered one of the greatest public health problems in many countries, since it is associated with a drop in quality of life. Although there are several factors corroborating with the development for this fact, eating habits are the relevant factor. Metabolic disorders can result in changes in liver function, and can develop Non-Alcoholic Fatty Liver Disease (NAFLD). As the number of obese and co-morbidities associated with overweight have increased steeply in recent decades, several models of experimental obesity have been proposed to investigate metabolic disorders involving their causes and consequences. Monosodium glutamate is widely used in cooking and also in food industries, but it acts on the central nervous system and promotes the degeneration of important areas of the hypothalamus which leads to satiety disorders and consequently excessive accumulation of abdominal fat. In order to study substances that present potential therapeutic activity in the control of metabolic disorders, N-acetyl-cysteine has antioxidant properties and exerts hepatoprotection. Thus, the objective of the present study was to evidence the induction of obesity by monosodium glutamate and to determine the effect of Nacetyl-cysteine on calorimetric parameters, energy metabolism, respiratory complex activity and oxidative stress in hepatic tissue. Thirty-two Winstar male mice were used at 21 days of age. Initially the animals were distributed in two experimental groups (n = 16). Grou... (Complete abstract click electronic access below) / Doutor

Pentachlorophenol, (PCP) is an organochlorine compound which was first developed in the 1930’s. PCP is said to be the most toxic of the chlorophenols and is classified as a hazardous substance and a probable human carcinogen. PCP has proven to be cytotoxic to a number of cell lines translating to its effect on various organs. The aim of the study was to assess organochlorine-induced hepatotoxicity in a mechanistic manner using an in-house developed procedure. Also, the possible hepatoprotective effect of methanolic extracts of the bark of two medicinal plants, Burkea africana (BA) and Syzygium cordatum (SC), as well as the known hepatoprotective agent, N-acetyl cysteine (NAC), were investigated. In addition to PCP, two of its major metabolites, tetrachloro-1,2-hydroquinone (TCHQ) and tetrachloro-1,4-benzoquinone (TCBQ) were also evaluated. A hepatocarcinoma cell line (HepG2) was used to investigate the effect of these compounds on different parameters of cellular function. Cytotoxicity was assessed using the neutral red uptake assay. Cytochrome P4501A1 (CYP1A1) activity was determined using ethoxy-resorufin-O-deethylation as surrogate. Generation of reactive oxygen species (ROS) was investigated by measuring dichlorofluorescein diacetate cleavage. Effects on mitochondrial membrane potential were determined using JC-1 staining, whilst necrosis was investigated by assessing plasma membrane integrity using propidium iodide (PI)staining. The degree of apoptotic death was determined by quantifying caspase-3 activity. Assays were repeated with an additional 1 h pre-treatment of the cells with either NAC, SC or BA in order to investigate whether these compounds were able to protect against the toxicity induced by PCP and its metabolites. The IC50 values of PCP, TCHQ and TCBQ were 68.0, 144.0 and 129.4 μM, respectively. All three test compounds induced CYP1A1 activity with PCP being the most potent. TCBQ produced extensive ROS generation. TCHQ also induced ROS generation, whilst PCP appeared to have no significant effect on ROS generation. All test compounds caused mitochondrial depolarization. None of the test compounds caused an increase in necrotic cell death. PCP, TCHQ and TCBQ had negligible effects on apoptosis. Both SC and BA alleviated the toxic effects observed in cells treated with PCP. Minor increases in viability occurred in cells pre-treated with plant extracts prior to exposure to both metabolites. NAC, as well as both plant extracts, greatly reduced CYP 1A1 activity induced by PCP. NAC, SC and BA exacerbated CYP1A1 induction in cells exposed to concentrations of TCBQ and TCHQ that initially produced little or no effect on CYP1A1 activity. Contrarily, decreased CYP1A1 activity was observed in cells exposed to concentrations of TCBQ and TCHQ where extensive induction of CYP1A1 occurred. NAC, as well as both plant extracts, suppressed ROS generation in cells exposed to all test compounds. In cells exposed to PCP and TCBQ more extensive mitochondrial depolarization was seen when pre-treated with NAC and plant extracts than when exposed to the compounds alone. Negligible effects were seen in pre-treated cells exposed to TCHQ. BA and SC caused increases in necrotic death in cells exposed to the test compounds. NAC, BA and SC had negligible effects on the changes in caspase-3 activity induced by the test compounds. From the results it is proposed that PCP induces its own metabolism by increasing CYP1A1 activity. It also causes mitochondrial insult which could lead to the opening of the mitochondrial permeability transition pore and subsequent release of cytochrome C, activation of caspases and eventually apoptotic cell death. With regard to TCHQ and TCBQ, results suggest that extensive ROS generation caused damage to various cellular macromolecules and that this could be the main cause of their toxicity. NAC, SC and BA appeared to alleviate toxicity in certain instances. Further investigation is required in order to assess them as possible hepatoprotective agents. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

Peripheral nerve trauma is a common cause of considerable functional morbidity, and healthcare expenditure. Particularly in the ~15% of injuries unsuitable for primary repair, standard clinical management results in inadequate sensory restitution in the majority of cases, despite the rigorous application of complex microsurgical techniques. This can largely be explained by the failure of surgical management to adequately address the neurobiological hurdles to optimal regeneration. Most significant of these is the extensive sensory neuronal death that follows injury, and which is accompanied by a reduction in the regenerative potential of axotomised neurons, and in the supportive capacity of the Schwann cell population if nerve repair is delayed. The present study aimed to accurately delineate the timecourse of neuronal death, in order to identify a therapeutic window during which clinically applicable neuroprotective strategies might be adopted. It then proceeded to investigate means to increase the regenerative capacity of chronically axotomised neurons, and to augment the Schwann cells’ ability to promote that regenerative effort. Unilateral sciatic nerve transection in the rat was the model used, initially assessing neuronal death within the L4&5 dorsal root ganglia by a combination of morphology, TdT uptake nick-end labelling (TUNEL), and statistically unbiased estimation of neuronal loss using the stereological optical disector technique. Having identified 2 weeks, and 2 months post-axotomy as the most biologically relevant timepoints to study, the effect upon neuronal death of systemic treatment with acetyl-L-carnitine (ALCAR 10, or 50mg/kg/day) or N-acetyl-cysteine (NAC 30, or 150mg/kg/day) was determined. A model of secondary nerve repair was then adopted; either 2 or 4 months after unilateral sciatic nerve division, 1cm gap repairs were performed using either reversed isografts, or poly-3-hydroxybutyrate (PHB) conduits containing an alginate-fibronectin hydrogel. Six weeks later nerve regeneration and the Schwann cell population were quantified by digital image analysis of frozen section immunohistochemistry. Sensory neuronal death begins within 24 hours of injury, but takes 1 week to translate into significant neuronal loss. The rate of neuronal death peaks 2 weeks after injury, and neuronal loss is essentially complete by 2 months post-axotomy. Nerve repair is incompletely neuroprotective, but the earlier it is performed the greater the benefit. Two clinically safe pharmaceutical agents, ALCAR & NAC, were found to virtually eliminate sensory neuronal death after peripheral nerve transection. ALCAR also enhanced nerve regeneration independently of its neuroprotective role. Plain PHB conduits were found to be technically simple to use, and supported some regeneration, but were not adequate in themselves. Leukaemia inhibitory factor enhanced nerve regeneration, though cultured autologous Schwann cells (SC’s) were somewhat more effective. Both were relatively more efficacious after a 4 month delay in nerve repair. The most profuse regeneration was found with recombinant glial growth factor (rhGGF-2) in repairs performed 2 months after axotomy, with results that were arguably better than were obtained with nerve grafts. A similar conclusion can be drawn from the result found using both rhGGF-2 and SC’s in PHB conduits 4 months after axotomy. In summary, these findings reinforce the significance of sensory neuronal death in peripheral nerve trauma, and the possibility of its` limitation by early nerve repair. Two agents for the adjuvant therapy of such injuries were identified, that can virtually eliminate neuronal death, and enhance regeneration. Elements in the creation of a bioartificial nerve conduit to replace, or surpass autologous nerve graft for secondary nerve repair are presented.

It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

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