Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 229-253.

Current approaches to treat neurodegenerative disease provide only mild symptomatic relief but do not modify the natural history of these conditions. A large body of evidence suggests that mitochondrial dysfunction is a key event in the pathophysiology of neurodegeneration. Supporting and improving mitochondrial function has a big potential as a strategy for neuroprotection. The goal of this dissertation was to test whether interventions that target mitochondrial function are effective at preventing neurodegeneration induced by mitochondrial failure in vivo. A rodent model of optic neuropathy induced by the mitochondrial toxin rotenone was used to test the neuroprotective effects of methylene blue (MB) and near-infrared light (NIL), two interventions with mechanisms of action localized to mitochondria. This work also tested the effects of memantine, an NMDA receptor blocker, to further characterize the relationship between excitotoxicity and mitochondrial dysfunction. Neuroprotective effects were evaluated via behavioral testing of visual function and histopathological analysis of the retina. The neurochemical effects of MB, NIL and memantine were analyzed in vitro and in vivo with indicators of oxidative stress, cell respiration and catalytic activity of respiratory enzymes, including NADH dehydrogenase and cytochrome oxidase. MB, a diaminophenothiazine with potent antioxidant and unique redox properties, prevented the changes in visual function and the retinal histopathology induced by rotenone. In vitro, MB increased oxygen consumption and prevented the increases in oxidative stress in brain tissue induced by rotenone. NIL prevented the behavioral impairment and the decrease in retinal and visual pathway metabolic activity, retinal nerve fiber layer thickness and ganglion cell layer cell density induced by rotenone in a dose-dependent manner. Whole-brain cytochrome oxidase and superoxide dismutase activities were also increased in NIL-treated subjects in a dose-dependent manner, suggesting an in vivo transcranial effect of NIL. Finally, uncompetitive NMDA receptor blockade with memantine displayed neuroprotection against rotenone-induced neurodegeneration in a dose-response manner, and this effect was associated with a decrease in retinal oxidative stress and a long-term increase in neuronal energy metabolism capacity. These data constitute a proof-of-principle that interventions that target the mitochondria and support the function of the respiratory chain are effective at preventing neurodegeneration in vivo. / text

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Malgré de très nombreuses études expérimentales et cliniques dans le domaine de l'arrêt circulatoire, seulement 2% à 12% des patients quittent l'hôpital avec une bonne récupération neurologique. Il est donc nécessaire de proposer de nouvelles thérapeutiques pour tenter d'augmenter la survie après un arrêt circulatoire. Pour atteindre ce but il semble indispensable d'améliorer la qualité du massage cardiaque durant la réanimation et de protéger le myocarde et le cerveau contre les phénomènes d'ischémie-reperfusion. Dans la première partie de ce travail, nous avons évalués dans une étude pré hospitalière l'utilisation d'un dispositif innovant de massage cardiaque interne par minithoracotomie et montré une amélioration de l'hémodynamique en comparaison avec le massage cardiaque standard. Dans la deuxième partie, nous avons testés les possibles effets protecteurs de l'EPO (érythropoïétine) dans deux types d'arrêt circulatoire. Dans un modèle d'arrêt cardiaque expérimental chez le rat nous avons démontré que lorsque l'EPO était injectée avant l'arrêt cardiaque, la réanimation initiale était améliorée et la survie des animaux augmentée ce qui pouvaient suggérer un effet cardio et/ou neuroprotecteur de l'EPO contre les effets délétères de l'ischémie reperfusion. Dans une étude clinique en chirurgie cardiaque sous circulation extra corporelle, nous n'avons pas pu démontré d'effet bénéfique de l'EPO ni sur l'ischémie myocardique, ni sur l'ischémie cérébrale ni sur les paramètres de l'inflammation. Sur la base de ces deux études, il est donc difficile de conclure sur le potentiel rôle bénéfique de l'EPO dans l'arrêt circulatoire. Néanmoins, sur la seule base des résultats expérimentaux, l'EPO pourrait faire partie de l'arsenal thérapeutique pour mieux protéger le myocarde et le cerveau contre les effets délétères de l'ischémie reperfusion après un arrêt cardiaque. / Despite extensive experimental and clinical research on cardiac arrest, only 2-12% of resuscitated patients are discharged from hospital in good neurological conditions. There is, therefore, a dear need for new therapies that improve survival after cardiac arrest. It ‘s necessary to improve the quality of cardiac massage and to protect against cardiac and cerebral ischemia occurring during cardiac arrest. In a first part, we evaluated the prehospital feasibility of performing a new method of minimally invasive direct cardiac massage (MID-CM®) and we suggested that better haemodynamic results can be obtained than with standard cardiopulmonary resuscitation. In a second part, we tested erythropoietin (EPO) against placebo in two model of cardiac arrest. In an experimental model of cardiac arrest, we demonstrated that EPO, when administrated before cardiac arrest, improved initial resuscitation and increased the duration of post-resuscitation survival. In a second model of circulatory arrest during cardiac surgery with cardiopulmonary bypass, EPO administration did not protect against cerebral ischemia and inflammatory response occurring during cardiac surgery with CPB. It is difficult to make definitive conclusion on the potential role of EPO in myocardial and cerebral protection after circulatory arrest. We can hope that EPO administration will represent pharmacological approach in upcoming years to additional myocardial salvage of the reperfused myocardium after cardiac arrest.

Introdução: Muitos estudos veem sendo realizados com a finalidade de identificar agentes que possam ter efeito benéfico no tratamento ou prevenção das lesões causadas nos neurônios devido à isquemia. A hipotermia já demonstrou resultados consistentes em estudos experimentais e a Estimulação Magnética Transcraniana (EMTr) já foi usada visando reduzir danos em neurônios hipocampais de animais submetidos a isquemia cerebral. Com a propriedade de aumentar ou diminuir a excitabilidade cortical a partir do estímulo magnético, estima-se que ocorra uma interferência na produção de alguns neurotransmissores e receptores de membrana, que promoveriam efeito protetor a estas células. Neste estudo avaliamos a capacidade da EMTr de proteger os neurônios de uma lesão por hipóxia, e sua possível interferência no efeito protetor da hipotermia, tentando identificar alguns mecanismos que possivelmente estariam envolvidos neste fenômeno. Métodos: Como modelo de isquemia, foram utilizados Gerbils previamente submetidos a uma avaliação de comportamento e memória por meio do teste de esquiva. O protocolo de EMTr foi a partir de sessões diárias com 25 séries de 5 segundo a 25Hz, com um intervalo de 45 segundos entre as séries, por sete dias consecutivos, com um total de 21 875 pulsos com uma intensidade de 100% do limiar motor, e sendo realizada a indução da isquemia logo após o término da última sessão, ou na isquemia após a EMTr, em sessões diárias com 25 séries de 5 segundos a 25Hz, com um intervalo de 45 segundos entre as séries, durante 3 dias consecutivos, começando imediatamente após a cirurgia. Foi mantida a temperatura de 36 °C durante o período de oclusão do vaso e os 30 minutos consecutivos, ou 31 a 32 °C quando em hipotermia. O preparo das lâminas teve cortes envolvendo a região do hipocampo, corados com hematoxilina e eosina, além de outros preparos, a marcação de TUNEL e Caspase, que visam evidenciar a ocorrência de apoptose. Resultados: Embora sem significância estatística, os animais que receberam EMTr aparentemente tiveram uma melhor performance no teste da esquiva, principalmente se aplicado após a indução da isquemia. A hipotermia demonstrou uma eficiência significativa, tanto na análise histológica quanto no teste da esquiva, associado ou não à EMTr, e nestes animais submetidos a isquemia durante a hipotermia, os que receberam EMTr tiveram área de sobrevida no hipocampo significativamente maior na análise histológica com hematoxilina e eosina. Nos animais submetidos à isquemia durante a temperatura normal, a EMTr não demonstrou aumentar a área de sobrevida das células do hipocampo. Conclusões: A EMTr (ativa ou placebo, prévia ou posterior à isquemia) pareceu ter um efeito positivo no teste de esquiva. O procedimento de estimulação pareceu bastante traumático e estressante para os animais, tendo ocorrido alguns óbitos durante a imobilização, provavelmente por asfixia. A EMTr apresentou efeito protetor significativo apenas nos animais submetidos a isquemia durante hipotermia / Introduction: Over the time many researches have been conducted with the aim of identifying agents that may have beneficial effects in the treatment or prevention of cerebral ischemia, hypothermia has shown consistent results in experimental trials and Repetitive Trans Cranial Magnetic Stimulation (rTMS) has been used in a study attempting to reduce damage in hippocampal neurons. With the property to increase or decrease cortical excitability from the repetitive magnetic stimulus, it is estimated that an interference occurs in the production of some neurotransmitters and receptors of neuronal membrane, which therefore protects these cells from hypoxia. In this study we evaluated the ability of rTMS to protect neurons from injury due to hypoxia, and its possible interference in the protective effect of hypothermia and we tried to identify some mechanisms that possibly are involved in this phenomenon. Methods: Ischemia model was performed using Gerbil that was subsequently submitted to an evaluation of behavior and memory through passive avoidance task. The rTMS protocol was daily sessions with 25 series of 5 seconds at 25Hz with an interval of 45 seconds between series, for 7 consecutive days, with a total of 21 875 pulses with an intensity of 100% of motor threshold, and being carried through the induction of ischemia soon after the end of the last session, or rTMS after ischemia, in daily sessions with 25 series of 5 seconds at 25Hz with an interval of 45 seconds between series, for 3 consecutive days, starting immediately after surgery. The temperature of 36 °C was maintained during the period of vessel occlusion and subsequent 30 minutes, or 31 °C to 32 °C when in hypothermia. The preparation of the slices had sections of the region involving the hippocampus, stained with hematoxylin and eosin in addition to other preparations, TUNEL and caspase, which aim to evidence the occurrence of apoptosis. Results: Although not statistically significant, animals that received rTMS, apparently had better performance in passive avoidance task especially when applied after ischemia. The hypothermia demonstrated a significant efficiency, both in the histological analysis and in the passive avoidance task, associated or not to applications of rTMS and, in these animals undergoing ischemia during hypothermia, the ones who received rTMS had survival area in hippocampus significantly higher in histological analysis with hematoxylin and eosin. In animals undergone to ischemia during normal temperature, the rTMS has not shown to increase the area of hippocampal cell survival. Conclusions: rTMS (placebo or active, after or before the ischemia) seems to have a positive effect on passive avoidance task. The stimulation procedure appeared to be very traumatic and stressful for the animal, in which a few deaths occurred during the procedure, probably from asphyxiation due to restraint. The rTMS had a significant protective effect only in animals undergoing ischemia during hypothermia, as demonstrated in the histological analysis with hematoxylin and eosin

No description available.

Die vorliegende Arbeit beschäftigt sich mit dem Einfluss der NMDA-Rezeptorantagonisten, Memantin, MK-801, CGS und Ifenprodil auf die hypoxieinduzierte Zellschädigung an kortikalen Zellen der Ratte. Außerdem wurde der Einfluss von subtoxischen Konzentrationen von NMDA sowie von Roscovitin, einem Hemmer Cyclin-abhängiger Kinasen, auf die hypoxiebedingte Zellschädigung untersucht. Ziel dieser Arbeit war es, die neuroprotektive Wirkung dieser Substanzen zu erfassen. Zur Untersuchung der hypoxischen Schädigung wurden zwei 48-Well-Zellkulturplatten mit 15 Tage alten kortikalen Zellen der Ratte verwendet. Eine Kulturplatte wurde für vier Stunden mit HEPES(N-2-Hydroxyethylpiperazine-N’-2-Ethansulfonsäure)-Puffer (ohne Glucose) unter hypoxischen Bedingungen inkubiert. Die zweite Platte, mit glukorisiertem HEPES-Puffer, wurde für vier Stunden unter normoxischen Bedingungen inkubiert. Der HEPES-Puffer wurde nach vier Stunden entfernt, die Kulturplatten mit Dulbecco’s Modified Eagle Medium (DMEM) gewaschen und mit diesem Medium für 24 Stunden unter normoxischen Bedingungen inkubiert. Anschließend wurde das Medium ent¬fernt, durch NMDA, Memantin, Roscovitin, CGS und Ifenprodil ersetzt und die Ansätze für weitere 24 Stunden unter normoxischen Bedingungen inkubiert. Zur Beurteilung der Zellschädigung wurden der Aktivitätsanstieg der Laktat-Dehydrogenase (LDH), die Freisetzung freier Sauerstoffradikale und die Steigerung der Caspase-Aktivität bestimmt. Während die Bestimmung der LDH-Aktivität und die Freisetzung der freien Sauer¬stoff¬radikale nekrotische Veränderungen der Zellen charakterisiert, zeigt eine Zunahme der Caspase-Aktivität apoptotische Vorgänge an. LDH ist ein stabiles zytoplasmatisches Enzym, das in fast allen Körperzellen vorkommt. Beim Absterben der Zelle wird das Enzym durch die Schädigung der Plasmamembran aus der Zelle freigesetzt, so dass es zu einem Anstieg der LDH-Aktivität proportional zur Anzahl der toten Zellen kommt. Diese Aktivität wurde spektrophotometrisch mit einem Mikrotiterplatten-Lesegerät bestimmt. Die Ergebnisse des LDH-Tests zeigen, dass nach der 24-stündigen Behandlung der Zellen mit MK-801 die LDH-Aktivität um 11%, bei Roscovitin um 13%, bei Memantin (5 µM) um 56%, bei Memantin (0,5 µM) um 52% und mit NMDA (5 µM) um 44% signifikant vermindert wurde. Bei einer hypoxiebedingten Schädigung kortikaler Zellen kommt es auch zur Bildung freier Sauer¬stoff¬radikale. 2’,7’-Dichlorfluorescein Diacetat (2’,7’-H2DCF-DA) wird von den Zellen auf¬ge¬nommen und intrazellulär mit Sauerstoff- und Stickstoffspezies zum Fluoreszenz¬farb-stoff 2’,7’-Dichlorodihydrofluorescein (DCF) deacetyliert. DCF verbleibt dabei in den Zellen, so dass die Messung der Fluoreszenz der Zellen als Maß für intrazelluläre Oxidationsprozesse verwendet werden kann. Die DCF-Fluoreszenz-Änderung wurde mittels eines Fluorimeters gemessen und die daraus resultierenden Daten mit einer im Fluorimeter integrierten Software bearbeitet. Die Ergebnisse zeigen, dass die Freisetzung der freien Sauerstoffradikale, der hypoxiegeschädigten Zellen, signifikant durch Ifenprodil (10 µM) um 119%, Memantin (50 µM) um 88% und NMDA (5 µM) um 134% reduziert wurde. Die hypoxieinduzierte Zellmembranschädigung führt desweiteren zu einem Anstieg der Caspase-Aktivität. Mit Hilfe des Apo-One Homogeneous Caspase-3/7-Assays (Promega) wurde die Aktivität der Caspasen 3 und 7 fluorimetrisch bestimmt. Um die unterschiedliche Zelldichte in den Kulturschalen zu berücksichtigen, wurde eine Proteinbestimmung nach der Bicinchoninsäure-Methode (Smith et al. 1985) durchgeführt. Einen protektiven Effekt auf die Zellschädigung zeigen Memantin und NMDA in Bezug auf die Beeinflussung dieser Caspase-Aktivität. Der hypoxiebedingte Anstieg der Caspase-3-Aktivität konnte nach 24-stündiger Inkubation mit Memantin (5 µM) um 24%, mit Memantin (0,5 µM) um 28% und mit NMDA (5 µM) um 24% vermindert werden. CGS hat in diesen Versuchen keinen protektiven Einfluss auf die hypoxie¬induzierte Zellschädigung. Diese Arbeit zeigt, dass die Applikation niedriger NMDA-Konzentrationen neuroprotektive Effekte auf die Entwicklung der hypoxischen Schädigung von kortikalen Zellen der Ratte hat. Darüber hinaus wird vermutet, dass NMDA sogar einen trophischen Effekt auf das Über-leben der kortikalen Neurone ausübt. Dieser schützende Mechanismus von NMDA scheint denselben, wenn nicht sogar einen größeren protektiven Effekt wie Memantin zu induzieren. Um die Therapiemöglichkeiten der zerebralen Hypoxie durch neuroprotektive Medikamente zu optimieren, wären jedoch weitergehende Untersuchungen besonders als In-vivo-Modelle wünschenswert.

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

>Magister Scientiae - MSc / This study focused on the synthesis of a series of novel tricycloundecane derivatives and evaluation of these compounds for neuroprotection using the fluorescent ratiometric calcium assay that indicates the ability of the test compounds to inhibit NMDA receptors and VGCC. The cycloaddition reaction between p-benzoquinone and monomerised dicyclopentadiene yielded tricycloundeca- 4,9-diene-3,6-dione which was used as the base structure and further derivatised. These derivatives were conjugated with benzylamine to form a series of imines and amines. A total of 10 compounds were synthesised for evaluation of inhibition of calcium influx through NMDA receptor channels and voltage-gated calcium channels. The structures were confirmed using NMR, IR and MS. On the proton NMR, the characteristic AB-quartet system was observed in the region of 1-2 ppm for all the compounds and the aromatic moiety was observed between 6.5-7.5 ppm for the novel polycyclic amines. These, with other functional groups, were used to confirm the individual structures