Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Sepse induz exagerada produção de mediadores inflamatórios, como o óxido nítrico (NO), e causa alterações cardiovasculares, neuroendócrinas e de temperatura corporal (Tc). Na fase tardia da sepse existe liberação basal de vasopressina (AVP), apesar da hipotensão persistente. Uma hipótese para isso seria a inibição da síntese de AVP pelo aumento da produção de NO. Nosso objetivo foi investigar a possível participação do NO, produzido centralmente pelas isoformas de NO sintase (NOS), na expressão de AVP e ocitocina (OT), na resposta cardiovascular e de Tc durante sepse experimental. Ratos Wistar receberam injeção i.c.v. de L-NAME, inibidor não seletivo de NOS (250g/L), ou de aminoguanidina (AG,250g/L), inibidor seletivo da isoforma induzida (NOSi). Outro grupo recebeu inibidor da guanilato ciclase solúvel (ODQ,0,25g/L). Grupos controles receberam veículos (salina ou DMSO1%). Trinta minutos após as injeções, foi induzido sepse por ligadura e perfuração cecal (CLP) ou operação fictícia. Os animais foram divididos em 4 grupos: 1) avaliação da sobrevida, 2) determinação da Tc, 3) aferição da pressão arterial (PAM) e frequência cardíaca (FC) e 4) avaliação de parâmetros hidroeletrolíticos e secreção de AVP e OT. A CLP promoveu alta mortalidade, aumentou NO progressivamente, diminuiu PAM e aumentou FC. A concentração plasmática de AVP (AVPp) aumentou na fase inicial da sepse e por seu efeito antipirético pode ter contribuído para a hipotermia observada. A razão de expressão para ambos os hormônios diminuiu nos núcleos supraóticos (SON) e paraventriculares (PVN). Na fase tardia AVPp estava basal e sua expressão nos SON e PVN mais diminuída do que na fase inicial. A expressão de OT diminuiu somente no SON. O pré-tratamento com L-NAME aumentou a sobrevida, reduziu a produção de NO até 20h, aumentou PAM e Tc, e manteve a FC semelhante ao grupo veículo. AVPp aumentou simultaneamente à diminuição da razão de expressão em ambos os núcleos. Na fase tardia, o grupo L-NAME apresentou NO aumentado e expressão de AVP diminuída, aparentemente contribuindo para AVPp basal e hipotensão. O L-NAME diminuiu a razão de expressão de OT na fase inicial, mas aumentou na fase tardia. A inibição de NOSi pela AG aumentou ainda mais a sobrevida e Tc. Apesar de não bloquear a produção de NO a AG aumentou a expressão de AVP e OT e manteve AVPp constante e acima da basal. Injeção i.c.v de AG aumentou PAM somente na fase inicial da sepse. O pré-tratamento com ODQ foi o mais eficiente em aumentar a sobrevida e Tc após CLP. Entretanto, não alterou o aumento progressivo de NO, e ainda diminuiu a expressão de AVP e OT. A AVPp basal após ODQ contribuiu para a hipotensão observada durante todo o período. Esses resultados mostram que aumento central de NO após CLP inibe a expressão de AVP e OT independente de GMPc no SON e parcialmente dependente no PVN. A inibição da expressão de AVP na fase tardia da sepse resulta em concentração basal do hormônio contribuindo para a hipotensão. Em nossos experimentos o controle da temperatura corporal teve maior contribuição no aumento da sobrevida durante sepse polimicrobiana do que a regulação neuroendócrina e/ou cardiovascular. / Sepsis induces massive production of inflammatory mediators, such as nitric oxide (NO), and causes cardiovascular, neuroendocrine and body temperature (Tb) alterations. In the late phase of sepsis there is a basal vasopressin (AVP) release despite the persisting hypotension. One reason could be the inhibition of AVP synthesis by the increase in NO production. Our aim was to investigate the possible involvement of NO, produced centrally by NO synthase (NOS) isoforms, on AVP and oxytocin (OT) expression, cardiovascular response and Tb during experimental sepsis. Male Wistar rats received an i.c.v. injection of the non-selective NOS inhibitor L-NAME (250g/L), or of aminoguanidine (AG,250g/L), a selective inhibitor of its inducible isoform (iNOS). Another group received soluble guanylate cyclase inhibitor (ODQ,0.25µg/L). Control groups received vehicles (saline or DMSO1%). Thirty minutes after the injections, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 4 groups for: 1) assessment of survival, 2) determination of Tb, 3) measurement of blood pressure (MAP) and heart rate (HR), and 4) evaluation of hydroelectrolytic parameters and AVP and OT secretion. The CLP promoted high mortality and progressive increase in NO levels. It also decreased MAP and increased HR. The AVP plasma concentration (AVPp) increased in the early phase of sepsis and its antipyretic effect may have contributed to the observed hypothermia. The expression ratio of both hormones was reduced in the supraoptic (SON) and paraventricular (PVN) nuclei. In the late phase, AVPp was basal and its expression decreased in both nuclei more than in the initial phase. The OT expression decreased only in the SON. L-NAME pretreatment increased the survival and reduced the NO production until 20h. MAP and Tb were increased, while HR remained similar to that observed in the vehicle control group. AVPp increased simultaneously to the decrease of its expression ratio in both nuclei. In the late phase, the L-NAME group showed NO levels increased and decreased AVP expression, apparently contributing to basal AVPp and hypotension. The L-NAME decreased OT expression ratio in the initial phase, but increased in the late phase. Inhibition of iNOS by AG further increased the survival and Tb. Even though AG did not block NO production, it increased AVP and OT expression and kept AVPp constant and above the baseline. AG pretreatment increased MAP only in the initial phase of sepsis. The ODQ pretreatment was more efficient to increase survival and Tb after CLP. However it neither altered the progressive NO increase nor the decrease in AVP and OT expression ratio. The basal AVPp after ODQ contributed to hypotension observed during the studied period. These results show that the increased central NO levels observed after CLP inhibit cGMP-independent hormone expression in the SON and partially dependent in the PVN. Inhibition of AVP expression, in the late phase of sepsis, results in basal concentrations of this hormone further contributing to hypotension. In our experiments the control of body temperature during polymicrobial sepsis had greater contribution in survival than the neuroendocrine and/or cardiovascular regulation.

O presente estudo teve como objetivo avaliar o efeito da inibição das cadeias inflamatórias reguladas pela ação do interferon-gama (IFN-γ) e da enzima óxido nítrico sintase indutível (iNOS) no dano radioinduzido após exposição a dose letal (8 Gy) ou moderada a severa (4 Gy) nos tecidos hematopoiéticos (baço e medula) de modelos experimentais irradiados nestas doses. Grupos de camundongos isogênicos C57Bl/6j foram expostos à radiação correspondente a 4 ou 8 Gy em exposições de corpo inteiro em fonte panorâmica de 60Co. Da mesma forma, foram irradiados camundongos cuja expressão de IFN-γ ou iNOS é ausente ou indetectável. Outros grupos receberam via oral por toda a duração do experimento um inibidor atividade de iNOS, aminoguanidina, ou via intraperitoneal uma quimiocina primordial promotora da hematopoiese, CXCL12, até o quarto dia após a exposição. Outra divisão experimental recebeu os dois agentes concomitantemente. Os animais foram sacrificados nos dias 2º, 4º e 8º após a irradiação, e fragmentos dos baços e fêmures foram preservados para histologia. Os esplenócitos e células não aderentes da medula óssea femoral foram removidos e divididos, fornecendo alíquotas para posterior RT-PCR e suspensões celulares apropriadas para ensaios de citometria de fluxo específicos para a detecção da freqüência de populações CD34+. Nestes mesmos dias de experimento, alíquotas de sangue caudal foram coletadas para contagem de hemácias e plaquetas periféricas. Os resultados mostraram que a ausência da produção de interferon-gama no local irradiado aumenta a sobrevivência e a quantidade de células progenitoras hematopoiéticas e que a ausência de iNOS ou seu bloqueio funcional diminuem a extensão do dano radioinduzido nos tecidos hematopoiéticos. Além disso, foi possível observar que a suplementação com CXCL12 sintética aumenta a freqüência do fenótipo CD34+ P.chave: radiação ionizante; medula óssea; óxido nítrico; aminoguanidina nos baços dos modelos testados, e que seu efeito parece antagonizar com a inibição da produção de NO pela aminoguanidina. / This study aimed to evaluate the effect of inhibition of the inflammatory chains governed by the action of interferon-gamma (IFN-γ) and the enzyme inducible nitric oxide synthase (iNOS) in damage after radiation exposure to lethal dose (8 Gy) or moderate /severe dose (4 Gy) in hematopoietic tissues (spleen and bone marrow) of experimental models irradiated at these doses. Groups of isogenic C57Bl/6j mice were exposed to radiation (4 or 8 Gy) in whole-body exposures in a 60Co panoramic source. Similarly, were irradiated mice whose iNOS or IFN-γ expression was absent or undetectable. Other groups received orally in all days of experiments an inhibitor of iNOS activity, aminoguanidine, or CXCL12, a primordial chemokine known as an hematopoeisis promoter intraperitonially by days 0 to 4th after radiation events Another groups received the two agents concomitantly. The animals were sacrificed on days 2nd, 4th and 8th after irradiation, and fragments of the spleens and femurs were preserved for histology. Splenocytes and non-adherent cells from femoral bone marrow were removed and divided, providing aliquots for subsequent RT-PCR and cell suspensions suitable for flow cytometry experiments specific to the detection of the frequency of CD34+ cell populations. In same days of experiment, tail blood samples were collected for counting of peripheral red blood cells and platelets. The results showed that the absence of the production of interferon-gamma at the irradiated sites increases the survival and the amount of hematopoietic progenitor cells and that the absence of iNOS or its functional blockade reduces the extent of radioinduced damage in tested hematopoietic tissues. Furthermore, it was possible to observe that supplementing with synthetic CXCL12 increases the frequency of CD34+ Keywords: ionizing radiation, bone marrow, nitric oxide, aminoguanidine phenotype in the spleens of tested models, and that its effect seems to antagonize the inhibition of the production of NO by aminoguanidine.

Sepse induz exagerada produção de mediadores inflamatórios, como o óxido nítrico (NO), e causa alterações cardiovasculares, neuroendócrinas e de temperatura corporal (Tc). Na fase tardia da sepse existe liberação basal de vasopressina (AVP), apesar da hipotensão persistente. Uma hipótese para isso seria a inibição da síntese de AVP pelo aumento da produção de NO. Nosso objetivo foi investigar a possível participação do NO, produzido centralmente pelas isoformas de NO sintase (NOS), na expressão de AVP e ocitocina (OT), na resposta cardiovascular e de Tc durante sepse experimental. Ratos Wistar receberam injeção i.c.v. de L-NAME, inibidor não seletivo de NOS (250g/L), ou de aminoguanidina (AG,250g/L), inibidor seletivo da isoforma induzida (NOSi). Outro grupo recebeu inibidor da guanilato ciclase solúvel (ODQ,0,25g/L). Grupos controles receberam veículos (salina ou DMSO1%). Trinta minutos após as injeções, foi induzido sepse por ligadura e perfuração cecal (CLP) ou operação fictícia. Os animais foram divididos em 4 grupos: 1) avaliação da sobrevida, 2) determinação da Tc, 3) aferição da pressão arterial (PAM) e frequência cardíaca (FC) e 4) avaliação de parâmetros hidroeletrolíticos e secreção de AVP e OT. A CLP promoveu alta mortalidade, aumentou NO progressivamente, diminuiu PAM e aumentou FC. A concentração plasmática de AVP (AVPp) aumentou na fase inicial da sepse e por seu efeito antipirético pode ter contribuído para a hipotermia observada. A razão de expressão para ambos os hormônios diminuiu nos núcleos supraóticos (SON) e paraventriculares (PVN). Na fase tardia AVPp estava basal e sua expressão nos SON e PVN mais diminuída do que na fase inicial. A expressão de OT diminuiu somente no SON. O pré-tratamento com L-NAME aumentou a sobrevida, reduziu a produção de NO até 20h, aumentou PAM e Tc, e manteve a FC semelhante ao grupo veículo. AVPp aumentou simultaneamente à diminuição da razão de expressão em ambos os núcleos. Na fase tardia, o grupo L-NAME apresentou NO aumentado e expressão de AVP diminuída, aparentemente contribuindo para AVPp basal e hipotensão. O L-NAME diminuiu a razão de expressão de OT na fase inicial, mas aumentou na fase tardia. A inibição de NOSi pela AG aumentou ainda mais a sobrevida e Tc. Apesar de não bloquear a produção de NO a AG aumentou a expressão de AVP e OT e manteve AVPp constante e acima da basal. Injeção i.c.v de AG aumentou PAM somente na fase inicial da sepse. O pré-tratamento com ODQ foi o mais eficiente em aumentar a sobrevida e Tc após CLP. Entretanto, não alterou o aumento progressivo de NO, e ainda diminuiu a expressão de AVP e OT. A AVPp basal após ODQ contribuiu para a hipotensão observada durante todo o período. Esses resultados mostram que aumento central de NO após CLP inibe a expressão de AVP e OT independente de GMPc no SON e parcialmente dependente no PVN. A inibição da expressão de AVP na fase tardia da sepse resulta em concentração basal do hormônio contribuindo para a hipotensão. Em nossos experimentos o controle da temperatura corporal teve maior contribuição no aumento da sobrevida durante sepse polimicrobiana do que a regulação neuroendócrina e/ou cardiovascular. / Sepsis induces massive production of inflammatory mediators, such as nitric oxide (NO), and causes cardiovascular, neuroendocrine and body temperature (Tb) alterations. In the late phase of sepsis there is a basal vasopressin (AVP) release despite the persisting hypotension. One reason could be the inhibition of AVP synthesis by the increase in NO production. Our aim was to investigate the possible involvement of NO, produced centrally by NO synthase (NOS) isoforms, on AVP and oxytocin (OT) expression, cardiovascular response and Tb during experimental sepsis. Male Wistar rats received an i.c.v. injection of the non-selective NOS inhibitor L-NAME (250g/L), or of aminoguanidine (AG,250g/L), a selective inhibitor of its inducible isoform (iNOS). Another group received soluble guanylate cyclase inhibitor (ODQ,0.25µg/L). Control groups received vehicles (saline or DMSO1%). Thirty minutes after the injections, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 4 groups for: 1) assessment of survival, 2) determination of Tb, 3) measurement of blood pressure (MAP) and heart rate (HR), and 4) evaluation of hydroelectrolytic parameters and AVP and OT secretion. The CLP promoted high mortality and progressive increase in NO levels. It also decreased MAP and increased HR. The AVP plasma concentration (AVPp) increased in the early phase of sepsis and its antipyretic effect may have contributed to the observed hypothermia. The expression ratio of both hormones was reduced in the supraoptic (SON) and paraventricular (PVN) nuclei. In the late phase, AVPp was basal and its expression decreased in both nuclei more than in the initial phase. The OT expression decreased only in the SON. L-NAME pretreatment increased the survival and reduced the NO production until 20h. MAP and Tb were increased, while HR remained similar to that observed in the vehicle control group. AVPp increased simultaneously to the decrease of its expression ratio in both nuclei. In the late phase, the L-NAME group showed NO levels increased and decreased AVP expression, apparently contributing to basal AVPp and hypotension. The L-NAME decreased OT expression ratio in the initial phase, but increased in the late phase. Inhibition of iNOS by AG further increased the survival and Tb. Even though AG did not block NO production, it increased AVP and OT expression and kept AVPp constant and above the baseline. AG pretreatment increased MAP only in the initial phase of sepsis. The ODQ pretreatment was more efficient to increase survival and Tb after CLP. However it neither altered the progressive NO increase nor the decrease in AVP and OT expression ratio. The basal AVPp after ODQ contributed to hypotension observed during the studied period. These results show that the increased central NO levels observed after CLP inhibit cGMP-independent hormone expression in the SON and partially dependent in the PVN. Inhibition of AVP expression, in the late phase of sepsis, results in basal concentrations of this hormone further contributing to hypotension. In our experiments the control of body temperature during polymicrobial sepsis had greater contribution in survival than the neuroendocrine and/or cardiovascular regulation.

O presente estudo teve como objetivo avaliar o efeito da inibição das cadeias inflamatórias reguladas pela ação do interferon-gama (IFN-γ) e da enzima óxido nítrico sintase indutível (iNOS) no dano radioinduzido após exposição a dose letal (8 Gy) ou moderada a severa (4 Gy) nos tecidos hematopoiéticos (baço e medula) de modelos experimentais irradiados nestas doses. Grupos de camundongos isogênicos C57Bl/6j foram expostos à radiação correspondente a 4 ou 8 Gy em exposições de corpo inteiro em fonte panorâmica de 60Co. Da mesma forma, foram irradiados camundongos cuja expressão de IFN-γ ou iNOS é ausente ou indetectável. Outros grupos receberam via oral por toda a duração do experimento um inibidor atividade de iNOS, aminoguanidina, ou via intraperitoneal uma quimiocina primordial promotora da hematopoiese, CXCL12, até o quarto dia após a exposição. Outra divisão experimental recebeu os dois agentes concomitantemente. Os animais foram sacrificados nos dias 2º, 4º e 8º após a irradiação, e fragmentos dos baços e fêmures foram preservados para histologia. Os esplenócitos e células não aderentes da medula óssea femoral foram removidos e divididos, fornecendo alíquotas para posterior RT-PCR e suspensões celulares apropriadas para ensaios de citometria de fluxo específicos para a detecção da freqüência de populações CD34+. Nestes mesmos dias de experimento, alíquotas de sangue caudal foram coletadas para contagem de hemácias e plaquetas periféricas. Os resultados mostraram que a ausência da produção de interferon-gama no local irradiado aumenta a sobrevivência e a quantidade de células progenitoras hematopoiéticas e que a ausência de iNOS ou seu bloqueio funcional diminuem a extensão do dano radioinduzido nos tecidos hematopoiéticos. Além disso, foi possível observar que a suplementação com CXCL12 sintética aumenta a freqüência do fenótipo CD34+ P.chave: radiação ionizante; medula óssea; óxido nítrico; aminoguanidina nos baços dos modelos testados, e que seu efeito parece antagonizar com a inibição da produção de NO pela aminoguanidina. / This study aimed to evaluate the effect of inhibition of the inflammatory chains governed by the action of interferon-gamma (IFN-γ) and the enzyme inducible nitric oxide synthase (iNOS) in damage after radiation exposure to lethal dose (8 Gy) or moderate /severe dose (4 Gy) in hematopoietic tissues (spleen and bone marrow) of experimental models irradiated at these doses. Groups of isogenic C57Bl/6j mice were exposed to radiation (4 or 8 Gy) in whole-body exposures in a 60Co panoramic source. Similarly, were irradiated mice whose iNOS or IFN-γ expression was absent or undetectable. Other groups received orally in all days of experiments an inhibitor of iNOS activity, aminoguanidine, or CXCL12, a primordial chemokine known as an hematopoeisis promoter intraperitonially by days 0 to 4th after radiation events Another groups received the two agents concomitantly. The animals were sacrificed on days 2nd, 4th and 8th after irradiation, and fragments of the spleens and femurs were preserved for histology. Splenocytes and non-adherent cells from femoral bone marrow were removed and divided, providing aliquots for subsequent RT-PCR and cell suspensions suitable for flow cytometry experiments specific to the detection of the frequency of CD34+ cell populations. In same days of experiment, tail blood samples were collected for counting of peripheral red blood cells and platelets. The results showed that the absence of the production of interferon-gamma at the irradiated sites increases the survival and the amount of hematopoietic progenitor cells and that the absence of iNOS or its functional blockade reduces the extent of radioinduced damage in tested hematopoietic tissues. Furthermore, it was possible to observe that supplementing with synthetic CXCL12 increases the frequency of CD34+ Keywords: ionizing radiation, bone marrow, nitric oxide, aminoguanidine phenotype in the spleens of tested models, and that its effect seems to antagonize the inhibition of the production of NO by aminoguanidine.

Orientador: Iguatemy Lourenço Brunetti / Resumo: O diabetes mellitus (DM) é uma síndrome metabólica crônica, caracterizada principalmente por hiperglicemia. A hiperglicemia crônica é um dos principais fatores envolvidos na instalação do estresse glico-oxidativo, o qual está implicado no desenvolvimento das complicações micro e macrovasculares do DM, as quais resultam em morbidade e mortalidade significativas. Portanto, o tratamento da doença tem como principal objetivo a manutenção dos níveis glicêmicos próximos a normalidade e a atenuação do estresse glico-oxidativo. A curcumina, apresenta potencial efeito antidiabético e antioxidante. Já a aminoguanidina é um dos agentes mais utilizados para prevenir a formação de produtos finais da glicação avançada (AGE), in vitro e em modelos animais. Esse estudo investigou em ratos, o potencial antidiabético da curcumina e/ou aminoguanidina incorporadas no iogurte, avaliando parâmetros relacionados às alterações do controle glicêmico, estresse glico-oxidativo (plasma, fígado e rim) e parâmetros da função neutrofílica (exsudato peritoneal). Ratos Wistar, não diabéticos e diabéticos (estreptozotocínico) não tratados, receberam apenas iogurte e os diabéticos tratados receberam: insulina (4U/dia); 90 mg/kg de curcumina; 50 mg/kg de aminoguanidina; 90 mg/kg de curcumina e 50 mg/kg de aminoguanidina; 100 mg/kg de aminoguanidina; e 90 mg/kg de curcumina e 100 mg/kg de aminoguanidina; os tratamentos foram realizados durante 45 dias. Os parâmetros analisados foram: (i) fisiológicos: peso corpo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Diabetes mellitus (DM) is a chronic metabolic syndrome characterized mainly by hyperglycemia. Chronic hyperglycemia is one of the main factors involved in the installation of glycoxidative stress, and it is implicated in the development of micro and macrovascular complications of DM, which result in significant morbidity and mortality. Therefore, the treatment of the disease has as main objective the maintenance of glycemic to levels near to normality and the attenuation of glycoxidative stress. Curcumin has potential antidiabetic and antioxidant effects. Aminoguanidine is one of the most used agents to prevent the formation of advanced glycation end products (AGE), in vitro and animal models. This study investigated in rats the antidiabetic potential of curcumin and/or aminoguanidine incorporated into yoghurt, evaluating parameters related to changes in glycemic control, glycoxidative stress (plasma, liver and Kidney) and parameters of neutrophil function (peritoneal exudate). Rats Wistar, non-diabetic and diabetic (streptozotocinic) untreated received alone yoghurt and treated-diabetic received: insulin (4U/day); 90 mg/kg curcumin; 50 mg/kg aminoguanidine; 90 mg/kg curcumin and 50 mg/kg aminoguanidine; 100 mg/kg aminoguanidine; and (vi) 90 mg/kg curcumin and 100 mg/kg aminoguanidine; the treatments were performed for 45 days. The parameters analyzed were: (i) physiological: body weight; (ii) biochemicals: glycemia, glycated hemoglobin (HbA1c), fructosamine, total cholestero... (Complete abstract click electronic access below) / Doutor

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Diabetes leads to vascular complications arising from biochemical changes generated by accumulation of advanced glycation end-products (AGEs). Aminoguanidine is an anti-AGE in clinical phase III studies, but its chronic use can lead to nephrotoxicity and hepatotoxicity. For this reason, structural changes were made in the aminoguanidine molecule, originating nineteen derivatives, with which were evaluated for their antidiabetic and/or antiglycation effects in diabetic rats. For this purpose, initially was performed the in vitro cytotoxicity assay using the colorimetric assay with MTT3-(4,5-dimethylthiazol-2-yl)-2,5 difeniiterazolium) in the J774A.1 macrophage cell line. To evaluate the antiglycation activity, AGES were produced in vitro by combining glucose with bovine serum albumin (BSA). The derivatives with the best outcome in vitro were submitted to acute toxicity tests in vivo at concentrations of 2 mg/kg, 10 mg/kg and 100 mg/kg oral administration and, thereafter the antidiabetic and antiglycation potential were checked in Wistar rats with diabetes induced by streptozotocin (20 mg/kg, i.p.). After euthanasia, heart, kidney and liver were extracted from the animals and submitted to histopathotological examination and blood for hemogram and biochemical tests. The outcomes obtained from MTT assay indicated that seventeen out of the nineteen derivatives did not show any toxic effect at the concentration of 10 puM, which means they kept 80% or more of viable cells. In antiglycation assay for in vitro activity, thirteen of these nineteen derivatives got above 50% of inhibition in the formation of advanced glycation end products in a concentration of 2 mg/ml, and significant when compared to a minoguanidine at the same concentration. In a dose range from 0.5 to 2.5 mg/m, with the reading of inhibition percentage every seven days for 49 days, the derivative LOM 13 reached 94.6% of inhibition, while aminoguanidine inhibited up to 74% of AGES, at concentration of 1.0 mg/ml. In studies in vivo the derivatives LOM3, LOM 13 and LOM15 were non-toxic, that means that the rats did not lose weight, neither showed behavioral, biochemical nor histological changes that could have been related to the use of derivatives. In tests for antidiabetic and antiglycation activity, these three derivatives were able to reduce blood glucose levels by up to 65%, being significant when compared to glimepiride (2 mg/kg, oral administration), the standard drug used. Furthermore, the levels of fructosamine and glycated hemoglobin remained lower in the groups treated with the derivatives. Based on that, it was possible to conclude that the derivatives studied have low cytotoxicity, have the antiglycation potential to AGES-BSA, they were non-toxic at the concentrations used and can reduce blood glucose, fructosamine levels and glycated hemoglobin in diabetic rats. Thus, they represent promising prototype of drugs, enabling the possibility of having in the future a product capable of reducing glycemia and at the same time having antiglycation action, protecting individuals from macro and microvascular complications of diabetes. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O diabetes leva a complicações vasculares provenientes de alterações bioquímicas geradas a partir do acúmulo dos produtos finais da glicação avançada (AGEs). A aminoguanidina é um anti-AGE em estudos clínicos de fase III, porém seu uso crônico pode levar à nefrotoxicidade e hepatotoxicidade. Por isso, modificações estruturais foram realizadas na molécula da aminoguanidina, originando dezenove derivados, os quais foram avaliados para a sua ação antidiabética e/ou antiglicante em ratos diabéticos. Para tanto, realizou-se inicialmente o teste de citotoxicidade in vitro pelo ensaio colorimétrico com MTT (3-(4,5-dimetiltiazol-2-yl)-2,5 difenilterazolium), em macrófagos da linhagem J774A.1. Para avaliação da atividade antiglicante, os AGEs foram produzidos in vitro a partir da junção de glicose com albumina sérica bovina (BSA). Os derivados com melhor resposta in vitro foram submetidos a testes de toxicidade aguda in vivo com concentrações de 2 mg/Kg, 10 mg/Kg e 100 mg/Kg p.o, e, posteriormente, verificou-se o potencial antidiabético e antiglicante em ratos Wistar com diabetes induzida por estrepzotocina (20 mg /kg, i.p.). Após eutanásia, foram retirados coração, rim e fígado dos animais para exames histopatológicos e o sangue para realização do hemograma e dosagens bioquímicas. Os resultados obtidos com o ensaio MTT indicaram que, dezessete dos dezenove derivados, não mostraram qualquer efeito tóxico na concentração de 10 M, ou seja mantiveram 80% ou mais de células viáveis. No ensaio para atividade antiglicante in vitro, treze desses dezenove derivados conseguiram um percentual acima de 50% de inibição na formação de produtos finais da glicação avançada na concentração de 2 mg/mL, sendo significativos quando comparados a aminoguanidina, na mesma concentração. Numa escala de doses, de 0,5 a 2,5 mg/mL, com leitura dos percentuais de inibição a cada sete dias por 49 dias, o derivado LOM 13 atingiu até 94,6% de inibição, enquanto que a aminoguanidina inibiu até 74% de AGEs, na concentração de 1,0 mg/mL. Nos estudos in vivo os derivados LOM3, LOM 13 e LOM15 não foram tóxicos, ou seja, os animais não perderam peso, nem apresentaram alterações comportamentais, nem bioquímicas ou histológicas que pudessem ser atribuídas ao uso dos derivados. Nos testes para atividade antidiabética e antiglicante, esses três derivados conseguiram reduzir os valores de glicemia em até 65%, sendo significativos quando comparados com a glimepirida (2 mg/kg, p.o.), droga padrão utilizada. Além disso, os níveis de frutosamina e hemoglobina glicada se mantiveram baixos nos grupos tratados com os derivados. A partir disso, foi possível concluir que os derivados estudados apresentam baixa citotoxicidade, têm potencial antiglicante para AGES-BSA, não foram tóxicos nas concentrações utilizadas e podem reduzir a glicemia e os níveis de frutosamina e hemoglobina glicada em ratos diabéticos. Portanto, estes representam protótipos de fármacos promissores, para que no futuro seja possível ter um produto capaz de reduzir os índices glicêmicos e, ao mesmo tempo ter ação antiglicante, protegendo os indivíduos das complicações macro e microvasculares do diabetes.