Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 104-122).

Abnormal homeostasis of intracellular Ca2+ plays a deleterious role in muscle pathologies by triggering processes that lead to dysfunction and necrotic or apoptotic cell death. One pathology where there is significant Ca2+ induced cell damage is ischemia, which initiates further damage (also mediated by Ca2+) generated by the required treatment process of revascularization; namely ischemia-reperfusion injury. Pharmacological agents used therapeutically for cell protection, especially for cardiac protection in ischemic heart diseases, have only directly targeted one of the elements regulating Ca2+ homeostasis, the L-type Ca2+ channels (calcium channel blockers). Other agents, like beta blockers, indirectly target various elements, including sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and ryanodine receptors (RyRs). However, there are no pharmacological agents that directly and specifically target these two crucial elements required for intracellular SR Ca2+ homeostasis. Dr. Julio A. Copello’s group has previously studied the cardioprotective agent CGP-37157 (CGP), a benzothiazepine (BZT) derivative of the benzodiazepine (BZD) clonazepam. CGP was previously thought to decrease intracellular SR Ca2+ by acting as a blocker of the mitochondrial Na+/Ca2+ exchanger (Omelchenko et al., 2003). They found, however, that CGP also activates RyRs and inhibits the SERCA, which could better explain the SR effects of the drug (Neumann et al., 2011). These results suggest that drugs inducing partial depletion of SR Ca2+ stores could provide cellular protection in stressful circumstances or processes. The aims of the dissertation were organized based on the two processes that cause damage to muscle cells during ischemia: ischemia and subsequent reperfusion (ischemia-reperfusion injury) (Ibanez et al., 2015). Aim one and two focused on drug-protective action during the ischemic event, while aim three focused on drug protective action in the reperfusion (early post-ischemia) process. In the first Aim, experiments were designed to test the hypotheses that RyRs and/or SERCA could also be the target of i) Drugs with structural similarities to CGP (i.e., other BZTs and some BZDs) and ii) Drug known to confer cellular protection under stressful cellular conditions such as antiepileptic agents. We found that some BZTs (K201, CGP analog) and antiepileptic agents (Sipatrigine and Pimozide) demonstrated potential to prevent SR Ca2+ overload by inhibition of SERCA and, in some cases also by inducing mild activation of RyR channels. These results provided potential mechanisms of action for agents with cell protective action: targeting SERCA and preventing Ca2+ overload in pre-ischemia process. From the results of the first aim, K201 had the most significant effects in both SERCA inhibition and RyRs activation. Therefore, Aim 2 experiments focused on exploring with greater detail the action of the compound K201 on RyRs, SERCA and Ca2+ signaling. We found that K201 is a more potent SERCA blocker than RyR agonist and that SERCA inhibition remains under acidosis mimicking ischemic conditions. In Aim 3, the focus was on testing drugs with potential to prevent the overloaded SR from leaking Ca2+ (via RyRs) upon reperfusion. For that, we have examined various classes of organic polycationic agents in their ability to act as fast and reversible RyRs blockers. Currently, no agent with these characteristics is availableas a therapeutic or has been well defined for use as an experimental drug. The membrane permeable cation DHBP was identified as a potent RyR inhibitor with potential for rapid and transient inhibition of spontaneous SR Ca2+ release during reperfusion. In summary, we have defined the ability of some BZTs and antiepileptic agents (K201, CGP analog, Sipatrigine and Pimozide) to prevent/slow down SR Ca2+ overload by inhibition of SERCA, which may play an important role in their mechanisms of cell protection in ischemic events. In the case of BZT, these drugs may help their cause by producing mild activation of RyR2 channels, In addition, we have identify DHBP as a reversible and fast acting RyR inhibitor with potential as template for development of transient inhibitors of spontaneous SR Ca2+ release which may have significant protective action against injury during early reperfusion of the heart.

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:31:10Z : No. of bitstreams: 1 cerqueira_bf_dr_botfmvz.pdf: 1499209 bytes, checksum: 7ff8a218fa7c9053994488fe2ecdb205 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A oclusão e a reperfusão das artérias esplâncnicas provoca alterações locais e sistêmicas derivadas principalmente da liberação de substâncias citotóxicas e da interação entre neutrófilos e células endoteliais. Buscando-se estudar os efeitos da pentoxifilina e da n-acetilcisteína em lesões provocadas em um modelo de isquemia e reperfusão (I/R), 60 ratos foram divididos em 6 grupos: SAL/CONT (salina/controle): animais submetidos à cirurgia, mas não à I/R, tratados com solução fisiológica; SAL/ISQ (salina/isquemia): animais submetidos à oclusão da artéria celíaca (AC), artéria mesentérica cranial (AMCr) e artéria mesentérica caudal (AMCa) durante 30 minutos, seguidos de 120 minutos de reperfusão, tratados com solução fisiológica; PTX/CONT: animais submetidos à cirurgia, mas não à I/R, tratados com pentoxifilina; PTX/ISQ: animais submetidos à I/R, tratados com pentoxifilina; NAC/CONT: animais submetidos à cirurgia, mas não à I/R, tratados com n-acetilcisteína; NAC/ISQ: animais submetidos à I/R, tratados com n-acetilcisteína. Os parâmetros avaliados foram pressão arterial média (PAM) carótida, pressão venosa jugular, fluxo sangüíneo na aorta e na AMCr, temperatura esofágica, hematócrito, hemogasometria, histologia intestinal, dosagem de espécies reativas ao ácido tiobarbitúrico (TBARS) em duodeno, jejuno e íleo e de malondialdeído (MDA) plasmático e do íleo. Nos grupos submetidos à I/R, a PAM teve queda significativa após 120 minutos de reperfusão; a pressão venosa não mostrou alterações no decorrer do experimento; houve queda do fluxo sangüíneo na aorta após os 30 minutos de isquemia, com queda mais acentuada ao final do período de reperfusão; o fluxo sangüíneo na AMCr diminuiu significativamente após 120 minutos de reperfusão, porém, em menor grau no grupo NAC/ISQ; a freqüência... . / Splanchnic artery occlusion and reperfusion result in local and remote tissue destruction caused by toxic factors released into the circulation and by neutrophil-endothelial cell interactions. In order to evaluate the effects of pentoxifylline (PTX) and n-acetilcysteine (NAC) on ischemia/reperfusion model (I/R), sixty rats were allocated into six groups: 1) SAL/CONT: Sham operation + saline; 2) SALI/ISQ: 30 min celiac, cranial and caudal mesenteric arteries occlusion + 120 min reperfusion + saline; 3) PTX/CONT: Sham operation + PTX (50 mg/kg); 4) PTX/ISQ: I/R + PTX; 5) NAC/SAL: Sham operation + NAC (430 mg/kg) and 6) NAC/ISQ: I/R + NAC. In all groups above mean arterial blood pressure (MABP), vein pressure, aorta and cranial mesenteric artery blood flow, heart rate, esophagus temperature, hematocrit, blood gas determination, histology, thiobarbituric acid reaction species (TBARS) and malondialdehyde (MDA) levels were determined. In I/R groups, MABP decreased significantly 120 minutes after reperfusion (p<0,05); as no difference in vein pressure were observed. Aorta blood flow decreased 30 minutes after ischemia, decreasing dramatically following restoration of blood flow. Cranial mesenteric artery blood flow decreased significantly after reperfusion, although the fall in NAC/ISQ group was attenuated. During the assays, heart rate was reduced, temperature decreased progressively and metabolic acidosis took place. Pentoxiffyline prevented histopathologic signs of injury in duodenum, jejunum and ileum, as well as n-acetilcysteine prevented ileum histopathologic signs of injury. I/R caused TBARS increase in NAC group jejunum and saline group ileum, but no difference was observed in plasmatic and ileal MDA. The present study allowed concluding that PTX was more efficient in preventing histophatologic injury than NAC, but neither PTX nor NAC... (Complete abstract, click electronic address below).

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os Ãcidos graxos insaturados Ãmega-3 (&#969;-3) e Ãmega-9 (&#969;-9) possuem aÃÃo anti-inflamatÃria e antioxidante, enquanto os Ãmega-6 (&#969;-6) sÃo prÃ-inflamatÃrios. Este estudo verificou os efeitos do prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, em modelo experimental de isquemia-reperfusÃo cerebral. Foram utilizados 42 ratos Wistar, divididos em dois grupos: Controle (n=24) e Teste (n=18). O grupo Controle foi subdividido em 4 grupos de 6 animais, cada: Simulado - Ãgua (Sim-Ãgua), Isquemia-ReperfusÃo - Ãgua (IR-Ãgua), Simulado - IsolipÃdico (Sim-IsolipÃdico) e Isquemia-ReperfusÃo - IsolipÃdico (IR-IsolipÃdico). Os animais receberam Ãgua ou uma mistura isolipÃdica com relaÃÃes &#969;-6/&#969;-3 = 8:1 e &#969;-9/&#969;-6 = 0,4:1 por via orogÃstrica, durante sete dias, conforme seus grupos. O grupo Teste foi subdividido em 3 grupos de 6 animais: IR-Mix1, IR-Mix2 e IR-Mix3. Os animais do grupo Teste receberam misturas oleosas com relaÃÃes &#969;-6/&#969;-3 = 1,4:1 e &#969;-9/&#969;-6 = 3,4:1 , diferindo apenas na fonte de &#969;-3: Mix1, contendo o Ãcido &#969;-3 &#945;-linolÃnico; Mix2, contendo os Ãcidos &#969;-3 &#945;-linolÃnico, eicosapentaenÃico e docosaexaenoico, e Mix 3, contendo os Ãcidos &#969;-3 &#945;-linolÃnico e docosaexaenÃico, administradas por via orogÃstrica, durante sete dias. No sÃtimo dia, os animais dos grupos IR-Ãgua, IR-IsolipÃdico, IR-Mix1, IR-Mix2 e IR-Mix3 foram submetidos à isquemia cerebral com oclusÃo bilateral das artÃrias carÃtidas comuns por 1 hora, seguida de reperfusÃo por 3 horas. Os animais dos grupos Sim-Ãgua e Sim-IsolipÃdico foram submetidos à operaÃÃo simulada. Ao final do experimento, todos os animais foram decapitados e seus cÃrebros fatiados para anÃlise histopatolÃgica da Ãrea CA3 do hipocampo. A morte neuronal foi quantificada pela contagem de neurÃnios vermelhos (NV). Constatou-se que a quantidade de NV no grupo IR-Ãgua (36,83  9,79) foi maior (P = 0,0046) que a observada do grupo Sim-Ãgua (17,67  8,48), bem como a quantidade de NV no grupo IR-IsolipÃdico (29,83  12,19) foi maior (P = 0,0459) que a observada no grupo Sim-IsolipÃdico (14,17  11,62). NÃo foi constatada diferenÃa na quantidade de NV entre os grupos Sim-Ãgua (17,67  8,48) e Sim-IsolipÃdico (14,17  11,62), ou entre os grupos IR-Ãgua (36,83  9,79) e IR-IsolipÃdico (29,83  12,19). A quantidade de NV no grupo IR-Mix1 (12,33  6,31) foi menor que a verificada nos grupos IR-Ãgua (36,83  9,79; P < 0,01) e IR-IsolipÃdico (29,83  12,19; P < 0,05). As quantidades de NV nos grupos IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47) tambÃm foram menores que as verificadas nos grupos IR-Ãgua (36,83  9,79; P < 0,001) e IR-IsolipÃdico (29,83  12,19; P < 0,01). NÃo foram constatadas diferenÃas nas quantidades de NV entre os grupos IR-Mix1 (12,33  6,31), IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47), entre si. Conclui-se que, independentemente da fonte de &#969;-3, o prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, protege os neurÃnios contra as lesÃes de isquemia-reperfusÃo cerebral em modelo experimental. / Omega-3 (&#969;-3) and omega-9 (&#969;-9) unsaturated fatty acids are anti-inflammatory and antioxidant, while omega-6 (&#969;-6) fatty acids are pro-inflammatory. This study investigated the preconditioning effects of fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, in a brain ischemia-reperfusion experimental model. Forty-two Wistar rats were aleatory assigned to two groups: Control (n=24) and Test (n=18). Control group was divided into 4 groups, each with 6 animals: Water-Simulated (Water-Sim), Water - Ischemia-Reperfusion (Water-IR), Isolipid-Simulated (Isolipid-Sim) and Isolipid - Ischemia-Reperfusion (Isolipid-IR). The animals received water or a isolipid mix with &#969;-6/&#969;-3 ratio of 8:1 and &#969;-9/&#969;-6 ratio of 0,4:1 by gavage, for 7 days, according to their groups. Test group was divided into 3 groups of 6 animals: Mix1-IR, Mix2-IR, and Mix3-IR. All animals from Test group received oil mixes with &#969;-6/&#969;-3 ratio of 1,4:1 and &#969;-9/&#969;-6 ratio of 3,4:1 , differing only on the &#969;-3 source: Mix1, with &#969;-3 linolenic acid; Mix2, with &#969;-3 linolenic, eicosapentaenoic and docosahexaenoic acids, and Mix 3, with &#969;-3 linolenic and docosahexaenoic acids, by gavage, for 7 days. At the 7th day, animals from Water-IR, Isolipid-IR, Mix1-IR, Mix2-IR, and Mix3-IR groups were subjected to 1-hour brain ischemia by occlusion of both common carotid arteries, followed by a 3-hour reperfusion. Animals from Water-Sim and Isolipid-Sim groups were submitted to a simulated operation. At the end of the experiment, all animals were decapitated and their brains were sliced and sent to histological analysis of the CA3 hippocampal region. Neuronal death was quantified by the red neurons (RN) count. It was found that the number of RN in Water-IR group (36.83  9.79) was higher (P = 0.0046) than the number observed in Water-Sim group (17.67  8.48), and similarly, the number of RN in Isolipid-IR group (29.83  12.19) was higher (P = 0.0459) than the number observed in Isolipid-Sim group (14.17  11.62). There was no difference between the amount of RN from Water-Sim (17.67  8.48) and Isolipid-Sim (14.17  11.62) groups, nor between Water-IR (36.83  9.79) and Isolipid-IR (29.83  12.19) groups. The number of RN in Mix1-IR group (12.33  6.31) was lower than the number seen in Water-IR (36.83  9.79; P < 0.01) and Isolipid-IR (29.83  12.19; P < 0.05) groups. The amounts of RN in Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups were also lower than the amounts observed in IR-Water (36.83  9.79; P < 0.001) and IR-Isolipid (29.83  12.19; P < 0.01) groups. There were no differences between the Mix1-IR (12.33  6.31), Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups. In conclusion, regardless of the source of &#969;-3, preconditioning with fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, protects the neurons against brain ischemia-reperfusion injuries in this experimental model.

Introdução: A torção testicular permanece como uma emergência urológica, despertando grande interesse em fármacos que podem minorar a lesão testicular e suas repercussões na fertilidade e produção hormonal. No entanto, não há fármaco aprovado para uso clínico rotineiro. Uma droga estudada em isquemia celular é a clorpromazina, sendo conhecidos seus efeitos protetores na função e estrutura da membrana celular e mitocondrial. Objetivos: Avaliar a diferença na lesão de células germinativas após 1 e 6 horas de torção e a ação da clorpromazina administrada previamente à resolução da torção no testículo isquêmico. Materiais e Métodos: 54 ratos Wistar, machos, com peso corporal entre 220 e 260 gramas distribuídos em 5 grupos: sham, controle com isquemia de 1 hora(A), controle com isquemia de 6 horas(B), experimental com isquemia de 1 hora(C) e experimental com isquemia de 6 horas(D). Em 48 animais foi realizada torção unilateral do cordão espermático com duas voltas em torno do seu eixo (720 graus), fixando-se o testículo nessa posição, após o que cada subgrupo foi separado em avaliação imediata (orquiectomia bilateral ao final do período de torção = 1) e tardia (orquiectomia bilateral, uma semana após a resolução da torção = 2). O grupo experimental recebeu 3 mg/kg de clorpromazina administrada via endovenosa, 30 minutos antes da resolução da torção. O grupo controle recebeu apenas solução salina a 0,9% por via endovenosa. Outros 6 animais formaram o grupo sham, onde foi realizada apenas a manipulação do cordão espermático. Após retiradas as gônadas, foram preparadas para análise histológica pela microscopia de luz e imunohistoquímica.Um pequeno fragmento de cada testículo foi separado para avaliação por microscopia eletrônica de transmissão (MET). Resultados: Na análise por microscopia de luz foram notadas alterações devido à isquemia como, necrose de coagulação e edema intersticial, principalmente nos grupos com isquemia mais prolongada (6h - B e D). Na avaliação por imunohistoquímica, houve maior expressão da caspase-3 nas células e túbulos dos testículos com 6 horas de isquemia, quando comparados com o grupo sham. No entanto, a expressão de bcl-2 não foi expressiva em nenhum grupo. Os grupos B e D também demonstraram alterações mais expressivas na análise por MET. Em nenhuma das avaliações foi observado superioridade do grupo da clorpromazina em relação ao grupo controle. Conclusão: As lesões celulares intratubulares induzidas pela isquemia e reperfusão testicular foram semelhantes após 1 e 6 horas, as diferenças foram relacionadas à sua maior intensidade no grupo com 6 horas e a clorpromazina não foi efetiva na prevenção da lesão por reperfusão. / Introdution: Testicular torsion remains as a urology emergency arousing interest about medicine which can reduce testicular injury and its impact on fertility and hormone production. However, there is no drug approved for routine clinical use. A drug studied in cell ischemia is chlorpromazine, being known its protective effects on the function and structure of cellular membrane and mitochondrial. Objective: To evaluate the difference in lesion of germ cells after 1 and 6 hours and the action of chlorpromazine administered before the resolution of ischemic testicle due torsion. Materials and methods: 54 male Wistar rats weighing between 220 to 260 grams divided into five groups: sham, control with one hour of ischemia (A) control with six hours of ischemia (B) experimental with one hour of ischemia (C) and experimental six hours of ischemia (D). In 48 animals was performed unilateral torsion of the spermatic cord with two laps around its axis (720 degrees), keeping the testicle in this position. After that, each subgroup was divided into immediate evaluation (bilateral orchiectomy at end of the torsion period = 1) or later (bilateral orchiectomy after one week of torsion resolution = 2). The experimental group received 3 mg / kg chlorpromazine administered intravenously 30 minutes before the resolution of torsion. The control group received only saline 0.9% intravenously. Other 6 animals were in the sham group, which was held just handling the spermatic cord. After withdrawal, the gonads were prepared for histological analysis by light microscopy and immunohistochemistry. A small piece of each testis was separated for evaluation by electron microscopy. Results: In analysis by light microscopy, ischemic changes were rated as coagulative necrosis and interstitial edema mainly in groups with prolonged ischemia (6h - B and D). When analyzed by immunohistochemistry, there was greater expression of caspase-3 in cells and tubules of the testes with 6 hour of ischemia compared to the sham group. However, bcl-2 expression was not impressive in either group. B and D groups also showed more significant changes in the analysis by electron microscopy. None of the ratings has been shown superiority of chlorpromazine group over the control group. Conclusion: The germ cell damage induced by ischemia and reperfusion was similar after 1 and 6 hours, the differences were related to its greatest intensity in the group with 6 hours and chlorpromazine was not effective in preventing reperfusion injury.

Ischemic stroke causes irreversible damage to the brain. The hippocampus is a vulnerable region and plays an important role in cognition and locomotor activity. Puerarin is a phytoestrogen that has beneficial effects in treating neurological disorders. How puerarin protects against hippocampal injury and its molecular mechanisms remain to be elucidated. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with puerarin alone or together with LY294002 (an PI3K inhibitor) before ischemia/ reperfusion (I/R). The open-and closed-field tasks and Morris water maze (MWM) test were used to assess the effects of puerarin on anxiety-like behavioral and cognitive impairment following I/R. Hematoxylin-eosin staining(HE) was used to examine the survival of hippocampal CA1 pyramidal neurons, and immunoblotting was performed to examine the expression of the related proteins. By using the rat model for transient I/R, we demonstrated that puerarin pretreatment significantly increased the travelling distance and number of crossings in the open-and closedfield tests, reduced latency and increased the proportion of distance and time in zone IV in the MWM. The number of live cells in the hippocampus is sharply increased by puerarin pretreatment. We further observed that the levels of phosphorylated Akt1, GSK-3 beta and MCL-1were elevated and those of cleaved-caspase-3 were reduced in the puerarin-treatment group. Notably, the PI3K inhibitor LY294002 counteracted all of the effects of puerarin. Our findings suggest that puerarin protects the hippocampus from I/R damage by activating the PI3K/Akt1/GSK-3 beta/MCL-1 signaling pathway.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM