Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

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.

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.

A lesão renal induzida pela I/R é a principal causa de IRA nos rins nativos e nos rins transplantados e estudos enfatizam a participação de células inflamatórias na sua patogênese, através da caracterização de lesão endotelial, infiltração leucocitária e a geração de mediadores inflamatórios pelas células epiteliais tubulares. Evidências recentes mostram que as células T CD4+ exercem um papel fundamental como mediadoras da agressão renal na I/R, ressaltando-se o envolvimento do paradigma Th1/Th2 como um possível mecanismo efetor. O presente estudo foi realizado com o objetivo de estudar a participação de algumas citocinas Th1 e Th2 no desenvolvimento da lesão de I/R renal. Para tanto, nós nos propusemos a desenvolver um modelo experimental de I/R renal em camundongos deficientes em IL-12, IFN-, e duplo deficientes em IFN- e IL-12 (representando defeito da via de ativação Th1), camundongos deficientes em IL-4 e IL-10 (representando defeito da via de ativação Th2) e duplo deficientes em IL-10 e IL-12, tendo como controles camundongos normais (selvagens). Todos os animais foram submetidos a uma lesão de I/R por ligadura reversível do pedículo renal por 45 minutos seguidos de 24 horas de reperfusão. Após a indução da isquemia, nós analisamos as alterações funcionais (creatinina e uréia por método bioquímico colorimétrico) e morfológicas renais (morfometria renal), além de investigar a expressão molecular de HO-1 (um gene de proteção tecidual), de t-bet (um transcrito envolvido na diferenciação Th1), de GATA-3 (um transcrito envolvido na diferenciação Th2), citocina pró-inflamatória IL-6 e de uma quimiocina pró-inflamatória MCP-1, visando caracterizar a influência da polarização Th1/Th2 da resposta imune na lesão renal induzida pela I/R. Nós mostramos que os camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10 apresentaram uma disfunção renal importante, caracterizada por altos níveis séricos de creatinina e uréia e por um alto grau de agressão morfológica renal, avaliada pela percentagem de área de necrose tubular. Todos estes resultados foram significativamente mais acentuados que obtidos nos camundongos deficientes em IL-12 e IFN-/IL-12, sendo comparáveis aos animais selvagens. Por outro lado, a capacidade de reparação renal, medida pela percentagem de área de regeneração tubular, foi mais precoce nos camundongos deficientes em IFN-, quando comparada com os camundongos deficientes em IL-12 e IFN-/IL-12. A análise molecular quantitativa, utilizando-se o PCR em tempo real mostrou uma expressão significantimente maior de RNAm de HO-1, IL-6 MCP-1 e do fator transcricional pata Th2 (GATA-3) nos camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10, em comparação com os camundongos deficientes em IL-12, IFN-/IL-12 que apresentaram baixa expressão do fator transcricional para Th1 (T-bet), em 24 horas após a I/R renal. Quando fomos fazer a transferência adotiva de médula óssea de animais deficientes em IL-12 e IL-4 para animais selvagens previamente com depleção subletal celular, esses animais vieram apresentar resultados semelhantes aos animais deficientes em IL-12 e IL-4, onde os animais selvagens reconstituídos com medula óssea dos animais IL-12 apresentaram menor níveis de uréia sérica e menor expressão de RNAm IL-6, enquanto que os animais selvagens recontituídos com medula óssea dos animais IL-4 apresentaram maior níveis de uréia sérica e maior expressão de RNAm IL-6, esses dados comprovam que realmente a citocina IL-12 parece ser a principal citocina envolvida no processo inflamatório desencadeado pela lesão de isquemia e reperfusão renal. Tais achados favorecem a hipótese dos efeitos deletérios das células com per_l Th1 e o papel protetor das células Th2 na lesão isquêmica renal. Estes novos episódios podem fornecer a base para uma melhor compreensão fisiopatológicos, bem como para o desenvolvimento de métodos preventivos e terapêuticos para a insuficiência renal aguda isquêmica. / Renal ischemia/reperfusion injury (I/R) is the major cause of acute renal failure (ARF) in the native as well as in the transplanted kidneys, with a complex pathogenesis that involves many components of inflammatory response such as leukocyte infiltration and generation of inflammatory mediators by tubular cells. Recent evidences show a critical role of the CD4+ T cell, with the Th1/Th2 paradigm as a possible ejectors mechanism. The present study has the objective to investigate the participation of some main Th1 and Th2 cytokines in the development of the renal of I/R. For that, we developed an experimental model of renal I/R in deficient in IL-12, IFN-, double deficient IFN- and IL-12 (representing defect of the pathway Th1), IL-4 and IL-10 (representing defect of the pathway Th2) and in double deficient IL-10 and IL-12 knockout mice, having wild-type mice as controls. All animals were submitted to renal I/R by reversible ligation of the renal pedicles for 45 minutes followed by 24 hours of reperfusion. After induction of the ischemia, we analyzed renal function and morphometric histological analyzes. Furthermore, we quantified the expression of HO-1 (cytoprotection gene), t-bet (transcription factor involved in the differentiation Th1), GATA-3 (transcription factor involved in the differentiation Th2), and cytokine pro-inflammatory IL-6 and chemokine pro-inflammatory MCP-1, aiming to characterize the influence of Th1/Th2 polarization in the immune response driven by the renal I/R. We showed that the IL-4, IFN-, IL-10 and IL-10/IL-12-de_cient mice presented an important kidney dysfunction, characterized by higher levels serum creatinine and tubular necrosis, being comparable to the wild-type animals. The molecular analyses, utilizing the real-time PCR, showed a significantly higher expression of HO-1, IL-6, MCP-1 and transcription factor involved in the differentiation Th2 (GATA-3) in the in IL-4, IFN-, IL-10 and IL-10/IL-12-defficient mice, in comparison with the in IL-12, IFN-/IL-12-de_cient mice showed lower expression transcription factor involved in the differentiation Th1 (T-bet) . Moreover, the renal dysfunction seen in Th2-de_cient mice was followed by a higher expression of IL-6. When we make the transfer adoptive of bone marrow of animals deficient in IL-12 and IL-4 to wild animals previously with sublethal cell depletion, these animals produce similar results to animals deficient in IL-12 and IL-4, where the animals reconstituted wild animals with bone marrow IL-12 showed lower levels of serum urea and lower expression mRNA of IL-6, while the wild animals reconstituted with bone marrow of animals IL-4 showed higher levels of serum urea and greater expression of mRNA IL-6. These data show that really the cytokine IL-12 appears to be the main cytokine involved in the inflammatory process triggered by the injury of ischemia and reperfusion kidney. Such _ndings favor the hypothesis of the deleterious effects of Th1 cells with prole and the protective role of Th2 cells in ischemic renal injury. These novel insights may provide basis for the better pathophysiological understanding, as well as for development of preventive and therapeutic methods for the ischemic acute renal failure.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

No description available.

Peripheral artery disease (PAD) and osteoporosis have recently been shown to be associated; with the parallel occurrence suggesting that PAD related ischemia may cause or enhance the onset of osteoporosis. In order to determine the mechanism linking osteoporosis and PAD this paper will examine the effects of ischemia and mechanical stimulation on bone remodeling. An immunohistochemistry protocol for vessel marking in bone was also developed. Ischemia was induced in a mouse model to determine vascular and mechanical property changes in bone in response to hypoxia, and mechanical loading-induced remodeling was analyzed for vascular changes. Both ischemia and mechanical loading increased bone vessel density, with ischemic bone increases seen at day 7 and 14. Bone stiffness increased after induced ischemia at day 28. These results point to resultant hypoxia from ischemia drives bone mechanical property changes, likely through stimulation of bone remodeling. We also conclude that an increase in vessel density is seen after induced mechanical loading of bone. Establishing the vascular contribution to the remodeling process may reveal treatment opportunities for remodeling-dependent pathologies such as osteoporosis.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 183-273).