Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 53-61). Also available in print.

Thesis (Ph. D.)--University of Louisville, 2005. / Department of Physiology and Biophysics. Vita. "May 2005." Includes bibliographical references (leaves 114-131).

München, Techn. Univ., Diss., 2004.

Made available in DSpace on 2014-06-11T19:30:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-24Bitstream added on 2014-06-13T18:40:40Z : No. of bitstreams: 1 caetano_amm_dr_botfm.pdf: 904781 bytes, checksum: 71fb65bf25040c2baff3cda7362c5ead (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A isquemia e reperfusão (I/R) é a principal causa de lesão renal aguda após transplante renal. Hiperglicemia está associada a diminuição da tolerância à isquemia e aumento da gravidade da lesão renal da I/R. Eritropoetina administrada antes da isquemia/reperfusão renal (pré-condicionamento da eritropoetina) exerce efeito renoprotetor em animais normoglicêmicos, mas, este efeito, ainda não foi estudado em animais com hiperglicemia transitória. O objetivo desta pesquisa foi investigar o efeito da eritropoetina na lesão de isquemia/reperfusão renal em ratos com hiperglicemia transitória. Vinte e oito ratos Wistar machos (>300g) foram anestesiados com isoflurano a 3%, intubados e submetidos à ventilação mecânica com isoflurano a 1,5%. Artéria carótida e veia jugular foram cateterizadas. Dióxido de carbono em final de expiração, concentração inspirada e expirada de gás anestésico, pressão arterial invasiva e temperatura retal foram continuamente monitorizados (Datex, AS3). A temperatura retal foi mantida entre 36oC-38oC. Os animais foram divididos aleatoriamente em quatro grupos e todos receberam glicose 2,5 g.kg-1 por via intraperitoneal, sendo submetidos a laparotomia mediana e nefrectomia direita (Grupo S – sham - n=6). Os demais animais foram submetidos a 25 minutos de isquemia renal esquerda por clampeamento da artéria renal esquerda (Grupos ISO, EA e EM). 30 minutos antes da isquemia renal esquerda o Grupo ISO (n=6) recebeu soro fisiológico, o Grupo EA (n=8) eritropoetina 5000 UI.kg- 1 e o Grupo EM (n=8) eritropoetina 600 UI.kg-1por via intravenosa. A pressão arterial média (PAM) e a temperatura foram avaliadas a cada 10 minutos. Os valores plasmáticos da glicose e da creatinina foram determinados no inicio (M1) e no final do experimento (M2). Vinte e quatro horas após o final do experimento (M3) os animais retornaram ao laboratório e foram anestesiados... / Ischemia and reperfusion (I/R) injury is the leading cause of acute renal injury following renal transplantation. Hyperglycemia is associated with decreased tolerance to ischemia and increases the severity of renal I/R injury. Erythropoietin administered before renal ischemia/reperfusion (erythropoietin preconditioning) may exert some renoprotective effect in normoglycemic animals. However, such effect has not been studied in transiently hyperglycemic animals. This study aimed to examine the effect of erythropoietin preconditioning on renal ischemia /reperfusion injury in transiently hyperglycemic rats. Twenty- eight male Wistar rats weighting more than 300g were anesthetized with 3% isoflurane. After tracheal intubation, the animals were mechanically ventilated with air and 1.5 % isoflurane. Carotid artery and jugular vein were cannulated. End-tidal carbon dioxide partial pressure, inspired and expired anesthetic gas concentrations, direct arterial pressure and rectal temperature were continuously measured. Glucose 2,5 g.kg-1 was administered intraperitoneally to induce hyperglycemia. Rectal temperature was kept between 360C-380C. Animals were submitted a midline laparotomy and right nephrectomy. Animals were randomly allocated into four groups: S, sham, (n=6) underwent only right nephrectomy, no left kidney ischemia/reperfusion. Group ISO (n=6), underwent a 25-minute period of left renal artery clamping, not preceded by erythropoietin preconditioning dose, Group EH (n = 8), received high-dose erythropoietin (5000 UI.kg-1 i.v.), 30 minutes before a 25 minutes period of left renal artery clamping; Group EL (n = 8) received low-dose erythropoietin (600 UI.kg-1, i.v.) 30 minutes before a 25-minute period of left renal artery clamping. Creatinine and glucose serum levels were determined at the start (M1), at the end (M2), and 24 hours after the experiment (M3). Rats were then anesthetized... (Complete abstract click electronic access below)

La reperfusion complète et précoce est le moyen le plus efficace pour limiter l'extension de l'infarctus cérébral et les séquelles neurologiques. Le traitement de l'infarctus cérébral a été révolutionné par la thrombectomie mécanique intra-artérielle en permettant une recanalisation dans plus de 70% des cas et une réduction significative de la morbidité comparativement à la thrombolyse seule pour le territoire carotidien. Le pronostic des occlusions basilaires reste dramatique et aucun essai n'a démontré le bénéfice de l'approche intra-artérielle à l'heure actuelle. Dans la première partie du travail, nous avons réalisé une méta-analyse sur la thrombectomie par «stent-retriever» des occlusions basilaires, à partir des résultats publiés dans MEDLINE entre novembre 2010 et avril 2014: recanalisation angiographique (TICI≥2b)=81% (IC 95%: 73-87); hémorragie cérébrale symptomatique à 24 heures=4% (IC 95%: 2-8); évolution neurologique favorable (mRS≤2 à 3 mois)=42% (IC 95%: 36-48); mortalité=30% (IC 95%: 25-36). L'approche intra-artérielle ouvre une nouvelle ère thérapeutique, cependant un modèle animal adapté et pertinent est nécessaire pour l'évaluation pré-clinique. Dans la deuxième partie du travail, nous avons caractérisé l'évolution spatio-temporelle précoce de l'infarctus par IRM multimodale dans un modèle d'ischémie cérébrale focale transitoire réalisé par occlusion sélective intra-artérielle de l'artère cérébrale moyenne chez le rat adulte. Une occlusion complète de l'artère cérébrale moyenne proximale était observée dans 75% des 16 rats opérés, et un mismatch diffusion/perfusion dans 77% des cas. Le volume ischémique durant l'occlusion artérielle, définie sur la séquence de diffusion, était de 90±64 mm3 et de 57±67 mm3 à 24 heures sur la séquence T2. La recanalisation artérielle s'associe à une reperfusion tissulaire dans 36% des cas. L'hypoperfusion persistait chez la majorité des animaux 3 heures après recanalisation. L'infarctus était de localisation cortical dans 31%, striatale dans 25%, et cortico-striatale dans 44%. Tous les animaux étaient en vie à 24 heures confirmant le caractère mini-invasif de ce modèle. Bien que la reperfusion sauve incontestablement une partie du parenchyme ischémique, elle s'accompagne également de lésions irréversibles spécifiques, dites de reperfusion, s'ajoutant aux lésions initiales. Limiter l'importance des lésions de reperfusion représente un objectif thérapeutique majeur. Dans la troisième partie, nous avons testé l'effet neuroprotecteur de la Cyclosporine A sur la réduction du volume de l'infarctus cérébral et sur le pronostic clinique. Une procédure d'ischémie reperfusion cérébrale de 60 minutes a été réalisée chez 48 animaux, puis ont été randomisés en quatre groupes (groupe témoin, pré-conditionnement, postconditionnement intraveineux et intra-artériel avec la Cyclosporine A à la dose de 10 mg/kg dans les 30 secondes suivant la reperfusion). Sur les 43 animaux inclus dans l'analyse, il n'a pas été observé de réduction du volume ischémique ni une amélioration du pronostic après injection intraveineuse ou intra-artérielle de Cyclosporine A. La Cyclosporine A ne permet pas non plus de limiter l'extension des lésions de reperfusion au sein de la zone à risque à 24 heures de la reperfusion cérébrale / Early and complete reperfusion is the most effective therapy to limit the extent of brain infarction. The treatment of acute anterior ischemic stroke has been revolutionized by the intra-arterial mechanical thrombectomy allowing a 70% recanalization rate and a significant reduction of morbidity compared with thrombolysis alone. The prognosis of basilar artery occlusion remains catastrophic, and to date any trial has demonstrated the benefit of intra-arterial approach. In the first part of the work, we conducted a systematic review and meta-analysis of all previous studies of stent retriever thrombectomy in basilar artery occlusion patients between November 2010 and April 2014: recanalization (TICI≥2b)=81% (95% CI: 73-87); symptomatic intracranial haemorrhage at 24 hours=4% (95% CI 2-8); favorable neurological outcome (mRS≤2 at 3 months)=42% (95% CI: 36-48); mortality=30%(95% CI 25-36). Intra-arterial approach opens new avenues for the developement of treatments for brain infarction, but a relevant animal model of acute ischemic stroke is required for preclinical evaluation. In the second part of the work, we evaluated the spatiotemporal evolution of cerebral ischemia by sequential multimodal MRI in a new minimally invasive model of transient focal ischemia by selective intra-arterial occlusion of the middle cerebral artery in rat. A complete occlusion of the proximal portion of the middle cerebral artery was observed in 75% of 16 operated rats, and a mismatch diffusion/perfusion in 77% of cases. Acute stroke volume during arterial occlusion was 90±64 mm3 on diffusion-weighted imaging, and 57±67 mm3 at 24 hours on T2-weighted imaging. Recanalization is associated with tissue reperfusion in 36% of cases. The hypoperfusion persisted in the majority of animals 3 hours after recanalization. Brain infarction was cortical in 31%, striatal in 25%, and corticalstriatal in 44% of cases. All animals were alive at 24 hours, confirming the minimally invasive nature of the model. Although reperfusion saves a portion of ischemic tissue, it also carries specific irreversible damage, called reperfusion injury, in addition to initial damage caused by ischemia. Limiting the size of infarction is a major objective. In the third part, we tested the neuroprotective effect of Cyclosporine A in reducing the lesion volume and functional outcome. A total of 48 adult rats underwent the intra-arterial ischemia reperfusion procedure, and were randomly assigned to four treatment groups (control, preconditioning, intravenous and intra-arterial postconditioning with Cyclosporine A). Intravenous or intra arterial injection of Cyclosporine A at reperfusion does not either reduce the volume of stroke or improve the neurological outcome. Administation of Cyclosporin A at reperfusion does not limit the extension of reperfusion injuries within the ischemic risk area at 24 hours

Orientador: Pedro Thadeu Galvão Vianna / Banca: Luiz Antonio Vane / Banca: Norma Suelei Pinheiro Modolo / Banca: Antonio Fernando Carneiro / Banca: Oscar César Pires / Resumo: A isquemia e reperfusão (I/R) é a principal causa de lesão renal aguda após transplante renal. Hiperglicemia está associada a diminuição da tolerância à isquemia e aumento da gravidade da lesão renal da I/R. Eritropoetina administrada antes da isquemia/reperfusão renal (pré-condicionamento da eritropoetina) exerce efeito renoprotetor em animais normoglicêmicos, mas, este efeito, ainda não foi estudado em animais com hiperglicemia transitória. O objetivo desta pesquisa foi investigar o efeito da eritropoetina na lesão de isquemia/reperfusão renal em ratos com hiperglicemia transitória. Vinte e oito ratos Wistar machos (>300g) foram anestesiados com isoflurano a 3%, intubados e submetidos à ventilação mecânica com isoflurano a 1,5%. Artéria carótida e veia jugular foram cateterizadas. Dióxido de carbono em final de expiração, concentração inspirada e expirada de gás anestésico, pressão arterial invasiva e temperatura retal foram continuamente monitorizados (Datex, AS3). A temperatura retal foi mantida entre 36oC-38oC. Os animais foram divididos aleatoriamente em quatro grupos e todos receberam glicose 2,5 g.kg-1 por via intraperitoneal, sendo submetidos a laparotomia mediana e nefrectomia direita (Grupo S - sham - n=6). Os demais animais foram submetidos a 25 minutos de isquemia renal esquerda por clampeamento da artéria renal esquerda (Grupos ISO, EA e EM). 30 minutos antes da isquemia renal esquerda o Grupo ISO (n=6) recebeu soro fisiológico, o Grupo EA (n=8) eritropoetina 5000 UI.kg- 1 e o Grupo EM (n=8) eritropoetina 600 UI.kg-1por via intravenosa. A pressão arterial média (PAM) e a temperatura foram avaliadas a cada 10 minutos. Os valores plasmáticos da glicose e da creatinina foram determinados no inicio (M1) e no final do experimento (M2). Vinte e quatro horas após o final do experimento (M3) os animais retornaram ao laboratório e foram anestesiados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Ischemia and reperfusion (I/R) injury is the leading cause of acute renal injury following renal transplantation. Hyperglycemia is associated with decreased tolerance to ischemia and increases the severity of renal I/R injury. Erythropoietin administered before renal ischemia/reperfusion (erythropoietin preconditioning) may exert some renoprotective effect in normoglycemic animals. However, such effect has not been studied in transiently hyperglycemic animals. This study aimed to examine the effect of erythropoietin preconditioning on renal ischemia /reperfusion injury in transiently hyperglycemic rats. Twenty- eight male Wistar rats weighting more than 300g were anesthetized with 3% isoflurane. After tracheal intubation, the animals were mechanically ventilated with air and 1.5 % isoflurane. Carotid artery and jugular vein were cannulated. End-tidal carbon dioxide partial pressure, inspired and expired anesthetic gas concentrations, direct arterial pressure and rectal temperature were continuously measured. Glucose 2,5 g.kg-1 was administered intraperitoneally to induce hyperglycemia. Rectal temperature was kept between 360C-380C. Animals were submitted a midline laparotomy and right nephrectomy. Animals were randomly allocated into four groups: S, sham, (n=6) underwent only right nephrectomy, no left kidney ischemia/reperfusion. Group ISO (n=6), underwent a 25-minute period of left renal artery clamping, not preceded by erythropoietin preconditioning dose, Group EH (n = 8), received high-dose erythropoietin (5000 UI.kg-1 i.v.), 30 minutes before a 25 minutes period of left renal artery clamping; Group EL (n = 8) received low-dose erythropoietin (600 UI.kg-1, i.v.) 30 minutes before a 25-minute period of left renal artery clamping. Creatinine and glucose serum levels were determined at the start (M1), at the end (M2), and 24 hours after the experiment (M3). Rats were then anesthetized... (Complete abstract click electronic access below) / Doutor

Artemisinin is herbal drug with a wide range of biological and physiological function. It is currently administered in the treatment against uncomplicated F.Palcifarum infections. It has also been shown to be cytotoxic against a variety of cancer cells. Despite the promise of many anti cancer drugs, drug induced cardiotoxicity has constantly threatened drug applicability especially in patients with co-morbities. Artemisinin has been shown to be cardioprotective, although the intracellular pathways remain to be elucidated. In this study, isolated perfused rat hearts were subjected to 35 minutes of ischaemia and 120 minutes reperfusion or primary cardiac myocytes subjected to 120 minutes hypoxia and 120 minutes reoxygenation where artemisinin (4.3μM) was administered in presence and absence of the PI3K inhibitor (wortmannin) (0.1μM), p70S6K inhibitor (rapamycin) (0.1μM), non selective nitric oxide synthase inhibitor (L-NAME) (100μM) and inducible nitric oxide synthase inhibitor (aminoguanidine) (100μM). At the end of the experiment, hearts underwent infarct size to risk ratio assessment via tri-phenyltetrazolium chloride staining or western blot analysis for p-Akt and p70S6K. Cardiac myocytes were assessed for either MTT analysis, cleaved-caspase 3 or for eNOS/iNOS or p-BAD activity using flow cytometry. In isolated hearts, artemisinin (0.1μM-100μM) showed a significant dose dependent decrease in infarct size (P<0.01-0.001 vs. I/R control). It was also shown to significantly improve cellular viability (66.5±6.3% vs. 29.3±6.1% in H/R, P<0.01) and decrease the levels of cleaved caspase-3 compared to the H/R control group (17.1±2.0% vs. 26.8±2.0% in H/R, P<0.001). Artemisinin was shown to confer protection via the activation of the PI3K-Akt-p70S6k cell survival pathway and presented an upregulation in p-eNOS and iNOS expression. Furthermore, co-administering artemisinin with doxorubicin showed artemisinin reverses I/R or H/R injury as well as doxorubicin-induced injury via the nitric oxide signalling pathway. Additionally, in HL-60 cells, the co-administration doubled artemisinins cytotoxicity while also implicating the nitric oxide pathway. This is the first study to shows that artemisinin ameliorates doxorubicin mediated cardiac injury whilst enhancing its cytotoxicity in HL-60 in a nitric oxide dependent manner. This study concluded that artemisinin was both anti apoptotic and protective against myocardial I/R injury via the PI3K-Akt-BAD/P70S6K and via the nitric oxide cell survival pathway as well as pro-apoptotic against HL-60 in a nitric oxide dependent manner.

Hypothesis: Glucose is known to be protective in moderate low flow ischaemia due to the production of glycolytic ATP. However, it is questioned whether glucose would still be protective in ultra-low flow ischaemia. Firstly, glycolysis is thought to be inhibited, and secondly, deleterious glycolytic metabolites accumulate. Our hypothesis was that in ultra-low flow ischaemia, glucose utilisation is not inhibited at the level of glycolysis, but by delivery. Increased delivery of glucose should result in increased production of protective glycolytic ATP, but the rate of metabolite accumulation would also increase. Using ultra low flow rates, I wished to investigate how to achieve optimal rates of glycolysis, and how such rates would be balanced by any detrimental component of metabolite accumulation. Methods: The isolated Langendorff-perfused rat heart, with a left ventricular balloon to record ischaemic contracture and reperfusion stunning, was used, with severe flow restriction. Glucose concentrations were changed and pre-ischaemic glycogen contents were altered by perfusion with different substrates (acetate - depletion~ glucose + insulin - loading) or by preconditioning, with 5 min ischaemia and 5 min reperfusion prior to sustained ischaemia. Results: Analysis of glucose uptake relative to delivery showed that in severe low flow ischaemia, the extraction of glucose was increased, and glycolysis was thus limited more by substrate supply than by enzyme inhibition. Analysis of metabolites confirmed this concept. The optimal glucose concentration during severe low flow ischaemia was 11 mM, giving maximal recovery on reperfusion. Both lower and higher glucose concentrations increased ischaemic contracture. Changes in pre-ischaemic glycogen levels correlated with the time to onset of contracture, such that a reduction in glycogen accelerated contracture. Prior glycogen depletion or loading did not improve functional recovery. The benefits of preconditioning on reperfusion function following sustained total global ischaemia could not be related to glycogen depletion. If preconditioning were followed by sustained low flow ischaemia, glucose uptake was increased, but no benefit was found, possibly because a low residual flow abolished the effects of preconditioning. Many of the above results are consistent with the hypothesis that too low a rate of glycolysis results. in insufficient ATP production for protection, while excess glycolytic rates lead to excess metabolite accumulation with detrimental effects. Conclusions: Provision of glucose at the correct concentration, when the benefit associated with glycolytic ATP outweighs the detriment associated with moderate metabolite accumulation, is protective to the low-flow ischaemic myocardium, which can upregulate its ability to extract glucose. Improved residual flow enhances this benefit. Prior glycogen depletion is not beneficial, despite a reduced metabolite accumulation. This mechanism cannot be related to the protective effect of preconditioning.

Dexrazoxane (DEX) is clinically used to reduce cardiotoxic efects of anthracycline cytostatics. Its cardioprotective efect is caused by chelatation of free iron and defends myocard against dangerous hydroxyl radicals. This research finds out how dexrazoxane works in ischemic-reperfusion damages of rat's heart. Each rat was infused by DEX (50, 150, 450 mg/kg) or by control solution. Isolated perfused rat's hearts were exposed to local ischemia for 30 minutes than 10 minutes of reperfusion for studing ischemic arrhythmias followed by 15 minutes of local ischemia and 10 minutes of reperfusion to examine reperfusion arrhythmias. For evaluation of EKG (ventricular arrhythmias) was used software CAR and Lambeth convention. Global ischemias (15 min.) were induced in rat's hearts (DEX 150 mg/kg) and left ventricules were used for HPLC to determinate concentration of glutathion. In vivo experiments rats were infused by DEX 50, 150 mg/kg or control solution and were exposed for 20 minutes to local ischemia and for 3 hours to reperfusion. Infarct size was evaluated based on the cross section of heart (GIMP, Ellipse). Maximum total number of ischemic arrhytmias decreased by DEX 150 mg/kg (64% comparing to controls). Reperfusion score was reduced by DEX 150 to 48% and percents of ventricular fibrilation was...

Myocardial reperfusion injury is thought to be caused by reperfusion induced i) cytosolic Ca²⁺ overload and/or, ii) the formation of oxygen derived freeradicals. At the start of this study, data implicating cytosolic Ca²⁺ overload in the genesis of reversible reperfusion injury were inconclusive. Although several workers have approached this problem by measurements of cytosolic calcium ions, it was my aim to examine the potential sources of such calcium overload. The experiments reported in this thesis were therefore designed to examine the role of altered intracellular and transsarcolemmal Ca²⁺ fluxes in the genesis of reperfusion stunning and arrhythmias. The study was also aimed at elucidating the possible sources and entry pathways contributing to this proposed cytosolic Ca²⁺ overload. In order to investigate the possible role of altered reperfusion Ca²⁺ fluxes in reperfusion injury, we exposed the isolated working, and Langendorff perfused rat heart model to ischaemia and reperfusion to induce reperfusion stunning and arrhythmias. Hearts were pre-treated (before ischaemia) or reperfused with pharmacological compounds, or by interventions known to enhance or inhibit intracellular or transsarcolemmal Ca²⁺ fluxes. The severity of reperfusion stunning (mechanical dysfunction) was measured by reperfusion aortic output, coronary flow and left ventricular pressure. The incidence of reperfusion ventricular arrhythmias was measured by the incidence of ventricular tachycardia and/ or fibrillation. In selected studies, the metabolic status of hearts was evaluated using biochemical assays performed on myocardial tissue samples. Data obtained in these studies indicate that increased Ca²⁺ fluxes through sarcolemmal L-type Ca²⁺ channels during early reperfusion exacerbate stunning, while inhibition of these fluxes with the Ca²⁺ antagonist drug nisoldipine or by Mg²⁺ or Mn²⁺ improve reperfusion function. These data also suggest that although interventions increasing Ca²⁺ fluxes early in reperfusion exacerbate reperfusion stunning, these same interventions improve reperfusion function when performed later. The data also indicate that Ca²⁺ may enter the myocyte indirectly via activation of the Na⁺/H⁺ and Na⁺/Ca²⁺ exchanger during reperfusion. Inhibition of Na⁺/H⁺ exchange activity by HOE 694 during reperfusion attenuated reperfusion stunning and arrhythmias. Both activation of the Na⁺/H⁺ (and Na⁺/Ca²⁺) exchanger and Ca²⁺ influx via the Ca²⁺ channel could contribute to reperfusion induced Ca²⁺ overload and subsequent injury. The study also showed that altered intracellular Ca²⁺ oscillations play a role in reperfusion stunning and arrhythmias as shown by the use of the SR Ca²⁺ release channel blocker, ryanodine. Inhibition of the sarcoplasmic reticulum Ca²⁺ A TP-ase pump by two novel inhibitors, thapsigargin and cyclopiazonic acid, during ischaemia and early reperfusion improved reperfusion function and reduced the incidence of ventricular arrhythmias. function when unphysiologically high concentrations of the peptide were infused into the heart during reperfusion. Taken together, these data suggest that: 1) Ca²⁺ fluxes during early reperfusion (intracellular and transsarcolemmal) play a role in reperfusion injury, 2) that both the Ca²⁺ channel and Na⁺/H⁺ exchange activity contribute to reperfusion injury by possibly contributing to cytosolic Ca²⁺ overload and that, 3) altered intracellular Ca²⁺ oscillations through the SR play a role in both stunning and arrhythmias. Thus the proposal is that modulation of Ca²⁺ fluxes through either the sarcolemma or the sarcoplasmic reticulum, lessen reperfusion injury (stunning and arrhythmias). Although these data do not provide direct evidence of reperfusion Ca²⁺ overload, they support the concept that calcium ions play a role in the genesis of reversible reperfusion injury.