Many factors present during the perioperative period render patients susceptible in developing myocardial ischaemia reperfusion injury. Various mode of conditioning the heart against this type of injury has been discovered in animal models and involve powerful innate pathways that enhance cellular survival. These may be harnessed by applying a trigger either immediately before (preconditioning) or after (postconditioning) the lethal ischaemic injury, by physical or pharmacological means. Morphine was the first clinically used opioid shown to be cardioprotective but the intravenous dose required limited its use clinically. Remifentanil, an ultra-short acting opioid, was later also shown to be cardioprotective. A better understanding of how these opioids can protect the heart may enable the rational design of clinical regimens that best protect patients. The purpose of this thesis is to demonstrate and elucidate how these two agents provide cardiac protection. I first demonstrated the clinical efficacy of remifentanil preconditioning in reducing the release CKMB, cardiac troponin I, heart type fatty acid binding protein and ischaemia modified albumin following cardiopulmonary bypass. As opioids cannot be omitted completely from patients undergoing cardiac surgery due to ethical considerations, I then used a well-established animal model of ischaemia reperfusion injury to complete the remainder of the studies. I demonstrated that remifentanil postconditioning was also effective in reducing myocardial infarct size, an effect mediated through the activation of kappa and delta opioid receptor subtypes, and in part triggered at the level of the myocardium. I then confirmed previous findings showing the efficacy of intrathecal morphine preconditioning using clinically relevant doses. In addition, I demonstrated that all three opioid receptor subtypes were involved. This effect was comparable to that achievable by classical ischaemic or intravenous morphine preconditioning and is mediated by central but not peripheral opioid receptor activation. Intrathecal morphine reduces the degree of myocardial apoptosis, alters the phosphorylation of Akt and the expression of endothelial nitric oxide synthatase and opens the potassium ATP channels. It also involves spinal adenosine receptors, similar to spinal morphine mediated analgesia. Intrathecal morphine preconditioning can be abolished by the interruption of autonomic nervous system function and blockade of calcitonin gene related peptide (CGRP) and bradykinin receptors. Intrathecal morphine postconditioning also has an infarct sparing effect. It also involves the activation of central opioid receptors and peripheral adenosine and CGRP receptors. Finally I demonstrated a pivotal role of central opioid receptor in remote preconditioning by showing that selective blockade of these receptors abolished the protective effects of remote but not classical ischaemic preconditioning. Cumulatively, these results demonstrated the versatility of opioid mediated cardioprotection using morphine or remifentanil and the pivotal role of central opioid receptors in cardioprotection and revealed some of the mechanisms underlying these benefits. Not only does intrathecal morphine provide analgesia, it also generates signals that are transmitted through the autonomic nervous system resulting in changes in cellular function in the heart. This point to the possibility of a relationship between an organism’s intrinsic response to pain and the triggering of an innate organ protective response to ischaemia. / published_or_final_version / Anaesthesiology / Master / Doctor of Medicine

Coronary artery disease limits myocardial blood flow and results in myocardial infarction. Reperfusion therapies restore coronary flow, but may also cause myocardial ischemia reperfusion injury (MIRI). Multiple critical factors contribute to MIRI and among them, oxidative stress plays an important role. This burst of oxidative stress during reperfusion is caused by a variety of sources which collectively are called reactive oxygen species (ROS). Peroxynitrite is more cytotoxic than other ROSs, which at high concentration serves as a detrimental molecule with a variety of target. Peroxynitrite is largely produced during the early reperfusion due to the dramatically increased concentrations of superoxide (O2●-) and nitric oxide (NO). Current cardioprotective therapies against MIRI include exogenous antioxidant treatment and conditioning treatment that induced endogenous antioxidant signaling which upregulates heme oxygenase1 (HO-1), which confers its antioxidant effect in cells and tissues by degrading the latent oxidant heme and generating downstream antioxidant molecules. More importantly, peroxynitrite is closely related to HO-1 in pathogenesis of MIRI and pharmacological or genetic methods that induce over-expression of HO-1 in turn decrease the peroxynitrite generation. In this thesis we report the results of three studies designed to explore the interaction of peroxynitrite and HO-1 in cardioprotection against MIRI. In the first study we demonstrated that HO-1 plays an essential role in chronic antioxidant treatment against MIRI in 4-week diabetic rats. Chronic antioxidant treatment with two kinds of antioxidants that target different sources of ROSs was administrated in an in vivo study with streptozotocin (STZ)-induced type 1 diabetic rats. Antioxidant treatments synergistically attenuate MIRI and cardiac dysfunction in type 1 diabetic rats by enhancing HO-1 expression, and inhibition of HO-1 expression cancelled antioxidant cardioprotection. This finding was supported by in vitro experiments in a cardiomyocyte hypoxia-reoxygenation model. The second study explored the peroxynitrite/HO-1 interaction in propofol post-conditioning (PPC) in acute MIRI with both ex vivo and in vivo animal models. We showed that PPC conferred similar cardioprotection as an established intervention˗ischemic post-conditioning (I-PostC). PPC cardioprotection was achieved through down-regulating peroxynitrite formation and activation of HO-1 and its related signaling molecules. This finding indicates that anaesthetic post-conditioning treatment (such as PPC) can achieve similar cardioprotection as ischemic post-conditioning and can avoid potential mechanical injury that may be caused by I-PostC. Inhibition of peroxynitrite reduction and subsequent enhanced HO-1 expression may be the fundamental mechanism of PPC cardioprotection. Lastly, we further explored PPC cardioprotection against MIRI in diabetic rats. We found that the diabetic heart lost its sensitivity to PPC and the diminished effect of PPC in inducing HO-1 over-expression may be a key mechanism. Exogenous supplementation of adiponectin, an adipocyte-derived plasma protein with anti-diabetic and anti-inflammatory properties, restored diabetic heart sensitivity to PPC that is associated with restoration of HO-1 expression. This finding may provide a potential therapy rescuing diabetic patient challenged by myocardial infarction. The studies described in this thesis have enhanced our knowledge concerning the role of peroxynitrite in the pathogenesis of MIRI and the critical role of HO-1 in different cardioprotective therapies, in particular anaesthetic postconditioning cardioprotection. / published_or_final_version / Anaesthesiology / Doctoral / Doctor of Philosophy

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.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

published_or_final_version / Medicine / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 200.

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

Thesis (M.Med.Sc.)--University of Hong Kong, 2003. / Includes bibliographical references. Also available in print.

A growing body of evidence has now accumulated supporting the involvement of oxygen-derived free radicals in the development of myocardial ischemia/reperfusion (I/R) injury. We have, therefore, undertaken the present study to examine (1) I/R-related alterations in myocardial antioxidant capacity in pentobarbital anesthetized open-chest rabbits subjected to left circumflex coronary artery ligation followed by reperfusion; (2) the protective effects of pretreatrnent with allopurinol or the 21-aminosteroid U74006F; (3) alternative mechanisms to xanthine oxidase inhibition for allopurinol protection against I/R injury; and (4) the effect of allopurinol treatment on the antioxidant capacity of erythrocytes in pigs used in a heart-lung transplantation study. In the rabbit myocardium, a marked impairment in myocardial antioxidant capacity developed in association with the onset of irreversible injury, as reflected in the enhancement in glutathione (GSH) depletion and formation of thiobarbituric acid-reactive substances (TBARS) following in vitro incubation of tissue homogenate with tert-butylhydroperoxide (TBHP). During the course of post-ischemic reperfusion, the protracted time-course of alterations in antioxidant capacity dissociated them from the early burst of radical formation known to occur at the onset of post-ischemic reperfusion of the myocardium. When the time-dependent changes in functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) were analysed in relation to activities of antioxidant enzymes, evidence suggestive of functionally relevant impairments in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and glutathione reductase (GRD) activities was found. These results and our demonstration of significant decreases in the activity of GSH-dependent antioxidant enzymes under acidotic conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in triggering irreversible myocardial I/R injury. Repetitive brief episodes of I/R produced a progressive decrease in myocardial ATP levels, which was not associated with any detectable changes in myocardial antioxidant capacity. Ischemic preconditioning produced by brief episodes of I/R did not affect the severity of subsequently induced I/R injury. These results suggest that brief episodes of myocardial ischemia do not produce oxidative tissue damage and the ischemia-induced depletion in myocardial ATP level is at least partially dissociable from the I/R-related impairment in tissue antioxidant capacity. Isolated Langendorff-perfused rabbit hearts subjected to I/R did not show any changes in antioxidant capacity. However, when intact hearts were subjected to ischemia in vivo and a subsequent reperfusion in vitro, an impairment in myocardial antioxidant capacity became apparent. These results suggest that blood elements, possibly activated neutrophils, may be a crucial factor involved in the development of I/R-induced oxidant injury. Chronic allopurinol pretreatment (1 mg/ml in drinking water or approximately 75 mg/kg/day) for 7 days prior to ischemia provided significant protection against I/R-induced alterations in myocardial antioxidant capacity, but not the decrease in tissue ATP levels. This chronic allopurinol regimen was found to enhance myocardial GRD activity in nonischemic tissue. In addition, both allopurinol and oxypurinol inhibited the transition metal ion-catalysed ascorbate oxidation and lipid peroxidation in vitro, likely as a consequence of their metal chelating properties. Similarly, myoglobin-TBHP-catalysed oxidation of uric acid and lipid peroxidation were also suppressed by allopurinol. All these suggest that allopurinol may favorably alter myocardial antioxidant capacity directly by virtue of its transition metal chelating properties and its antioxidant actions in myoglobin-mediated oxidative processes. The acute administration of 21-aminosteroid U74006F (3 mg/kg, i.v) under conditions comparable to those known to protect against trauma-induced damage in the central nervous system failed to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive oxy-radicals have been implicated in both types of tissue damage, the observed difference in susceptibility to protection by this steroidal antioxidant suggests that the molecular mechanisms involved are not identical. In the heart-lung transplantation study, erythrocytes from allopurinol-treated pigs (given repeatedly at an oral dose of 50 mg/kg) showed a time/dose-dependent increase in antioxidant capacity as reflected in the decrease in malondialdehyde production following in vitro oxidative challenge. The extent of red cell protection in both donor and recipient animals correlated significantly with the functional viability of the transplanted lung tissue, as assessed by tissue water content. These results suggest that the measurement of erythrocyte antioxidant capacity may provide an useful assessment of generalized alterations in tissue antioxidant status produced by pharmacological interventions. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Orientador: Orlando Petrucci Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:20:22Z (GMT). No. of bitstreams: 1 Lima_FanySilva_M.pdf: 1729080 bytes, checksum: a3d4e3c77bc179cf859c845d5e3bac06 (MD5) Previous issue date: 2015 / Resumo: O infarto agudo do miocárdio (IAM) ainda permanece como umas das principais causas de morbimortalidade em indivíduos adultos, ocasionando lesões miocárdicas pela isquemia seguida da reperfusão. No presente trabalho estudamos a cardioproteção por meio de três diferentes estratégias: a utilização de um fármaco denominado - Piracar (Piracetam, L-carnitiva, glutamato e aspartato), a utilização de uma solução contendo eritropoietina, glicose, insulina e potássio (EG); e por meio da modulação humoral/neurológica denominada isquemia de pré-condicionamento remoto (IPCR). Para esta análise utilizou-se de modelo agudo de isquemia e reperfusão miocárdica em suínos onde foram avaliadas variáveis hemodinâmicas, quantificação da área de infarto, quantidade de troponina I liberada (TnI-C) e de adenosina trifosfato no músculo cardíaco (ATP). Também foram estudadas proteínas relacionadas a isquemia e reperfusão miocárdicas de duas vias conhecidas como a Survivor Activating Factor Enhancement (SAFE) e a Reperfusion Injury Salvage Kinase Pathway (RISK) utilizando Western Blotting. Foi observado maior ativação das proteínas ERK (p?0,05) e STAT (p?0,05) no grupo EG e IPCR comparados ao controle, quando comparados entre si o grupo que se apresentou melhor foi o EG, também com a quantidade de ATP significativamente maior. No grupo Piracar e IPCR a AKT (p?0,05) apresentou-se ativada comparada aos demais grupos. Não encontramos diferença nas análises hemodinâmicas e na porcentagem de área de infarto. Entretanto, a TnI-C apresentou-se elevada na fase de reperfusão nos grupo IPCR e EG; e reduzida no grupo Piracar. Dos tratamentos estudados, o grupo EG foi o que mais se destacou pelo aumento significativo das proteínas ERK e STAT, e aparente melhora na reserva metabólica pela quantidade elevada de ATP disponível, enquanto os demais grupos e nas demais formas de análises foram semelhantes ou com resultados inferiores / Abstract: Acute myocardial infarction (AMI) still the major cause of morbidity and mortality in adults, causing myocardial ischemia followed by lesions of reperfusion. In the present study, we studied cardioprotection by 3 different strategies: the use of a so-called drug - Piracar ( Piracetam, L- carnitiva, glutamate and aspartate) using a solution containing erythropoietin, glucose, insulin and potassium (EG); by humoral and/modulation neurological called remote ischemic preconditioning (IPCR) . For this analysis we used the model of acute myocardial ischemia and reperfusion in pigs where hemodynamic variables were evaluated, quantification of infarct area, amount of troponin I released (cTnI) and adenosine triphosphate in the heart muscle (ATP). Were also studied proteins related to myocardial ischemia and reperfusion two-way known as the Survivor Activating Factor Enhancement (SAFE) Reperfusion Injury and Salvage Kinase Pathway (RISK) using Western blotting. We found greater activation of ERK proteins (p= 0,05) and STAT (p= 0,05) in the EG group and IPCR compared to the control, when comparing between the group that performed best was the EG, also with the amount significantly higher ATP. In group Piracar AKT (p= 0,05) was significantly activated compared to the other groups. No differences in hemodynamic analysis and the percentage of infarcted area. However, cTnI showed up high in the reperfusion phase in IPCR and EG group; and reduced in Piracar group. Of the treatments, the EG group was the one that stood out the significant increase in ERK and STAT proteins, and apparent improvement in metabolic reserve by the high amount of ATP available, while the other groups and other forms of analysis were similar or results below / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências