Myocardial ischemia-reperfusion (I/R) injury is an important health concern in myocardial infarction and situations such as angioplasty and cardiac surgeries. Therefore, patients and physicians need therapeutic interventions that are applicable at the time of surgery. Flavonoids and ascorbate (vitamin C) are known for their antioxidant activity and may be involved in the currently known health benefits of plant based foods and drinks. The objectives of this study were to 1) determine the extent to which ascorbate or catechin alone at levels which could be in blood after dietary supplementation, can protect myocardial tissue in the reperfusion phase of I/R injury, and 2) evaluate the possible cooperative or synergistic protective effect of ascorbate and catechin when given together. Isolated rat hearts (n=48) were perfused in the retrograde mode with modified Krebs-Henseleit buffer, and following the induction of 30 min global ischemia, ascorbate (150 µM) and/or catechin (5 µM) were added directly into the perfusate during 90 min reperfusion. To determine the histopathological features, hematoxylin and eosin (H&E) stain was used in one heart per condition; while to assess the biochemical analysis, the heart tissues were assessed for apoptosis (caspase-3 activity), oxidative stress (thiobarbituric acid reactive substances (TBARS) and total malondialdehyde (MDA) levels), and redox status (reduced and oxidized glutathione tissue levels). A comparison of IR hearts with two controls, sham (perfused for a 15 min stabilization period) and continuous perfusion (perfused for 135 min), showed in most but not all measurements that this was a suitable model of IR injury. The treatment experiments showed that 150 µM ascorbate protected the heart against lipid peroxidation and cell apoptosis by 100%, while 5 µM catechin protected by 67% and 90% respectively. No cooperative protective effect could be observed when ascorbate and catechin were used together. None of the treatments significantly affected either reduced or oxidized glutathione levels. In conclusion, this study showed strong protection by ascorbate, which could be used in clinically relevant situations, and is the first to report the protection by catechin at this dose under conditions of myocardial ischemia-reperfusion injury.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.

The treatment of asthma still relies on primary therapy with bronchodilators; in particular β adrenergic receptor (bAR) agonists with a diverse range of short acting and long acting βARs available. An increase in the number of cardiovascular events with the use of bronchodilators have recently been reported including hypertrophy, heart failure, myocardial ischaemia and infarction. Several subtypes of βAR receptors exist including the β1 Adrenergic Receptor (β1AR) and β2 Adrenergic Receptor (β2AR), both located in the heart. The effects of selective β2AR agonists were investigated in the Langendorff model of myocardial ischaemia reperfusion injury, isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. The selective β2AR long acting β agonists Formoterol and Salmeterol had no significant effect on infarct to risk ratio or time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The non-selective β1AR agonist Isoproterenol has been show to induce myocardial ischaemia and infarction in rat hearts previously, here we demonstrated Isoproterenol (0.5μM) significantly decreased time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The short acting β2AR agonist Salbutamol (0.01μ-1μM) significantly increased infarct to risk ratio in the Langendorff in addition to significantly decreasing time to hypercontracture in cardiomyocytes in the oxidative stress model highlighting a potential role of the mitochondrial permeability transition pore (mPTP). Activation of phosphorylated Akt and phosphorylated Erk1/2 via the PI3K/Akt signalling pathway and p44/p42 MAPK pathway were investigated by western blot analysis. Salbutamol significantly elevated expression of p-Akt in rat hearts exposed to reperfusion for 20 and 120 minutes whilst reducing expression of p-Erk. Recorded elevated cleaved caspase 3 expression in Salbutamol treated hearts can be associated as a marker of increased in cardiomyocyte cell death. The β1AR antagonist CGP 20712 was administered in the presence of Salbutamol with minimal reduction in infarct size in rat hearts recorded and no significant change in time taken to hypercontracture in isolated cardiomyocytes suggesting that Salbutamol mediated toxicity is via β2AR activation. Confirmation of this was verified with the β2AR antagonist ICI 118, 551. Significant decrease in infarct size was recorded in addition to a significant increase in time to hypercontracture in the oxidative stress model. Further to this, caspase 3 expression was significantly reduced in addition with p-Akt expression. With a potential role of the mitochondria and the mPTP contributing to Salbutamol induced myocardial injury, the Cyclophilin D inhibitor Cyclosporin A was administered in hearts and cardiomyocytes in the presence of Salbutamol. Infarct size was significantly reduced whilst time taken to hypercontracture significantly increased, suggesting that CsA treatment inhibits Salbutamol mediated injury via Cyclophilin D inhibition of the mPTP. To conclude, our results demonstrated that Salbutamol caused cardiotoxicity at tissue, cellular and protein level in conditions of ischaemia reperfusion injury. Further to this, inhibition of Cyclophilin D by CsA, or the use of the β2AR antagonist ICI 118, 551 inhibits Salbutamol induced toxicity.

Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemia-induced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout (TLR4-/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-Ik{cyrillic}B) as well as activation of nuclear factor kappa B (NFk{cyrillic}B) increased in the HF of WT mice. In contrast, there were lower levels of p-Ik{cyrillic}B and NFk{cyrillic}B binding activity in TLR4-/- mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4-/- mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4-/- mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21415号 / 医博第4405号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

No description available.

Introduction. Free tissue transfer is the 'gold standard' of surgical care for patients requiring composite tissue reconstruction when local options are unavailable or unsuitable. It is a form of autologous transplant wherein composite tissue is harvested from a distant site and used to reconstruct the primary defect. The flap is rendered ischaemic following transection of its vascular pedicle until successful anastomosis with the recipient vessels is completed. Ischaemia depletes cellular ATP, lowers pH and strains cellular homeostatic mechanisms. The only way to halt the inevitable progression to cell death is by reperfusion. However, reperfusion per se initially worsens the injury through the influx of inflammatory cells and mediators. This biphasic injury is named ischaemia reperfusion injury (IRI) and is characterized by microcirculatory dysfunction primarily mediated by oxidative stress. This can lead to inadequate perfusion and ultimately tissue necrosis. IRI occurs in all transplants, is unavoidable and has no treatment. Preconditioning is an intervention performed before a known event that improves the outcome of that event. The elective nature of transplants permits such interventions to be executed. Haem-­oxygenase 1 (HO-­1) is a cytoprotective enzyme that is up-­regulated in response to diverse stressors including oxidative stress. Haem arginate (HA) is a potent inducer of this enzyme. Pharmacological preconditioning with HA has been shown to reduce IRI and improve clinical outcome in models of visceral IRI. Aim: to investigate whether HA could be used to improve outcome in myocutaneous flaps. Objectives: (1) to establish a reliable model of myocutaneous IRI (2) to assess the effects of pharmacological preconditioning with HA on clinical outcome measures and (3) to investigate the mechanisms underlying the effects of HA preconditioning demonstrated in the in vivo model by in vitro work. Methods. An in situ transverse rectus abdominis myocutaneous (TRAM) flap was developed. Forty male, Lewis rats were randomly assigned to receive IV: Control (NaCl); HA; HA + tin mesoporphyrin (SnMP, an HO-­1 inhibitor) and SnMP alone. Laser Doppler imaging (LDI) scans were performed to assess perfusion. Clinical outcome was assessed by percentage area flap necrosis and perfusion. In vitro adult human epidermal keratinocytes (HEKa) were treated in: Control medium; HA; SnMP and Desferrioxamine (DF) or combinations thereof. MTT and VialightTM plus ATP assays were used to assess cytotoxicity. Intracellular reactive oxygen species (ROS) concentration was determined by flow cytometry (CMH2DCFDA assay). Statistical analysis was performed by one-­way analysis if variance (ANOVA) followed by Tukey's test. Results. In vivo preconditioning with HA increased HO-­1 protein expression and level of bioactivity. This bioactivity was successfully inhibited by SnMP. In the skin, HO-­1 up-­regulation occurred in macrophages. HA based treatments resulted in significantly worse necrosis at 48 h: Control vs HA (p = 0.01). HA based treatments significantly decreased perfusion at: 24 h (Control vs HA, p = 0.0002) and 48 h (Control vs HA, p = 0.04). By contrast, SnMP did not affect either clinical outcome measure. In vitro preconditioning with HA was cytotoxic and increased intracellular ROS: both were reversed by co-­administration of DF but not SnMP. Conclusion. In contrast to data from visceral models, HA preconditioning proved deleterious in myocutaneous flaps. This is most likely due to the generation of ROS by free haem independent of HO-­1 up-­regulation.