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.

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.

Ischaemia-reperfusion (IR) injury is caused by the re-introduction of oxygen to organs, following periods of reduced blood flow (ischaemia). Whilst re-establishing blood flow (reperfusion) to the heart following myocardial infarction is vital for organ survival, this paradoxically leads to tissue damage. Mitochondria are at the heart of IR injury, with succinate dehydrogenase (SDH) a major player in orchestrating the damage. Succinate accumulates during ischaemia and is rapidly oxidised by SDH upon reperfusion, producing reactive oxygen species (ROS), leading to cellular death. I have investigated the development of drugs, aimed at targeting succinate metabolism to ameliorate IR injury. I firstly screened a range of compounds for their ability to inhibit SDH, having been chosen for their similar structures to succinate or the classical SDH inhibitor, malonate. Interestingly, only malonate and oxaloacetate showed potent SDH inhibition, thus were selected for further development. Malonate ester prodrugs with different properties were characterised. Hydrolysis rates of the esters differed greatly, with tuned, labile, malonate esters releasing malonate much more rapidly. Malonate esters were taken up into cells and hydrolysed to release malonate to different extents. Additionally, mitochondria-targetedmalonatemono and diesters were developed, each differing in mitochondrial and cellular uptake andmalonate release. Targeted and nontargeted malonate esters distributed into tissues in vivo, with preliminary in vivo work carried out on IR injury models, to assess for protective effects of the compounds. In addition, the physiological role of the tricarboxylic acid cycle metabolite, itaconate, was investigated. In lipopolysaccharide stimulated macrophages, itaconate has been reported to exert its effects by inhibition of SDH however, I found itaconate was a relatively poor SDH inhibitor, indicating other mechanisms of action. Current prodrugs of itaconate have many non-specific effects, not attributable to itaconate. I therefore characterised a novel itaconate prodrug and found it to be a much better surrogate, which could be subsequently used to elucidate roles for itaconate. Overall, I have shown the importance of ester selection for the prodrug delivery of dicarboxylate molecules and developed methods to improve their biological delivery.

"September 2002" Bibliography: leaves 207-230. Ch. 1. Literature review -- Ch. 2. General methods -- Ch. 3. Dietary n-3 PUFAs and reperfusion injury in isolated cardiomyocytes -- Ch. 4. The effect of dietary n-3 PUFAs on cardiomyocyte membrane fluidity, intracellular ROS and Ca 2+ levels during oxidative stress -- Ch. 5. The effect of dietary fish oil supplementation on antioxidant enzyme gene expression in rat myocardium -- Ch. 6. The effect of dietary lipids on ischaemia-reperfusion injury in rat myocardium -- Ch. 7. General discussion -- Ch. 8. Appendices. The broad aims of this thesis were to develop a cellular model for studying reperfusion injury, in order to investigate the reported protective effects of n-3 PUFAs, and to examine the underlying mechanisms associated with such protection.

"September 2002" / Bibliography: leaves 207-230. / x, 230 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The broad aims of this thesis were to develop a cellular model for studying reperfusion injury, in order to investigate the reported protective effects of n-3 PUFAs, and to examine the underlying mechanisms associated with such protection. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2002

Primary graft non-function is one of the major limitations of organ transplantation increasing the risk of rejection and early graft failure. A major cause of primary non-function is ischemia reperfusion injury (IRI), an obligatory insult in transplantation. During procurement, the donor is subjected to a period of ischemia inducing the release of tissue-damaging factors such as nitric oxide and reactive oxygen species. Upon engraftment and reperfusion with the recipient blood, these ischemia-induced factors cause rapid cell death and amplification of the inflammatory response leading to further tissue damage. / CD39 is an integral vascular and immune ectonucleotidase. CD39 hydrolyses extracellular nucleotides ATP and ADP into AMP, which is then hydrolysed into adenosine by CD73. Extracellular adenosine produced by the concerted action of CD39 and CD73 has potent anti-inflammatory and anti-coagulation effects acting principally via the purinergic adenosine receptor A2a. / NKT cells have only recently been recognised and constitute an important subset of T lymphocytes that display both effector and suppressive functions. NKT cells are found in high proportion in the liver of mice and are implicated by depletion studies in protection against hepatic IRI. / We have generated mice transgenic for human CD39 (hCD39) and have shown they have an anti-coagulant phenotype. As CD39 is also critical to immune regulation we hypothesised that transgenic expression of hCD39 would modify lymphocyte development and/or function and consequently impact on ischemia reperfusion injury. / Flow cytometric analysis was used to assess the number and phenotype of lymphocytes within the thymus and in the periphery of hCD39 transgenic mice. In vitro and in vivo assays were used to test the function of CD4+ T cells and invariant NKT cells from hCD39 transgenic mice. Bone marrow adoptive transfers experiments defined the role of hCD39 expression on bone marrow progenitor cells in comparison to tissue expression. The importance of adenosine signalling through the A2a receptor was studied by crossing hCD39 transgenic mice with A2a receptor knock-out (KO) mice. The effect of hCD39 expression on ischemia reperfusion injury was evaluated in a model of murine liver transplantation / A high level of hCD39 expression in the transgenic thymus resulted in lymphocyte maturation blockade and peripheral lymphopenia of CD4+ T cells and invariant NKT cells. Both lymphocyte populations were functionally deficient. The observed phenotype resulted from the expression of hCD39 on bone marrow progenitor cells but was independent of A2a receptor signalling. Over-expression of hCD39 in transgenic livers was protective against ischemia reperfusion injury induced by cold storage and liver transplantation.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.