Dissertations / Theses on the topic 'Ischaemia and reperfusion damage'

The overall aim of this Thesis was to characterise the energetic properties of the mouse papillary muscle as this preparation could become a useful model to study alterations of energetic aspects of cardiac pathologies and heart-focussed genetic changes. Measurements of resting and active metabolism of the papillary muscles were made in vitro using the myothermic technique. In the first study the mechanism underlying impaired contractility of post-ischaemic rat papillary muscle was investigated. The rat preparation is well established and was used to develop protocols and approaches that could later be used as the basis for studies with mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min and change in energetics was studied 30 min into the reperfusion phase. The work output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination with studies of Ca2+ handling, suggests that the reduced work output was caused by attachment of fewer cross-bridges in each twitch, but with no change in work generated by each cross-bridge. The following two studies involved characterisation of the energetics of the mouse papillary muscle and included measurements of resting and active metabolism. The resting metabolic rate varied with muscle size but the mean initial value was tilda 25 mW g-1 and the estimated steady value tilda 5 mW g-1 . The resting metabolic rate declined exponentially with time towards a steady value, with a time constant of 18 ± 2 min. There was no alteration in isometric force output during this time. The magnitude of resting metabolism depended inversely on muscle mass, more than doubled following a change in substrate from glucose to pyruvate and was increased 2.5-fold when the osmolarity of the bathing solution was increased by addition of 300 mM sucrose. Addition of 30 mM BDM affected neither the time course of the decline in metabolic rate nor the eventual steady value. The energy requirements associated with contractile activity were tilda7 mJ g-1 twitch-1 at a contraction frequency of 1 Hz. The enthalpy output was not affected by changing substrate from glucose to pyruvate but did decrease with an increase in temperature. The enthalpy output was partitioned into force-dependent and force-independent components using BDM to selectively inhibit cross-bridge cycling. The force-independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle initial efficiency was &tilda32% and net efficiency tilda 17% when shortening at a realistic velocity. The enthalpy output decreased with increased contraction frequency but was independent of shortening velocity. On the basis of these values, it was calculated that the twitch energetics were consistent with ATP splitting by half the cross-bridges and the pumping of one Ca 2+ into the SR for every three cross-bridge cycles. The lack of influence of shortening velocity on energy cost supports the idea that the amount of energy to be used is determined early in a twitch and is not greatly influenced by events that occur during the contraction. The suitability of the mouse papillary muscle as a model to study ischaemia and reperfusion damage was also assessed. This preparation is excellent for studying muscle specific changes in work and enthalpy output; however, due to the long-term instability and variability amongst preparations, the suitability of this preparation in prolonged experiments remains uncertain.

The overall aim of this Thesis was to characterise the energetic properties of the mouse papillary muscle as this preparation could become a useful model to study alterations of energetic aspects of cardiac pathologies and heart-focussed genetic changes. Measurements of resting and active metabolism of the papillary muscles were made in vitro using the myothermic technique. In the first study the mechanism underlying impaired contractility of post-ischaemic rat papillary muscle was investigated. The rat preparation is well established and was used to develop protocols and approaches that could later be used as the basis for studies with mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min and change in energetics was studied 30 min into the reperfusion phase. The work output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination with studies of Ca2+ handling, suggests that the reduced work output was caused by attachment of fewer cross-bridges in each twitch, but with no change in work generated by each cross-bridge. The following two studies involved characterisation of the energetics of the mouse papillary muscle and included measurements of resting and active metabolism. The resting metabolic rate varied with muscle size but the mean initial value was tilda 25 mW g-1 and the estimated steady value tilda 5 mW g-1 . The resting metabolic rate declined exponentially with time towards a steady value, with a time constant of 18 ± 2 min. There was no alteration in isometric force output during this time. The magnitude of resting metabolism depended inversely on muscle mass, more than doubled following a change in substrate from glucose to pyruvate and was increased 2.5-fold when the osmolarity of the bathing solution was increased by addition of 300 mM sucrose. Addition of 30 mM BDM affected neither the time course of the decline in metabolic rate nor the eventual steady value. The energy requirements associated with contractile activity were tilda7 mJ g-1 twitch-1 at a contraction frequency of 1 Hz. The enthalpy output was not affected by changing substrate from glucose to pyruvate but did decrease with an increase in temperature. The enthalpy output was partitioned into force-dependent and force-independent components using BDM to selectively inhibit cross-bridge cycling. The force-independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle initial efficiency was &tilda;32% and net efficiency tilda 17% when shortening at a realistic velocity. The enthalpy output decreased with increased contraction frequency but was independent of shortening velocity. On the basis of these values, it was calculated that the twitch energetics were consistent with ATP splitting by half the cross-bridges and the pumping of one Ca 2+ into the SR for every three cross-bridge cycles. The lack of influence of shortening velocity on energy cost supports the idea that the amount of energy to be used is determined early in a twitch and is not greatly influenced by events that occur during the contraction. The suitability of the mouse papillary muscle as a model to study ischaemia and reperfusion damage was also assessed. This preparation is excellent for studying muscle specific changes in work and enthalpy output; however, due to the long-term instability and variability amongst preparations, the suitability of this preparation in prolonged experiments remains uncertain.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Physiotherapy and Exercise Science
Full Text

Thesis (MScMedSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications.
AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

Kidney transplants from both living-related (LRD) and living unrelated (LURD) donors have superior function and survival than transplants from cadaver donors. This may be unsurprising as kidneys from living donors are procured under optimal conditions, from healthy donors with minimal ischaemia times. In contrast, cadaver kidneys are obtained from traumatised donors and may experience extended periods of cold ischaemic storage before transplantation. An immunohistochemical analysis has been performed on biopsies obtained before, and immediately after transplantation, to investigate the potential causes of early inflammatory events associated with cadaver renal transplantation that may influence subsequent graft outcome. An immunohistochemical analysis of biopsies obtained before transplantation demonstrated upregulated expression of endothelial E-selectin and proximal tubular expression of ICAM-1, VCAM-1 and HLA Class II antigens in cadaver donor kidneys. Analysis of donor parameters demonstrated that traumatic physiological events experienced in intensive care around the time of brain death were significantly associated with the induction of proinflammatory antigens. Antigen induction in cadaver donor kidneys before transplantation was significantly associated with early acute rejection. Furthermore, in cadaveric kidneys with long cold ischaemia times, glomerular neutrophil infiltration and deposition of activated platelets expressing P-selectin on intertubular capillaries were detected following reperfusion, in association with impaired short and long term graft function. Expression of inflammatory mediators were absent in all LRD renal allografts before and after reperfusion. A clinical trial was performed to determine whether ischaemia/reperfusion injury may be ameliorated by reflushing cadaver kidneys after cold storage to remove harmful products that may have accumulated in the vessel lumen. Reflushing did not prevent the inflammatory events observed after reperfusion or improve graft function. Therefore, a novel, oxygen free radical scavenger (lec-SOD) was obtained to assess its potential efficacy in preventing ischaemia/reperfusion injury. Lec-SOD bound with high affinity to macro- and microvascular endothelial cells under cold hypoxic conditions following incorporation into Marshall's preservation solution, significantly inhibiting cold hypoxia induced cell death, adhesion molecule induction and neutrophil adhesion. Furthermore, preservation of kidneys with lec- SOD for 18 hr in an experimental model of chronic renal allograft rejection, significantly attenuated neutrophil infiltration and MHC Class I induction day 1 post-transplant, with improved long term renal function. The results presented in this Thesis demonstrate that donor factors and cold ischaemia/ reperfusion injury elicit an early inflammatory response that may influence graft outcome of cadaver kidneys. Refinements in donor management and organ preservation may limit the deleterious effects of ischaemia/reperfusion injury in cadaver renal allografts, increasing graft survival to that observed in living donor transplantation.

Liver Ischaemia Reperfusion Injury (IRI) leads to production of reactive oxygen species and cytokines, which affects hepatocellular function following liver resection and transplantation. This thesis examines 2 hypotheses: 1) The role of intravenous glycine in amelioration of liver IRI in a in vivo animal model of partial lobar liver IRI. 2) Does prophylactically administered N-acetylcysteine prevent liver IRI in patients undergoing elective liver resection. Materials and Methods 1) A rabbit model of hepatic lobar IRI was used to evaluate glycine. 3 groups (n=6) Sham group (laparotomy alone), ischaemia reperfusion (I/R) group (1 hour ischaemia and 6 hours of reperfusion), and glycine I/R group (IV glycine 5 mg/kg prior to the I/R protocol) were used. Portal blood flow, bile flow and bile was analysed by H1NMR spectroscopy. Hepatic microcirculation, intracellular tissue oxygenation, serum TNFα, IL-8, ALT, AST were measured at 1, 2, 4 and 6 hours following reperfusion. 2) A randomised double blind clinical trial was conducted to assess the effect of NAC on liver IRI following liver resections. The main outcomes were: morbidity and mortality, ICAM-1 expression in liver tissue, liver function tests. Patients were randomised to receive NAC as IV infusion (NACG) or a placebo group (PG) which received 5% dextrose only. Immunohistochemistry for ICAM-1 was carried out on perioperative liver biopsies. Results 1) Glycine normalised the bile flow, reduced phosphatidylcholine shedding, lactate surge, and stimulated bile acid, pyruvate, glucose and acetoacetate release. Glycine improved portal blood flow, hepatic microcirculation by the 2nd hour, and hepatic intracellular tissue oxygenation by the 4th hour of reperfusion. Glycine ameliorated serum TNFα at 1, 2 and 4 hours and serum Il- 8, AST and ALT up to 6 hours post reperfusion as compared to the I/R alone group. 2) Of the 43 patients, 15 received NAC, 16 were randomised to the PG, 12 were excluded due to inoperable tumours. Serum ALT was reduced in NACG (p=0.001), while serum ALP was higher in the NACG (p=0.003). ICAM-1 expression was up-regulated in 6/16 patients in the PG and in 3/15 patients in NACG. ICAM-1 was down-regulated in 1/15 patients in the NACG and none in the PG, the difference was not significant. Conclusions 1) Glycine ameliorated liver IRI, improved bile flow and composition. 2) NAC ameliorated parenchymal liver injury and enhanced liver regeneration in patients undergoing elective liver resection.

Restoring cerebral blood flow using reperfusion treatment is a common method in treating ischaemic stroke. Reperfusion treatment should be given within 4.5 hours from stroke onset. However, reperfusion beyond this time window poses the risk of reperfusion injuries such as intracranial haemorrhage and cerebral tissue swelling. The focus of this thesis is to study the effect of cerebral tissue swelling after reperfusion as it can occur in a few hours after the treatment. Cerebral tissue swelling may cause brain structure movement and cerebral microvessel compression; the latter may then lead to secondary ischaemia occurrence. In this thesis, two mathematical models are presented. The first model investigates the effect of ischaemia-reperfusion in the formation of cerebral tissue swelling. This model provides the understanding of suitable reperfusion conditions to reduce the effect of tissue swelling and also becomes the basis for the subsequent model. Meanwhile, the second model studies the role of a water-transporting protein, aquaporin-4 in ischaemia-reperfusion and its potential as part of treatments for cerebral tissue swelling. In addition, the ionic concentration may change during ischaemia which may be a factor contributing to cerebral tissue swelling. Thus, the effect of ionic concentration on the swelling formation is also investigated. Finally, validations of these models are achieved by developing patient-specific geometries from available ischaemic stroke patient MRI data and utilising finite element analysis. Comparison between the simulation results and the MRI data is done by quantifying the brain ventricles movement during cerebral tissue swelling.

Cardiac hypertrophy may be associated with an enhanced susceptibility to ischaemic/reperfusion injury but the mechanisms remain unresolved. There is evidence for an increased dependence on glucose metabolism in cardiac hypertrophy, which may be beneficial in normoxia but detrimental in ischaemia. The role of glycogen, the major endogenous substrate during ischaemia, to the enhanced susceptibility of the hypertrophied heart to ischaemic/reperfusion injury is unclear. Work in this thesis investigates the role of glycogenolysis to the severity of ischaemia, and assesses oxidative substrate utilisation following reperfusion, in the hypertrophied heart. Pressure overload cardiac hypertrophy was induced surgically in male Sprague-Dawley rats by intra-renal constriction. A moderate hypertrophy was observed nine weeks post surgery as evidenced by between a 4 and 25 % increase in heart weight: tibia length ratio. Hearts were perfused in an isovolumic mode, and function was recorded. ¹³C-NMR spectroscopy was performed on extracts from hypertrophied and control hearts reperfused with ¹³C labelled substrates to determine the profile of substrate use. Glycogen content was unchanged in hypertrophied hearts compared to control hearts and there was no evidence for glycogen loading in the presence of physiological substrates and insulin. In addition, no further glycogen loading occurred when insulin concentrations were increased to pharmacological levels. Provision of other carbohydrate substrates, such as lactate, did result in a further increase in myocardial glycogen content. Hypertrophied hearts experienced the same extent of ischaemia as controls with no evidence of increased ischaemic injury, implying that a compensated model of hypertrophy was generated in this study. Myocardial function decreased during low flow ischaemia and stopped during global ischaemia, but contracture was not observed. The severity of ischaemia was the determining factor in the degree of glycogen degradation. Increased glycogen degradation during ischaemia did not correlate with increased ischaemic injury, suggesting that the availability of glycogen for energy provision limited ischaemic injury. Recovery on reperfusion was markedly improved in the presence of insulin. This improvement appeared to be mediated by the inotropic actions of insulin rather than by alterations in substrate provision. The profile of substrate use in hypertrophied hearts during reperfusion was found to be the same as that in controls. No metabolic alterations were observed in the hypertrophied heart that enhanced susceptibility to ischaemic/reperfusion injury, implying that compensated hypertrophy is a beneficial response of the heart.

Background - Ischaemia/reperfusion accounts for a large proportion of fatalities in the developed world. Even if death is avoided, the patient suffers a deterioration in their quality of life. Erytriropoietin (EPO) has been examined in clinical studies investigating its effect in anaemic chronic heart failure patients with any positive effect attributed to the correction of anaemia. Given the recent discovery of the EPO receptor on the myocyte surface, this thesis examined whether EPO could have a direct effect on the myocardium and limit ischaeinia/reperfUsion injury and the mechanism by which any protection occurs. Methods and results - Using an isolated perfused rat model of ischaemia/reperfusion, we demonstrated that EPO could mimic preconditioning in a P1-3-Kinase (PI3K)- dependent manner. Adrninistration of EPO at reperfusion limited infarct size by activation eNOS and could be abolished by inhibitors of NOS, PI3K and ERK 1/2. This thesis also showed that EPO could delay mitochondrial permeability transition pore opening (mPTP) in an oxidative stress myocyte model of mPTP opening, an effect that was suppressed by inhibitors of NOS and PBK. A 3 week treatment of EPO reduced injury in a NOS-dependent manner that was independent of haematocrit. Finally this thesis demonstrated that administration of EPO as late as 30 minutes after commencement of reperfusion could still reduce infarct size. Conclusion - This thesis demonstrated that EPO can be used in a variety of settings to elicit a protective effect against ischaemia/reperfusion injury. This variety of effective time points promises an important future role for EPO in the treatment of ischaemic heart disease.

CaMKII acts as a second messenger to Ca2+ signals within the cardiac myocyte. Cellular stresses such as ischaemia and subsequent reperfusion perturb the normal physiological oscillations of Ca2+ to cause an escalating concentration which damages the cell. CaMKII has been implicated as an injury signal during such cellular conditions. However, there are discrepancies as to whether CaMKII is a possible mechanism of ischaemic preconditioning as its inhibition can abrogate or improve the protective effect of preconditioning. This thesis investigated the effects of CaMKII inhibition in models of ischaemia-reperfusion (I-R) injury. It was hypothesised that CaMKII promotes irreversible injury caused by acute myocardial infarction (AMI), but would also have a beneficial role in mediating cardioprotection by ischaemic preconditioning. This work has demonstrated that: i) in an ex vivo rat heart model of regional I-R injury, CaMKII promoted irreversible injury but is not a feasible target for reperfusion therapy as only a pre-ischaemic intervention reduced myocardial infarction; ii) CaMKII activation was not a pre-requisite for protection with ischaemic preconditioning, although an additive protective effect of CaMKII inhibition and ischaemic preconditioning was possible; iii) models of simulated I-R or oxidative stress in the H9c2 cells did not involve CaMKII activity; iv) isolated cardiac myocytes paced at 1Hz and subjected to simulated I-R do not engage a significant amount of CaMKII activity. These studies substantiate the involvement of CaMKII during ischaemic injury and establish that it does not play a substantial role in ischaemic preconditioning. It highlights the characteristics of the kinase within in vitro models of I-R injury. Understanding CaMKII role in I-R may underpin the development of future therapeutic strategies for the management of AMI.

Intestinal ischaemia and reperfusion (I/R) contributes to the pathogenesis of numerous clinical conditions in all age groups. Many of these diseases, including neonatal necrotizing enterocolitis (NEC), result in significant morbidity and mortality through multiple organ dysfunction, and available treatment is currently limited to supporting vital functions. My aims were: to investigate novel therapeutic strategies such as moderate hypothermia and peroxynitrite decomposition catalyst FeTMPyP [5,10,15,20- tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III)] in experimental models of adult and infant intestinal I/R; and to characterise the inflammatory process in human NEC, evaluating its relationship with clinical outcome. In an adult rat model, total-body moderate hypothermia applied throughout ischaemia and reperfusion counteracts oxidative stress in both the intestine and distant organs. This suggests that hypothermia could be beneficial as a preventative measure when intestinal ischaemia can be foreseen. However, in clinical practice therapy can usually be commenced only after ischaemia has occurred. In two sets of experiments, I showed that rescue hypothermia applied after mesenteric ischaemia improves outcome in both adult and neonatal rats, and this benefit is maintained after rewarming. Hypothermic protection could result from prevention of multiple organ dysfunction through several different pathways, including modulation of hepatic phosphoenergetics, pulmonary inflammatory infiltrate, cardiac energy metabolism, and systemic oxidative stress. Administration of peroxynitrite decomposition catalyst FeTMPyP as a rescue therapy at reperfusion also exerts a protective effect in neonatal rats, possibly via inhibition of adhesion molecule expression, leukocyte recruitment, and lipid peroxidation in the intestine, leading to prevention of systemic oxidative stress. In a study conducted on human specimens from neonates with NEC, tissue injury seems to be mediated via increased expression of endothelial adhesion molecules ICAM-1 and P-Selectin, leading to macrophage and neutrophil infiltration. Endothelial E-Selectin is expressed exclusively in NEC patients, and appears to be a marker of rapidly evolving disease and distant organ failure.

The aim of these studies was to investigate the changes in gene expression that occur in H9c2 myoblast cells subjected to the metabolic stresses associated with myocardial ischaemia/reperfusion. The expression of Vascular Endothelial Growth Factor-A (VEGF-A) and Myocyte Stress 1 protein (ms1) were examined. VEGF-A has a physiological role in stimulating angiogenesis following cardiac ischaemia/reperfusion in vivo, whereas ms1 expression is induced following left ventricular hypertrophy, but has not been examined previously during ischaemia/reperfusion. No increase in VEGF-A, or ms1 mRNA was detected during simulated ischaemia but both were upregulated following reperfusion. The induction in VEGF-A mRNA was caused by an increase in mRNA stability regulated by ERK and JNK pathways. A reporter system was used to demonstrate that simulated ischaemia/reperfusion exerts its effect on both the 5' and 3'UTR of VEGF-A mRNA. The induction in ms1 mRNA was caused by an increase in transcription that was dependent on activation of the JNK pathway. An increase in the amount of ms1 protein expression was also detected during reperfusion. Regulation of translation also influences the expression of proteins during ischaemia/reperfusion. The effect of simulated ischaemia on message translation was investigated using cDNA microarrays. Simulated ischaemia inhibited CAP dependent translation and induced eIF2a phosphorylation. Some messages were more highly represented in the polysome fraction from ischaemic cells, compared to that from resting cells, including FAD oxidoreductase and a sub complex of NADH dehydrogenase, proteins involved in electron transport.

Warm ischaemic injury occurs when an organ or region of the body is starved of oxygenated blood under normothermic conditions. Two important clinical examples of warm ischaemia are donation after circulatory death (DCD) kidney transplantation and abdominal aortic aneurysm (AAA) repair. The tissue injury that results from warm ischaemia can lead to organ dysfunction, which has important clinical consequences. In kidney transplantation warm ischaemic injury can lead to delayed graft function, increased rates of primary non-function and poorer long-term outcomes. In open AAA repair occlusion of the abdominal aorta leads to remote injury to organs such as the kidneys. Renal failure following AAA repair can cause significant morbidity including the need for renal replacement therapy. Hydrogen sulphide (H2S) is an endogenously produced gas that been shown to be protective against ischaemia-reperfusion injury. The aims of this thesis were twofold. First, the effect of H2S on ischaemia-reperfusion injury in a porcine model of direct renal ischaemia was assessed. Second, the effect of H2S on inflammation and remote renal injury was assessed using a rodent model of aortic occlusion. This research demonstrated that hydrogen sulphide, delivered as an exogenous agent, preserved renal function and reduced inflammation in a large animal model of renal warm ischaemia. Furthermore, in a rodent model of remote renal injury hydrogen sulphide also reduced systemic and renal inflammation but had no effect on renal function. In addition, there were no significant side effects from the administration of hydrogen sulphide.

Myocardial infarction is the greatest cause of mortality worldwide, and a source of considerable morbidity. Treatment of STCelevation MI (STEMI) has improved enormously with the advent of primary percutaneous coronary intervention (PPCI), but ischaemia/reperfusion (I/R) injury remains an important complication. Evidence from animal studies points to a role for lymphocytes, and in particular T cells, in myocardial I/R injury, but this has not yet been studied in humans. The goal of my PhD was to investigate this phenomenon in human patients treated with PPCI, with particular emphasis on T cell kinetics, their relationship to I/R injury, and the potential mechanisms involved. I retrospectively analysed a large database of MI patients treated with PPCI. I demonstrated that lymphopaenia during admission was an independent predictor of increased longCterm mortality, confirming the prognostic relevance of lymphocytes in this setting for the first time. I then studied a prospectively recruited cohort of STEMI patients, determining lymphocyte subset kinetics with detailed flowCcytometric analysis. T cells were acutely depleted from the circulation within minutes of reperfusion, with highly differentiated effector cells showing the greatest changes. TransCcoronary gradients suggested some cells were sequestered into the reperfused myocardium. Cardiac MRI analysis revealed a significant relationship between postCreperfusion effector T cell kinetics and microvascular obstruction (MVO), a component of I/R injury, raising the possibility of a mechanistic link. This discovery was driven primarily by positive findings in cytomegalovirus seropositive patients, who had higher percentages of highly differentiated T cells. Analysis of chemokine receptors subsequently identified CX3CR1, with its ligand fractalkine, as the prime candidate for a key role in effector T cell kinetics postCreperfusion, potentially influencing MVO. These findings identify a possible therapeutic target in I/R injury postCPPCI, opening up a new avenue for further research and future treatment development.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p<0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p<0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p<0.001), leading to a 60% survival rate at 48 hours IRI (p<0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p<0.001), with a survival benefit of 75% (p<0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p>0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Activation of the PI3K/AKT pathway protects the heart from ischaemia-reperfusion injury. Phosphatase and Tensin Homolog deleted on Chromosome10 (PTEN) is a negative regulator of this pathway. The hypothesis on which this thesis was based stated that inhibition of PTEN would confer protection against ischaemia-reperfusion injury. PTEN was reduced using: 1) a PTEN inhibitor, bpV(HOpic), 2) a mouse model of PTEN haploinsufficiency and 3) PTEN siRNA. The effects of PTEN reduction on ischaemia-reperfusion injury were investigated by using: 1) an isolated perfused heart model of ischaemia-reperfusion injury, 2) an isolated cardiomyocyte model of ROS induced mitochondria damage and 3) a cellular model of hypoxia-reoxygenation injury. No protection against ischaemia-reperfusion was observed in isolated perfused myocardium from C57BL/J6 mice, which were perfused with bpV(HOpic), or from PTEN+/-mice. Likewise, no protection against ROS induced mitochondrial damage was observed in isolated cardiomyocytes from the PTEN+/- mice. In these models an increase in AKT activity was recorded, however, this was not sufficient to confer cardioprotection. Similarly, H9c2 rat myoblast cells, silenced for PTEN expression using siRNA, were not protected against hypoxia-reoxygenation injury. Nevertheless, in isolated C57BL/J6 hearts perfused with bpV(HOpic) and in myocardium from PTEN+/- mice, when the PI3K/AKT pathway was stimulated by the cardioprotective intervention of ischaemic preconditioning a reduced threshold for protection was achieved. To conclude, the level of PTEN inhibition achieved in this study was not sufficient to bestow protection against simulated ischaemiareperfusion injury. However, it appears that reductions in PTEN can increase the sensitivity towards cardioprotection.

Thesis (MScMed)--Stellenbosch University, 2008.
ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential.
AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.

Hypothesis: That infrainguinal arterial bypass procedures are associated with an ischaemia-reperfusion injury and that this may be reduced by administration of sodium nitroprusside, a nitric oxide donor. First study: 28 subjects, with critical limb ischaemia, undergoing infrainguinal bypass were randomised to receive sodium nitroprusside (0.1 μg/kg body weight/min) (n=15) or placebo (n = 13), infused into the operated limb, during the performance of the second or distal anastomosis and the infusion continued for twenty minutes after. Blood sampling and a doppler studies were performed. 9 patients undergoing varicose vein operations were also examined. The results indicated that application of the proximal clamp resulted in ischaemia and reperfusion with associated neutrophil activation. Sodium nitroprusside resulted in vasodilatation but had no effect on either parameter. Bypass patients had higher baseline neutrophil activation than the varicose vein group. Second Study: 30 further bypass patients ere randomised (treatment n = 15, placebo n = 15) with the infusion commencing at the time of the proximal anastomosis. Blood and urine sampling and doppler studies were performed. Changes in plasma antioxidant status, microalbuminuria and enzymuria occurred only in the placebo group indicating that, in addition to vasodilating the critically ischaemic limb, the nitric oxide donor may have been protective. Paradoxically, myeloperoxidase levels rose only in the treatment group but there was no evidence of increased free radical activity or permeability changes associated with this. Conclusion: Infrainguinal bypass is associated with an ischaemia-reperfusion injury which begins at the time of first clamp application. These procedures also result in a small but significant decrease in antioxidants and an increase in permeability. Sodium nitroprusside, a nitric oxide donor, administered into the operated limb appears to be protective against these changes and is an effective peripheral vasodilator.

The purpose of the work described in this thesis was twofold. Firstly, to determine what role nitric oxide (NO) played in renal warm ischaemia reperfusion injury and secondly, once the role was clarified, to assess if this injury could be ameliorated by the pharmacological manipulation of renal NO. The introduction to this thesis comprises two parts. Chapter 1 reviews the pathophysiology of renal ischaemia reperfusion and outlines some of the treatment modalities that have been used in response. Chapter 2 focuses on the physiology of NO and the pathophysiological role it plays in renal reperfusion injury and contrasts some apparent differences between the kidneys and other organs. The three main experimental Chapters are then presented. The first of these describes the development and verification of a NO assay for use with renal tissue homogenate. The next Chapter describes the development of a new model of renal warm ischaemia reperfusion injury in a rodent model which allows the collation of renal NO levels with comparative renal functional, pathological and pathophysiological data. The final experimental Chapter details both the effect of warm ischaemia reperfusion injury on renal NO generation and the subsequent effect of pharmacologically manipulating NO on the degree of renal injury sustained. In the final Chapter the experimental results are reviewed and possible avenues of further investigation are discussed.

Ischaemia-reperfusion injury is a common clinical event, still associated with high mortality and morbidity, and lacks a specific therapy. It is well established that postischaemic reflow is associated with the generation ofoxygen radicals, and that oxidant stress occurring during reperfusion may produce deleterious effects in reperfused tissue. A consistent feature oflocal tissue injury that follows ischaemiareperfusion injury is the histological fmding ofan acute inflammatory response mediated in part by increased vascular permeability, mast cell degranulation, plugging ofthe microvasculature by circulating leukocytes, with a concomitant recruitment of adherent and emigrating leukocytes, release ofreactive oxygen species and proteolytic enzymes, increased albumin extravasation in downstream postcapillary venules, and cell death. Ofthe different circulating cells activated, experimental models ofischaemia-reperfusion have identified the polymorphonuclear leukocyte (neutrophil) as mediating the initiation reperfusion-induced tissue injury. Research on ischaemia-reperfusion injury has focused on either animal studies or patients who are hospitalised. There is limited research on ischaemia-reperfusion in healthy individuals. Therefore, the aim ofthe series ofstudies set out was to investigate the functional response ofleukocytes ofhealthy individuals in response to ischaemia-reperfusion and from the results obtained to develop a working model of the mechanism involved in ischaemia-reperfusion. Male Participants (n = 125) (mean age 22 ± 4) all considered healthy were recruited from the university student population and the general public to participate in the study. Participants were exposed to ischaemia-reperfusion for various durations. Venous whole blood samples taken from the anticubital vein were subjected to various assays including the use ofluminol-enhanced chemiluminescence, and enzyme-linked immunosorbent assay with the aim ofdetermining the role, if any, that neutrophils play in ischaemia-reperfusion. From the results obtained from the series of studies it was demonstrated that reactive oxygen species production and neutrophil priming activation occur as a result ofischaemia-reperfusion, interacting possibly with each other and with other elements such as endothelial cells and other leukocytes. Further investigation is required to elucidate the mechanisms involved that lead to neutrophil activation, which will allow for the development ofnovel treatment to be developed.

Ischaemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI) and predisposes to the development of chronic kidney disease (CKD). The role of TGF-β in extracellular matrix deposition and renal fibrosis has been well established. This study was designed to establish an in vitro model of renal tubular IRI, evaluate the role of TGF-β in IRI in human proximal tubular epithelial cells (HKC8 and HK2 cells) and further determine the role of αvβ6 integrin in IRI. Initially an in vitro model of hypoxia and free radical stress by treating HKC8, HK2 and fibroblasts (MRC-5 cells) with CoCl2 and H2O2 respectively was established. These treatments led to pro-fibrotic changes characterised by increased expression of fibrotic marker α-SMA and reduced expression of epithelial cell marker E-Cadherin at mRNA and protein level. Binding of TGF-β to its receptor leads to activation of the kinase ALK5. ALK5 inhibition prevented the changes induced by H2O2 or CoCl2 suggesting the involvement of TGF-β to the cellular response to IRI. To confirm that TGF-β is released after treatment of cells to mimic IRI, media transfer and co-culture studies were performed. These experiments confirmed that bioactive TGF-β was being released. Lastly, the role of αvβ6 integrin was studied post H2O2 or CoCl2 treatment. Expression of αvβ6 integrin was elevated in conditions mimicking IRI in vitro and in biopsy samples acquired from patients with acute tubular necrosis and in mouse kidney following IRI. Knockdown of αvβ6 integrin in HKC8 cells decreased the bioavailability of active TGF-β following CoCl2 or H2O2 treatment and therefore the pro-fibrotic changes that were seen. This study confirms that bioactive TGF-β is produced following IRI and αvβ6 plays an important role in its release.

Glucagon-Like Peptide-1 (GLP-1) is an incretin hormone released by enteroendocrine cells lining the intestine in response to the presence of nutrients. GLP-1 is known to cause increased secretion of insulin from the pancreas and has been identified as one of the crucial components of insulin and in turn glucose homeostasis. GLP-1 has a very short half life of 1-2 minutes, being rapidly degraded by a ubiquitous enzyme called dipeptidyl dipeptidase IV and also undergoing renal excretion. Interestingly GLP-1 mRNA transcripts have been identified in several organs outside of the expected enteropancreatic axis including the heart. Insulin has been shown to reduce cell death in the ischemic-reperfused rat myocardium and in isolated rat myocytes via its ability to activate prosurvival kinase signalling pathways. We propose that GLP-1 could protect the myocardium against ischaemia-reperfusion injury by activating similar prosurvival signalling pathways. Both in-vivo (open chest) and in-vitro (Langendorff perfused) rat heart models of regional ischaemia and reperfusion were used. In-vivo treatment with GLP-1 produced a significant reduction in infarction (% infarct/risk zone) compared to valine pyrrolidide (VP), (an inhibitor of the enzyme dipeptidyl peptidase), and control groups (20.0 2.8, vs. 47.3 4.3, and 44.3 2.4, respectively PO.001). In isolated perfused hearts (where there is no circulating insulin) GLP-1 significantly reduced infarct size compared to VP and control (26.7 2.7 vs. 52.6 4.7 and 58.7 4.1, PO.001) groups respectively. Protection was abolished in the presence of the PI3kinase inhibitor, LY294002 (58.6 4.1), the ERK 1/2 MAPK inhibitor, U0126 (48.3 8.6), the p70s6K inhibitor, Rapamycin (57.1 4.9%) and by the GLP-1 receptor antagonist exendin-9-39 (57.3 3.8). GLP-1 protects the myocardium against ischaemic - reperfusion injury when given throughout ischaemia - reperfusion or when given just five minutes prior to the onset of reperfusion or as a preconditioning mimetic. To further elucidate the mechanism of GLP-1 mediated myocardial preservation we carried out Western blot studies examining the phosphorylation of components of the RISK pathway which showed an increase in the phosphorylation of BAD. The increased phosphorylation of the pro-death peptide BAD, confirmed the potential anti- apoptotic effect of GLP-1. In conclusion we have demonstrated for the first time that GLP-1 protects the rat myocardium against ischaemia-reperfusion injury, both in vivo and in vitro. GLP-1 appears to protect via the up regulation of specific prosurvival kinase pathways. This may represent a new therapeutic potential for this class of drugs currently undergoing trials in the treatment of non-insulin dependent diabetes.