Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by Subject "Anoxemia"

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    Cell death in hyppxic injury : signaling mechanisms and dynamics in the decision making process
    (Stellenbosch : University of Stellenbosch, 2009-12) Loos, Benjamin; Engelbrecht, Anna-Mart; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseased myocardium, type I, better known as apoptotic cell death, which is characterized by cell shrinkage and chromatin condensation, type II, or cell death with autophagy, presents a morphology with intracellular accumulation of autophagic vacuoles and type III, better known as necrosis, is characterized by cellular swelling and rapid loss in cellular membrane integrity. However, recent literature strongly argues against rigid classifications in the context of cell death mechanisms but rather suggests to adopt a view of cell death as a dynamic and integrative cellular response. Furthermore, the contribution of autophagy in cell death or cell survival is still poorly understood. Therefore the aims of this study were twofold: (i) to characterize the contribution of each cell death type in context of the severity and duration of an ischaemic insult and (ii) to determine whether manipulation of the autophagic pathway affects the contribution of cell death and translates into protection of the heart. Rodent derived cardiac myoblast cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs of simulated ischaemia (SI) under hypoxic conditions in a humidified environment containing 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusion respectively. We employed a modified ischaemic buffer containing either 2-deoxy- D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult of mild (mild SI), moderate (moderate SI) and severe (severe SI) character respectively. We evaluated the contribution of each cell death mode using a combination of viability- and ATP assays. Molecular markers for each cell death process such as LC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescence techniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MA respectively, and similar parameters were evaluated after 2 hours of mild or moderate SI. Propidium Iodide exclusion and Fluorescence Resonance Energy Transfer (FRET) in combination with mitochondrial inner membrane depolarization were employed to assess the onset of cell death dynamically. Flow cytometry was employed to evaluate the degree of protection. In addition, the ATP levels and reactive oxygen species (ROS) were evaluated. Our results strongly indicate a differential induction of cell death, which is dependent on the severity and duration of the ischaemic insult. Mild SI led to the induction of autophagy and apoptosis, whilst moderate or severe SI induced both apoptotic and necrotic cell death without an indication of autophagy. Only mild SI, but not moderate and severe SI, resulted in an ATP surge. Moreover, our data provide direct evidence that increased autophagy delays the loss of cellular membrane integrity and delays caspase-3 activation as well as mitochondrial depolarization in ischaemic cardiomyocytes. Our results show a profound effect of increased autophagy on the onset of apoptosis as well as necrosis under simulated ischaemic conditions, providing cellular protection. This ATP surge observed during mild SI was abolished with increased autophagy. Furthermore, our results indicate a profound effect of autophagy on ROS generation. Under normoxic conditions, increased autophagy induced a significant decrease in ROS while the inhibition of autophagy significantly increased ROS generation. However, when increasing or decreasing autophagy prior to the ischaemic insult, ROS increased significantly in both scenarios. The results suggest that the severity of ischaemia determines the mode of cell death differentially. An increase in autophagic responsiveness and flux, as induced through rapamycin treatment, provides a selective advantage for tissue against injury, possibly by maintaining intracellular ATP levels through the provision of metabolic substrates. Autophagy is described as an inherent cellular mechanism v which affects the onset of cell death and exhibits protective effects in the ischaemic myocardium when upregulated prior to the ischaemic insult. The protective effect of increased autophagy was mirrored in the isolated perfused rat heart model, reflected by improved functional recovery during ischaemia/reperfusion.