The Langendorff perfused murine heart has become an increasingly important research model in cardiovascular physiology and pharmacology. However, the model remains relatively poorly characterised when compared with the widely employed rat preparation. The purpose of the research within this thesis was initially two-fold: 1) to characterise the functional and substrate-dependent properties of the murine model; and 2) to characterise the relationships between glycolysis, ischaemic tolerance and adenosine-mediated cardioprotection in the mouse. Initial studies, confirmed by simultaneous/subsequent work in other laboratories, revealed the frequent occurrence of regular cyclic oscillations in contractile function and coronary flow in glucose-perfused isovolumically contracting hearts. This phenomenon (labelled 'cycling') was unaltered by inhibition of ?-adrenergic receptors, prostaglandins, and nitric oxide synthase. However, A1/A2 adenosine receptor agonism did abolish the oscillations in flow and reduced contractile oscillations by 50%. Importantly, cycling was eliminated by addition of 50 IU/l insulin to perfusion fluid, or provision of 5 mM pyruvate as a co-substrate with glucose. These data suggest that functional 'cycling' in glucose-perfused murine hearts likely occurs as a result of a mismatch between substrate metabolism (energy supply) and myocardial energy demand. It may be that glycolysis with exogenous glucose is insufficient to ensure appropriate matching of myocardial energy supply and demand. For this reason, it is advisable to employ a co-substrate such as pyruvate in studies of murine hearts. Further studies performed within this thesis generally employ this co-substrate addition. Addition of pyruvate as co-substrate removes 'cycling' but is also known to inhibit/modify glycolysis, which may affect ischaemic tolerance and/or cardioprotection mediated by adenosine. Experiments throughout this thesis demonstrated that pyruvate-perfusion improved tolerance to both ischaemia (delayed time to onset of ischaemic contracture; TOC) and reperfusion (reduced diastolic dysfunction and cell death). The delay in TOC as a result of pyruvate-perfusion also suggests that contracture is not solely influenced by anaerobic glycolysis (as outlined in current paradigms). To test the relevance of glycolysis to ischaemic injury hearts were subjected to various forms of glycolytic inhibition. Glycolysis was inhibited by use of 10 mM pyruvate, (iodoacetic acid) IAA treatment, and glycogen depletion by pre-ischaemic substrate-free perfusion (all groups employing pyruvate as sole-substrate). Each form of glycolytic modification resulted in significant delays in TOC, in complete contrast to findings from other models and species. Glycogen depletion also reduced the peak level of contracture. These findings indicate that the mouse is either unique in terms of substrate metabolism and mechanisms of contracture (an unlikely possibility), or raise serious questions regarding current models of contracture development during ischaemia (theorised to be delayed by prolonging anaerobic glycolysis). Modification of glycolysis also altered post-ischaemic outcome, with pyruvate perfusion and glycogen depletion both enhancing functional recoveries. However, IAA treated hearts, despite near-identical ischaemic tolerance (ie contracture development) to pyruvate-perfused hearts, displayed very poor functional recovery, which was below that for all other groups. These data clearly reveal that blocking glycolysis improves tolerance to ischaemia (as evidenced by reduced contracture), provide evidence of dissociation of ischaemic injury or contracture from post-ischaemic recovery, and confirm the key importance of glycolysis in enhancing recovery from ischaemia. Since tolerance to ischaemia/reperfusion was shown to be glycolysis dependent, and since it has been theorised that adenosine protects hearts through modulating glycolysis, the relationships between glycolytic inhibition and adenosine-mediated cardioprotection was tested. In a number of studies, exogenously applied adenosine was shown to protect both glucose- and pyruvate-perfused hearts (supporting no dependence of adenosinergic protection on glycolysis). However, to more equivocally test the role of glycolysis effects of IAA were studied and were shown to markedly limit protection with adenosine. The effects of adenosine during ischaemia were abolished by IAA treatment, and effects on post-ischaemic recovery were reduced (but not eliminated). Similar results were acquired for protection with endogenous adenosine (using iodotubercidin to block adenosine phosphorylation). Collectively, these data reveal that adenosinergic protection during ischaemia depends entirely upon glycolysis while protection during reperfusion likely involves glycolysis dependent and independent processes. However, glycolysis is required for full recovery of function during reperfusion. Further studies assessed the involvement of glycolysis in cardioprotection afforded by transgenic A1 adenosine receptor (A1AR) overexpression. It was found that pyruvate-perfusion provided the same protection as A1AR overexpression, and the two responses (to pyruvate and A1AR overexpression) were not additive. Thus, it is probable that common mechanisms are targeted in both responses (likely glycolysis). Finally, the effects of adenosine and pyruvate on oxidant injury were studied, testing whether interactions between adenosine and pyruvate observed in prior work within this thesis could be explained by alterations in anti-oxidant responses. It was found that adenosine has quite profound anti-oxidant responses in glucose-perfused hearts, with very selective effects on markers of damage. Pyruvate also had some anti-oxidant effects but interestingly it reduced the anti-oxidant effects of adenosine. In conclusion, the work entailed within this thesis demonstrates that the isolated mouse heart model may possess unique properties and should be further characterised by potential users in order to improve its utility, and the reliability of experimental findings (chiefly when studying ischaemia-reperfusion). Other work within thesis demonstrates that modification of glycolysis is important in dictating recovery from ischaemia-reperfusion, and also impacts on adenosine-mediated protection (principally but not exclusively during ischaemia itself). The manner in which glycolysis is modified and contributes to protection remains unclear.

While previous research into the pathogenesis of ischaemic and reperfusion injuries has focussed on the cardiac myocyte, there is increasing evidence for a crucial role for coronary vascular injury in the genesis of the post-ischaemic phenotype [1-3]. Post-ischaemic vascular injury may be manifest as a transient or sustained loss of competent vessels, impairment of vascular regulatory mechanisms, and ultimately as the 'no-reflow' phenomenon (inability to sufficiently reperfuse previously ischaemic tissue despite the removal of the initial obstruction or occlusion). It is now appreciated that the earliest distinguishing feature of various forms of vascular injury (including atherosclerosis and infarction) is 'endothelial dysfunction', which is the marked reduction in endothelial-dependent relaxation due to reduced release or action of endothelial nitric oxide (NO). Importantly, vascular injury may worsen myocardial damage in vivo [4,5], significantly limiting tissue salvage and recovery. The pathogenesis of post-ischaemic vascular injury and endothelial dysfunction is incompletely understood, but has generally been considered to reflect a cardiovascular inflammatory response, neutrophils playing a key role. However, while blood-borne cells and inflammatory elements are undoubtedly involved in the 'progression' of vascular injury [6,7], accumulating evidence indicates that they are not the primary 'instigators' [8]. It should be noted that a wealth of controversial findings exists in the vascular injury literature and mechanisms involved remain unclear. Indeed, multiple mechanisms are likely to contribute to post-ischaemic vascular injury. Adenosine receptors are unique in playing a role in physical regulation of coronary function, and also in attenuating injury during and following ischaemia. While the adenosine receptor system has been extensively investigated in terms of effects on myocardial injury [9,10], little is known regarding potential effects of this receptor system on post-ischaemic coronary vascular injury. This thesis initially attempts to further our understanding of the role of adenosine in normal coronary vascular function, subsequent chapters assess the effect of ischaemia-reperfusion on vascular function, and adenosine receptor modification of vascular dysfunction in the isolated asanguinous mouse heart. Specifically, in Chapter 3 the receptor subtype and mechanisms involved in adenosine-receptor mediated coronary vasodilation were assessed in Langendorff perfused mouse and rat hearts. The study revealed that A2A adenosine receptors (A2AARs) mediate coronary dilation in the mouse vs. A2B adenosine receptors (A2BARs) in rat. Furthermore, responses in mouse involve a sensitive endothelial-dependent (NO-dependent) response and NO-independent (KATP-mediated) dilation. Interestingly, the ATP-sensitive potassium channel component predominates over NO-dependent dilation at moderate to high agonist levels. However, the high-sensitivity NO-dependent response may play an important role under physiological conditions when adenosine concentrations and the level of A2AAR activation are low. In Chapter 4 the mechanisms regulating coronary tone under basal conditions and during reactive hyperaemic responses were assessed in Langendorff perfused mouse hearts. The data support a primary role for KATP channels and NO in mediating sustained elevations in flow, irrespective of occlusion duration (5-40 s). However, KATP channels are of primary importance in mediating initial flow adjustments after brief (5-10 s) occlusions, while KATP (and NO) independent processes are increasingly important with longer (20-40 s) occlusion. Evidence is also presented for compensatory changes in KATP and/or NO mediated dilation when one pathway is blocked, and for a modest role for A2AARs in reactive hyperaemia. In Chapter 5 the impact of ischaemia-reperfusion on coronary function was examined, and the role of A1 adenosine receptor (A1AR) activation by endogenous adenosine in modifying post-ischaemic vascular function was assessed in isolated buffer perfused mouse hearts. The results demonstrate that ischaemia does modify vascular control and signficantly impairs coronary endothelial dilation in a model devoid of blood cells. Additionally, the data indicate that post-ischaemic reflow is significantly determined by A2AAR activation by endogenous adenosine, and that A1AR activation by endogenous adenosine protects against this model of vascular injury. Following from Chapter 5, the potential of A1, A2A and A3AR activation by exogenous and endogenous agonists to modulate post-ischaemic vascular dysfunction was examined in Chapter 6. Furthermore, potential mechanisms involved injury and protection were assessed by comparing effects of adenosine receptors to other 'vasoprotective' interventions, including anti-oxidant treatment, Na+/H+ exchange (NHE) inhibition, endothelin (ET) antagonism, and 2,3-butanedione monoxime (BDM). The data acquired confirm that post-ischaemic endothelial dysfunction is reduced by intrinsic A1AR activation, and also that exogenous A3AR activation potently reduces vascular injury. Protection appears unrelated to inhibition of ET or oxidant stress. However, preliminary data suggest A3AR vasoprotection may share signalling with NHE inhibition. Finally, the data reveal that coronary reflow in isolated buffer perfused hearts is not a critical determinant of cardiac injury, suggesting independent injury processes in post-ischaemic myocardium vs. vasculature. Collectively, these studies show that adenosine has a significant role in regulating coronary vascular tone and reactive hyperaemic responses via NO and KATP dependent mechanisms. Ischaemia-reperfusion modifies vascular control and induces significant endothelial dysfunction in the absence of blood, implicating neutrophil independent injury processes. Endogenous adenosine affords intrinsic vasoprotection via A1AR activation, while adenosinergic therapy via exogenous A3AR activation represents a new strategy for directly protecting against post-ischaemic vascular injury.

Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

Ischaemic stroke occurs as a result of a blockage in one of the brain’s arteries, leading to neuronal injury and death. Although stroke is a major cause of death and disability, there is no widely available treatment. Inflammation occurs in the brain and in the periphery following stroke, and both contribute to the ischaemic damage. Leukocytes such as neutrophils are key mediators of brain damage and inflammation, particularly in the presence of systemic inflammatory challenges such as interleukin-1 (IL-1). Tumor necrosis factor-stimulated gene 6 (TSG-6) is a potent inhibitor of neutrophil migration, and also modulates the immune response by dampening expression of cytokines and stabilising the extra-cellular matrix (ECM). Mesenchymal stem cells (MSCs) have shown immunomodulatory actions in many inflammatory conditions, and their benefit has often been attributed to the production of TSG-6. This work aimed to evaluate the potential of TSG-6 and TSG-6-expressing MSCs as therapies in cerebral ischaemia, and to investigate the expression profile of endogenous TSG-6 in response to stroke. Mice were subjected to middle cerebral artery occlusion (MCAo) followed by reperfusion. We investigated whether IL-1-induced acute brain injury after stroke is reversed by TSG-6, and long-term recovery was evaluated in mice treated with TSG-6 or MSCs. Functional outcomes were assessed, and brains were sectioned and stained for analysis of lesion volume, haemorrhagic transformation, blood-brain barrier (BBB) disruption and neutrophil infiltration. The expression profile of TSG-6 was evaluated in mice allowed to recover for 4h, 24h, 3, 5 or 7 days. TSG-6 expression was determined by quantitative PCR and immunohistochemistry. Treatment with TSG-6 reduced IL-1-induced neutrophil infiltration into the striatum, and led to decreased BBB disruption and haemorrhagic transformation at 24h. Treatment with TSG-6 in the absence of a systemic inflammatory challenge had no significant effect on lesion volume, BBB disruption or haemorrhagic transformation after 7 days reperfusion, however thalamic neutrophil infiltration was significantly reduced. Treatment with human MSCs had no significant effect on behavioural or histological outcomes, however a heightened inflammatory response in MSC-treated mice suggested rejection of the cells by the murine immune system. TSG-6 expression peaked in the ischaemic hemisphere at 5 days post-reperfusion, and was associated with astrocytes in the glial scar surrounding the infarcted tissue. TSG-6 might be a promising therapy for the treatment of stroke in the presence of systemic inflammation. TSG-6-expressing MSCs might provide a broader therapeutic potential, and further work should optimise experimental conditions to prevent rejection of the cells. Expression of TSG-6 within the glial scar suggests a potential role in repair and recovery following ischaemic stroke. Modulating the peripheral immune response remains an attractive and accessible therapeutic target for the treatment of cerebral ischaemia.

Transient ischaemic attack (TIA) and minor stroke are associated with a high risk of recurrent stroke which can be predicted with a clinical rule and reduced with urgent treatment. Delay in accessing assessment and vascular risk factor modification should therefore be as short as possible, yet little is known in the UK about where patients seek care and the key influences of the time to contact healthcare services. However, using cohort studies to answer questions on healthcare access requires an assessment of how well such cohorts represent the wider population. Within the primary care consultation, the recognition of TIA is an important step in the care pathway as definitive treatment is initiated by specialists, yet TIA presentations are not common for individual GPs and difficulties in diagnosis may be due to low clinical exposure in routine practice or inadequacies in training. For patients where GPs suspect that TIA may be the cause of symptoms, inaccurate risk prediction and diagnosis of TIA can result in delay to definitive care and the existing tools for prognosis and diagnosis have been exclusively derived from clinical assessments in secondary care rather than primary care.

Le pronostic à long-terme actuel après un accident ischémique transitoire (AIT) ou un infarctus cérébral reste mal connu. Ainsi, j’ai déterminé les risques absolus à long-terme de récidive d’infarctus cérébral et d’évènement coronarien aigu après un AIT ou un infarctus cérébral et identifié les individus qui restent à haut risque absolu de récidive ischémique malgré la prévention secondaire actuelle.Dans une cohorte populationnelle contemporaine de patients ayant eu un AIT ou un infarctus cérébral (OXVASC study, 2002-2014), les risques absolus de récidive d’infarctus cérébral et d’infarctus du myocarde après un AIT/infarctus cérébral ont significativement diminué au cours de la période d’étude, très probablement du fait de l’amélioration de la prévention secondaire avec le temps. Cependant, malgré la prévention secondaire actuelle les sous-groupes de patients avec un antécédent de pathologie coronarienne et ceux sans antécédent coronaire mais avec un score Essen 4 sont exposés à un risque absolu de récidive d’évènement ischémique suffisamment élevé pour justifier d’une intensification du traitement. Néanmoins, les thérapeutiques de prévention secondaire futures nécessitent de permettre d’obtenir une réduction absolue du risque de récidive d’évènement ischémique importante pour compenser un risque augmenté d’effets indésirables ou de surcoût par rapport aux thérapeutiques actuelles. En effet, chez ces sous-groupes de patients à haut risque de récidive ischémique, une réduction plus intensive du taux de cholestérol avec les inhibiteurs des PCSK-9 parait tout à fait justifiée, cependant nous avons montré que le coût de ces traitements excède la limite du rapport coût-efficacité généralement accepté tandis que le bénéfice d’une majoration du traitement antithrombotique semble contrebalancé par l’augmentation du risque hémorragique extracrânien. / The current long-term prognosis after transient ischaemic attack (TIA) or ischaemic stroke is not well known. I aimed to determine the long-term absolute residual risks of recurrent stroke and coronary events after TIA or ischaemic stroke and identify individuals who remain at high absolute risk of recurrent ischaemic events despite current secondary prevention management.In a population-based cohort of consecutive TIA or ischaemic stroke patients (OXVASC study, 2002-2014), the overall absolute risks of recurrent ischaemic stroke and coronary events after TIA/ischaemic stroke have decreased over the study period, and are likely to be explained by the improvement of secondary prevention over time. However, despite current secondary prevention, the subgroups of patients with prior coronary artery disease and those without prior coronary artery disease but with an Essen score of 4 remain at sufficiently high absolute risk of recurrent ischaemic events to justify more intensive treatment. Nevertheless, future secondary prevention therapies would need to achieve a substantial absolute risk reduction to outweigh increased side effects or cost compared to current therapies. Indeed, in these high-risk subgroups, more intensive lipid-lowering therapies might be justified, but we showed that the total cost of PCSK-9 inhibitors seems to exceed the generally accepted cost-effectiveness threshold while benefit from increased antithrombotic treatment might be offset by the higher risk of extracranial bleeding.

Ischaemic stroke represent more than 80% of the total stroke instances. The location of the occlusion and the amount of brain tissue involved determines the effect of the stroke. Stroke can result in paralysis, memory loss, speech impairment and even a “lock-in” state. The amount of neuronal damage will determine whether these symptoms will be temporary or permanent. Stroke is deemed the second leading cause of death for individuals over the age of 60. According to the World Stroke Organization (WSO) every six seconds stroke claims a life, regardless of age or gender. Stroke is a global burden and the medical costs and disability related to stroke in America for 2010 was projected at almost $73.7 billion. The morphology of platelets, fibrin networks and erythrocytes as well as the differential white blood cell counts of 20 thrombo-embolic ischaemic stroke patients were investigated. Internal and external alterations were revealed in the platelets of stroke patients when compared to healthy controls. The decreased numbers of alpha granules in the platelets of the stroke patients indicated these platelets to be activated. Substances released by activated platelets promote fibrin network structure, specifically the formation of fibrin strands and accumulation of additional platelets. The fibrin network of healthy individuals consists of major, thick fibers with minor, thin fibers distributed between them. The fibrin network of stroke patients exhibited an abnormally layered and matted ultrastructure comprising of mainly thin, minor fibrin fibers packed closely together. An uncharacteristic circular morphology was also observed. These alterations in the fibrin network indicate the activated platelets to be actively involved in the thrombotic event. Neuronal damage related to stroke is also advanced by the vasoactive substances released by activated platelets. It can therefore be deduced that the morphology of the fibrin network is altered long before the concrete thrombotic event transpire. Large numbers of abnormal erythrocytes were distinguished in the blood of stroke patients. Among these abnormal forms of erythrocytes specifically codocytes, knizocytes, stomatocytes and echinocytes were identified. Abnormal erythrocyte forms were significantly increased in hypertensive patients and females independently. Alterations in the ultrastructure of erythrocytes disturb blood flow in the microcirculation and could possibly augment the ischaemic event. Inflammation is closely related to ischaemic stroke. An increased monocyte count and a reduced number of neutrophils were a significant feature among all the stroke patients of this study. Patients with hypertension as well as patients consuming aspirin on a daily basis showed the greatest influence on the observed differential white blood cell counts. These morphological alterations observed in the platelets, fibrin network and erythrocytes as well as the differential white blood cell count could be incorporated in an analysis regime that could probably indicate an impending thrombotic event. Therefore treatment could be initiated before the ischaemic event to possibly prevent the stroke. For future studies a larger study population, a more refined patient enrolment as well as the analysis of follow-up blood samples from patients could substantiate the above-mentioned findings and provide additional information concerning the thrombotic event and the effectiveness of treatment procedures. / Dissertation (MSc)--University of Pretoria, 2010. / Anatomy / Unrestricted