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Aging is a risk factor for stroke. Animal models of stroke have been widely used to study the pathophysiology of ischemic stroke, which in turn helped to develop numerous therapeutic strategies. Despite the considerable success of therapeutic strategies in animal models of ischemic stroke, almost all of them have been proved to be unsuccessful in the clinical trials. One of explanation is that data obtained from young animals may not fully resemble the effects of ischemic stroke in aged animals or elder patients, causing the discrepancy between animal experiments and clinical trials. To investigate these differences with regard to age, pathway specific gene arrays were used to identify and isolate differentially expressed genes in periinfarct following focal cerebral ischemia. The results from this study showed a persistent up-regulation of pro-apoptotic and inflammatory-related genes up to 14 days post stroke, a 50% reduction in the number of transcriptionally active stem cell-related genes and a decreased expression of genes with anti-oxidative capacity in aged rats. Also, it was observed that at day 3 post-stroke, the contralateral, healthy hemisphere of young rats is much more active at transcriptional level than that of the aged rats, especially at the level of stem cell- and hypoxia signaling associated genes. Next, protein levels between young and aged post-stroke rats in periinfarct were compared using proteomic tools. Among others, AnxA3 was identified as differentially regulated protein, but the expression of AnxA3 has no significant changes in periinfarct between these two age groups at day 3 and 14. Different from periinfarct, a strong upregulation of AnxA3 at day 3 in young rats plus a strengthened increase of AnxA3 at day 14 in aged rats using immunohistochemical quantification indicated a delayed microglial accumulation in infarct core of aged rats, suggesting that quick activation of microglia in infarct core of young rats might be beneficial for recovery. Colocalization with established microglial marker demonstrated that AnxA3 as a novel microglial marker is implicated in the microglial responses to the focal cerebral ischemia. In addition, it was found that AnxA3 positive microglial cells incorporated more proliferating cell marker BrdU. Third, the expression, localization and function of several transport proteins were investigated in young rats following focal ischemic stroke. P-gp staining was detected in endothelial cells of desintegrated capillaries and by day 14 in newly generated blood vessels. There was no significant difference, however, in the Mdr1a mRNA amount in the periinfarct region compared to the contralateral site. For Bcrp, a significant mRNA up-regulation was observed from day 3 to 14. This up-regulation was followed by the protein as confirmed by quantitative immunohistochemistry. Oatp2, located in the vascular endothelium, was also up-regulated at day 14. For Mrp5, an up-regulation was observed in neurons in the periinfarct region (day 14). In conclusion, reduced transcriptional activity in the healthy, contralateral sensorimotor cortex in conjunction with an early up-regulation of proapoptotic genes and a decreased expression of genes with anti-oxidative capacity in the ipsilateral sensorimotor cortex of aged rats, plus the delayed up-regulation of AnxA3 positive microglial cells in infarct core may contribute to diminished recovery in post-stroke old rats. In addition, it was demonstrated in this study that after stroke the transport proteins were up-regulated with a maximum at day 14, a time point that coincides with behavioral recuperation. The study further suggests Bcrp as a pronounced marker for the regenerative process and a possible functional role of Mrp5 in surviving neurons. This study provided several evidences for the different responses of young and aged rats using a focal ischemic stroke model. Understanding the effect of age is crucial for the development of relevant therapeutic drugs.
Multiple sclerosis (MS) and stroke share a number of mechanisms of neuronal damage. In both cases the balance between neurodestruction and neuroprotection appears modulated by the function of the adaptive immune system. MS is a chronic inflammatory disease of the central nervous system (CNS), leading to permanent disability. It seems certain that an autoimmune response directed against the CNS is central to the pathogenesis of the disease. While these CNS-specific T cells are activated in MS patients, they are inactive and naive in healthy. Therefore it is believed that an activation of autoreactive T cells by cross-reactivity with pathogens occurs outside of the CNS. In consequence T cells express adhesion molecules and proteinases which enable them to cross the blood-brain barrier. In stroke, however, the blood-brain barrier is disturbed in its integrity caused by the decreased blood flow. Cells can freely migrate from the periphery into the brain. CNS autoreactive cells from the periphery can be activated within the CNS and thus contribute to further tissue damage. While the local autoimmune response remains temporary in stroked brains, it is chronically destroyed in MS. The differences between the underlying mechanisms are not understood. This thesis investigated T cell responses in Multiple Sclerosis in response to the therapeutics Mitoxantrone and IFN-b. The induction of a TH1 to TH2 cytokine response appears to be a shared mechanism of action between both therapeutic agents. Primarily the post stroke immune response was investigated. Patients developed a stroke induced immune suppression characterized by monocytic dysfunction and lymphocytopenia explaining the high frequency of post stroke infections. Moreover early post stroke predictors of subsequent infections, like the CD4+ T cell count, were identified. The T cell response of stroke patients appeared primed to proinflammation and unsuppressed after mitogen stimulation. A detailed understanding of post stroke immune alterations may offer new avenues of intervention to improve the clinical fate of stroke victims. In addition, such knowledge could also further our understanding of Multiple Sclerosis, because, while increasing the infection risk, the dampening of the immune system could have an important protective function, if it limits autoimmune brain damage triggered by the massive release of brain antigens during stroke. If these two pathways could be modulated separately it would create the opportunity to develop distinct therapeutic approaches that inhibit autoimmunity and strengthen antibacterial defenses. To further delineate these mechanisms it is crucial to investigate the role of the innate immune system as compared to the adaptive immune system in stroke induced immune suppression.
Studies of stroke in experimental animals have demonstrated the neuroprotective efficacy of a variety of interventions; however, most such strategies have failed to show clinical benefits in aged humans. One possible explanation for this discrepancy between animal and clinical studies may be the role that age plays in the recovery of the brain following insult. For example, the poor functional recovery of aged rats after stroke may be caused by a decline in brain plasticity. Although the incidence of ischemic stroke increases dramatically with advancing age, relatively few studies have been conducted on aged animals, which would mimic most closely the context in which stroke occurs in humans. We have shown that, at one week following stroke, there was vigorous expression of MAP1B and its mRNA, as well as MAP2 protein, in the border zone adjacent to the infarct of 3 month- and 20 month-old male Sprague Dawley rats. Hypothesis: The decline in brain plasticity is caused by an age-related decline in the upregulation of factors promoting brain plasticity (MAP1B, ßAPP) and an age-related increase in astroglial scaring and in the expression of neurotoxins such as beta amyloid. Methods: Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague Dawley rats. The functional outcome was assessed in neurobehavioral tests at 3, 7, 14, and 28 days post-stroke. At these time points, brains were removed and analyzed for markers of (i) brain plasticity (microtubule-associated protein 1B, MAP1B, secreted forms of fi-amyloid precursor protein); (ii) neurogenesis (BrdU-positive cells, doublecortin, nestin); (iii) neurotoxicity (B-amyloid aggregates); (iv) inflammation (microglia, astrocytes, oligodendrocytes, endothelial cells). Results: (1) There was a non-significant tendency for blood pressure to be higher in old than in young rats. By post-stroke day 3 the infarct volume covered about 15% of the cortical neurons in young and 28% in aged rats. By day 7, infarct volumes were roughly equal in the two age groups. (2) Cell counting showed increases in the number of BrdU-positive cells in the infarcted area of old rats at day 3 post-stroke. This increase became even more dramatic at day 7 post-stroke in aged rats. There was no significant contribution of apoptosis to cell death. (3) Behaviorally, young rats recovered gradually and reached a maximum of 90% of baseline performance at day 14, post-stroke while the aged rats recovered only to a maximum of 70% of pre-surgery performance by week 2 post-stroke, and remained at that level. (4) The temporal pattern of recovery correlated well with the expression of growth-associated phenotype of ßAPP as well as with MAP1B accumulation in varicosities along axons (an indicator of growth) in cortical areas affected by stroke and was at maximum between days 14 to 28 in young rats. In contrast, aged rats showed delayed (day 28) and reduced axonal remodelling as well as a delayed (day 28) expression of growth-associated ßAPP. Instead, the neurotoxic carboxy-terminal form of ßAPP steadily accumulated over time and reached a maximum at day 14 in aged rats as compared to 28d for the young rats. Nestin, a marker for immature neurons, overlapped with BrdU-labelled cells at day 7 post-stroke in corpus callosum and at the infarct border in both young and aged rats, suggesting increased stroke-induced neurogenesis. (5) In young rats there was a gradual activation of both microglia and astrocytes that peaked by days 14 to 28 with the formation of a glial scar. In contrast, aged rats showed an accelerated astrocytic and microglial reaction that peaked in week 1 post-stroke. We also noted a strong activation of oligodendrocytes at early stages of infarct development in all rats that persisted in aged rats. Evolution of astrocytic and microglial reactivity closely paralled the time course of scar formation in both young and aged rats and coincided with the stagnation in the recovery rate of aged rats. Conclusions: The time course of functional recovery in young rats correlated well with the expression of plasticity proteins such as MAP1B and ßAPP while an early and persistent expression of the neuro toxic fragment AB in conjunction with a delayed expression of MAP1B and ßAPP may impede functional recovery in aged rats. The results also suggest that a temporally anomalous glial reaction to cerebral ischemia in aged rats leads to the premature formation of scar tissue that impedes functional recovery to stroke.