Open Access

Hintergrund: VPS ist ein bekannter Inhibitor der HDAC. In den zurückliegenden Jahren sind mehrere Publikationen erschienen, die VPS eine bedeutende Rolle in Bezug auf eine neuroprotektive Wirkung zugeschrieben haben. Jedoch sind in diesem Zeitraum auch gegenteilige Ergebnisse veröffentlicht worden. In bisherigen eigenen Voruntersuchungen sahen wir, dass VPS weder neuroprotektive noch zytotoxische Effekte hervorgerufen hatte. In der vorliegenden Arbeit untersuchten wir unter Verwendung von Dosierungen, die dem therapeutischen Wirkspiegel von VPS entsprachen, ob VPS die neurotoxische Wirkung von MPP+ inhibieren konnte. Hypothese: Die vorliegende Dissertation soll einen Beitrag zur Erweiterung des Verständnisses der Auswirkungen einer VPS-Gabe auf das Verhalten neuraler Vorläuferzellen liefern. Insbesondere sollte die postulierte neuroprotektive Wirkung von VPS gegenüber dem neurotoxischen Agens MPP+ evaluiert werden. Methoden: Die fmNPZ werden in einer feuchtigkeitsgesättigten Atmosphäre in einem Expansionsmedium propagiert. Die Differenzierung erfolgt auf PLL beschichteten Kulturplatten in P4-8F Medium mit ohne VPS, 100μg/ml VPS und 200μg/ml VPS für bis zu 96 Stunden. Zur Detektion neuroprotektiver Effekte von VPS verwendeten wir einen Zytotoxizitästest: neben der simultanen Gabe von MPP+ und VPS erfolgte eine 24-stündige Vorbehandlung mit VPS, bevor MPP+ appliziert wurde. Zur Auswertung der Tests kamen histochemische Verfahren (PI/bisBenzimid-Färbung) zur Anwendung. Zur Evaluation des Proliferationspotenzials der fmNPZ unter Anwendung von VPS nutzen wir immunhistochemische Verfahren nach Standardprotokollen unter Verwendung folgender Antikörper: Maus Anti-BrdU (Konzentration: 60μl 5mM Stocklösung/ml), Kaninchen Anti-Ki-67 (1:500) und entsprechende Floureszenz-gelabelte Sekundär-AK (1:500). Ergebnisse: 100μg/ml als auch 200μg/ml VPS - als Einzelsubstanz - bewirkten nach 96 Stunden Differenzierungszeit eine signifikante Zunahme an avitalen Zellen. MPP+ - als Einzelsubstanz - zeigte die erwartete konzentrationsabhängige signifikante Zunahme an nekrotischen Zellen. Nach Simultangabe von MPP+/VPS bewirkte VPS sowohl unter Expansions- als auch unter Differenzierungsbedingungen keinen signifikant neuroprotektiven Effekt gegenüber MPP+. Nach 24-stündiger Vorbehandlung mit VPS sahen wir unter Expansionsbedingungen nach Applikation von 200μg/ml VPS einen die Toxizität von MPP+ verstärkenden Effekt. Unter Differenzierungsbedingungen bewirkte erst die Zugabe von 60μM MPP+ und eine weitere gemeinsame Kultivierungszeit von 72 Stunden einen signifikanten Anstieg an avitalen Zellen. Die Proliferationskapazität der fmNPZ war nach Zugabe von 100μg/ml VPS nicht verändert. Schlussfolgerungen: Zur Klärung der Frage welche Effekte VPS auf das Verhalten prädopaminerger, neuraler Progenitorzellen auslöst, konnte diese Arbeit einen weiteren wichtigen Beitrag leisten. Das gesetzte Ziel konnten wir somit erreichen. Die Therapie neurodegenerativer Erkrankungen wie die des Morbus Parkinson besteht derzeit in einer rein symptomatischen Behandlung ohne die Progredienz der Erkrankungen deutlich verlangsamen oder gar stoppen zu können. Eine kausale Therapie wie zum Beispiel eine zellbasierte Ersatztherapie konnte bis dato noch nicht erfolgreich etabliert werden. Unsere Arbeit liefert einen weiteren Beitrag zum Verständnis der Wirkungen von VPS auf das Differenzierungs- und Expansionsverhalten prädopaminerger, neuraler Progenitorzellen. Unter Berücksichtigung des in dieser Arbeit verwendeten Versuchsaufbaus kann postuliert werden, dass VPS keinen neuroprotektiven Effekt auf die fmNPZ ausübt. In höherer Dosierung als die die dem therapeutischen Wirkspiegel von VPS entsprechen, konnte unter bestimmten Bedingungen ein zytotoxischer Effekt nachgewiesen werden. Diese Ergebnisse passen sowohl zu früheren Publikationen mit anderen Zellsystemen, die einen toxischen Effekt von VPS beobachtet hatten, als auch zu den Ergebnissen eigener Voruntersuchungen. Die gewonnenen Erkenntnisse sind unter dem Aspekt der zukünftigen Entwicklung Stammzell-basierter Zellersatztherapien als auch der pharmakologischen Beeinflussung der in vivo Neurogenese von Bedeutung, weil diese Erkenntnisse einerseits Wirkungen von VPS auf Ebene neuraler Vorläuferzellen aufzeigen und andererseits daraus folgend eine kritische Evaluation der Anwendung von VPS ermöglicht.

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.

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.

Age-related decline in interhemispheric connectivity between motor areas has been reported with both transcranial magnetic stimulation (TMS) and diffusion tensor imaging (DTI) measurements. However, not all studies were able to confirm these findings, and previous studies did not apply structural (DTI) and functional (TMS) measurements within each individual appropriately. Here, we investigated age dependency of the ipsilateral silent period (ISP) and integrity of fibers in the corpus callosum as operationalized by fractional anisotrophy (FA), using TMS and DTI, respectively, in 20 participants between 19 and 72 years of age. We found age-dependent increase for ISP, and decrease of FA, both indicating a decrease in interhemispheric inhibition, with a negative association between FA and ISP for the dominant hemisphere (r = 0.39, p = 0.043). Our findings suggest that aging leads to decline of interhemispheric motor connectivity, as evidenced in both structural and functional parameters, which should be taken into account when interpreting disease- or medication-related changes.