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Abstract: Ischemic stroke is an aging disease and causes high mortality or long-term disability. The reduced neurological recovery in aging is possibly associated with impairment of angiogenesis and non-specific enhancement of inflammatory reaction. To check this hypotheses, those events were compared within young and elder animals brain at day 14 following focal ischemic stroke. Moreover, it is of importance to investigate also the potential therapies of indomethacin for prolonging the therapeutic window using aged animal models. The focus of present study was on neurobiological and neurological differences between young and old rats modulated by indomethacin daily treatment beginning at four hours post-ischemic episode. The effectiveness of indomethacin treatment in young and elder rats was probed using immunohistochemistry, oligonucleotide microarray, Real Time PCR and neurological evaluation. Our results provide insight of several age-independent positive consequences of Cox non-specific inhibition by indomethacin including increased NeuN positive surviving neurons, reduced infarct volume and enhanced neuroprotective response of innate immune system evidenced by increased Iba1 and Anx3 immunoreactivities in moderately activated microglia in periinfarction. From gene level we observed in both age groups downregulation of Mdk and Cxcl1 chemokines, and Id3 transcription factor which might modulate inflammatory response and facilitate repair. Other several findings showed age-dependent drug effect. Indomethacin had reduced efficacy in aged ischemic brain. From a total of 34 genes differential regulated, we observed 43% in young and only 28% of genes in aged have tendency toward age-matched sham expression level. In aged rats, indomethacin is ineffective in inhibiting phagocytic activity which is probably due to no expression changes of several cytokines like Tnfá and Cxcl4. Also, at protein level we observed no change of lysosomal ED1 immunoreactivity under treatment. On the other hand aging is characterized by no expression changes of Plau, Timp1, Timp2 and Col18a1 after treatment resulting in no improvement of angiogenesis. In young rats, conversely, drug administration decreased phagocytic activity by downregulating several cytotoxic cytokines such as Tnfá and Cxcl4. Moreover, the observable decrease of proteases like MMP10, Plau and MMP inhibitor Timp2 employed in matrix remodeling together with downregulation of Col18a1 expression after treatment might sustain angiogenesis in young rat ischemic brain. Indomethacin improves the motor-sensory performance in ischemic stroke rats as compared with age-matched untreated animals. Young rats fully recovered while aged showed important recuperation but did not achieve the preoperative level. In view of all this, indomethacin treatment might be consider as adjuvant therapy following ischemic stroke, even if aging blunts the positive effect of indomethacin on altered angiogenic-related gene expression. Because of the small number of rats, the results obtained from this study show only a tendency to significance and that further studies with more animals need to be statistically validated before firmly conclusions can be drawn. KEY WORDS: indomethacin; aging; microglia; angiogenesis; gene expression; microarray; neurological recovery; reversible middle cerebral artery occlusion.
Der Hintergrund der vorliegenden Arbeit war, dass zwar mehrere epidemiologische Studien eine J- bzw. U-förmige Assoziation zwischen Alkoholkonsum und Gesamtmortalität bzw. der Mortalität und Morbidität von kardiovaskulären und cerebrovaskulären Erkrankungen gezeigt hatten, jedoch nur wenige Daten über eine Assoziation mit subklinischer Atherosklerose vorliegen. Ziel der Arbeit war, den Zusammenhang zwischen täglichem Alkoholkonsum und der Intima-Media-Dicke (IMD) der A.carotis an Teilnehmern der bevölkerungsbasierten Study of Health in Pomerania (SHIP) zu untersuchen. Die IMD ist nicht nur ein Indikator für subklinische atherosklerotische Läsionen in der Gefäßwand, sie ist darüber hinaus ein Surrogat-Marker für eine generalisierte Atherosklerose sowie für ein erhöhtes Risiko bezüglich kardiovaskulärer und cerebrovaskulärer Erkrankungen. Die Basiserhebung der SHIP ist eine Querschnittsuntersuchung, die nach Altersklassen zwischen 20 und 79 Jahren stratifiziert eine zufällig erhobene Bevölkerungsstichprobe aus den Städten Greifswald, Stralsund und Anklam sowie 29 umgebenden Gemeinden untersucht. Bei insgesamt 1230 Männer und 1190 Frauen, die jeweils älter als 45 Jahre waren, wurde die IMD der A. carotis communis mittels B-Bild-Sonographie gemessen. Der tägliche Alkoholkonsum wurde in Computer-untersützten Interviews anhand des Alkoholkonsums des Wochentags bzw. des Wochenendes vor dem Interviews ermittelt und geschlechtsspezifisch in Kategorien á 20 g bei Männern bzw. á 5 g bei Frauen eingeteilt. Die Verlässlichkeit der Angaben wurde anhand der CDT und GGT sowie des Lübecker Alkohol- und Abusus- Screening Tests (LAST) überprüft. Bei Männern kann die Assoziation zwischen der IMD der A. carotis communis durch einen J-förmigen Kurvenverlauf beschrieben werden, während hingegen bei Frauen keine signifikante Assoziation nachgewiesen werden konnte. Im absteigenden Schenkel der Kurve haben Studienteilnehmer mit zunehmendem täglichen Alkoholkonsum eine signifikante oder zumindest grenzwertig signifikante geringere IMD als Probanden, die keinen Alkohol trinken. Das Minimum der Kurve wird erreicht bei einem täglichen Alkoholkonsum von 61-80 g/d, während bei täglichem Alkoholkonsum von mehr als 80 g/d die Kurve wieder ansteigt. Der Kurvenverlauf bleibt nach Adjustierung im wesentlichen in seiner J-förmigen Konfiguration unverändert. Die Adjustierung erfolgte für die ebenfalls als Risikofaktor für Atherosklerose geltenden Co-Variablen: Alter, systolischer Blutdruck, Diabetes mellitus, Nikotinabusus, LDL-HDL-Cholesterin-Ratio und ungesundem Lebenswandel, definiert als ungünstige Ernährungsgewohnheiten und fehlende körperliche Betätigung in der Freizeit. Eine lineare Regressionsanalyse zeigt nach Adjustierung für die genannten Risikofaktoren einen statistisch signifikanten Abfall der IMD um 0,009 mm/Zunahme des Alkoholkonsums um 20 g/d. Diese Assoziation verliert ihre statistische Signifikanz, wenn eine weitere Adjustierung des Regressionsmodells für HDL-Cholesterin und Fibrinogen als Marker für inflammatorische Prozesse vorgenommen wird. Somit wurde gezeigt, dass bei Männern ein Alkoholkonsum invers assoziiert ist mit der IMD der A. carotis als Surrogat-Marker für generalisierte Atherosklerose bzw. eines erhöhten Risikos für kardiovaskuläre und cerebrovaskuläre Erkrankungen. Dabei liegt jedoch die Menge des täglich zu konsumierenden Alkohols deutlich oberhalb der Schwelle, bei der schwerwiegende alkoholbedingte Erkrankungen bzw. Organschäden zu erwarten sind.
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.