Contrary to previous beliefs, the brain has a potential to regenerate itself. Neurogenesis, i.e. the cell division of neuronal precursor cells in the adult brain, and their subsequent differentiation to neurons, has been observed particularly in two areas of the brain, the subgranular zone [SGZ] of the dentate gyrus and the subventricular zone [SVZ] 1 . Neurogenesis appears to be stimulated by various noxious insults, e.g. ischemia, seizures, suggesting that it is part of an endogenous repair mechanism of the brain. However, stress, e.g. inflammation, radiation, can also disrupt neurogenesis2, 3, and the overall significance of the phenomenon in relation to brain damage remains unclear. We investigated the differential effects of a well defined focal ischemic insult on neurogenesis in two remote germinal areas of murine brains.
Methods
Anesthetized male C57/Bl6 mice (n=4 per group) were subjected to 90-min of MCAO using the intraluminal filament technique. Continuous Laser-Doppler measurements verified ischemia and reperfusion. Body temperature was monitored and controlled to physiologic levels throughout the procedure. During reperfusion, animals were pulse-injected with the thymidine analogon BrdU (50 mg/kg; 3x/d) 24 hr before sacrifice. At designated times (48hr, 72hr, 96hr after MCAO) animals were transcardially perfused using 4% paraformaldehyde, brains were carefully removed and further processed for cryosections (40μm). Free-floating sections were immunostained using antibodies against BrdU and various cellular markers (doublecortin, Tuj1, NeuN, GFAP). Cy2- and Cy3-labeled secondary antibodies allowed evaluation of cell proliferation/maturation using confocal microscopy and digital image analysis (Image J, NHI). Differences in cell proliferation between the survival times were determined by ANOVA.
Results
With the applied insult, cell proliferation (BrdU+ cells) in ipsilateral SGZ increased continuously, beginning 48hr after MCAO to more than six-fold at 96hr (382±87/mm2 at baseline vs. 2092±1189/mm2 at 96hr, p<0.05). At the same time, the contralateral SGZ did not show a significant increase in BrdU+ cells. The majority of the proliferating cells were positive for doublecortin, but negative for any of the other markers used, thus representing newborn neurons. Cell proliferation in SVZ, in contrast, showed an opposite pattern: in ipsilateral SVZ cell proliferation progressively declined to about 50% of baseline during the first 96hr after MACO (7033±60/mm2 at baseline vs. 4076±118/mm2, p<0.05). The contralateral SVZ showed 25% more BrdU+ cells at 48hr than at baseline. Again, most BrdU+ cells were doublecortin co-labeled.
Conclusion
The cell proliferatory response to focal cerebral ischemia appears to differ between SGZ and SVZ early after the insult. One possible explanation is the relative distance between respective germinal areas and the infarct core. This may result in more severe stress and less trophic support in the SVZ, which is located closer to the infarct, after MCAO. Accordingly, the SGZ would show enhanced, and the SVZ suppressed proliferation. Different levels of cellular stimulation/stress after brain ischemia seem to have opposite effects on the neuroregenerative response to ischemia. This observation has relevance for future therapeutic strategies to enhance neurogenesis after brain insults, and warrants further investigation.