The main aim of this work was to analyse neuronal function and plasticity in the hippocampal formation after global ischemia in rats. Histological evaluations after global ischemia have shown selective death of CA1 pyramidal cells and no obvious damage in the dentate gyrus (DG). However, in vivo electrophysiological recordings of the population spike signal (PSA) in the DG revealed a significant and long-lasting impairment of this evoked response after global ischemia. The electrophysiological effects appeared as early as 1 day after the insult. Synaptic transmission (fEPSP) was much less influenced and did not show a significant decrease. Interestingly, in rats which showed no electrophysiological response at all, it was possible to induce a PSA by tetanization on day 10 post ischemia. In contrast to the in vivo results, electrophysiological recordings from post-ischemic ex vivo hippocampal slice-preparations showed no functional impairments at all. Further experiments were conducted according to the hypothesis that detailed immunohistochemical analysis would reveal morphological changes in the post-ischemic DG. In line with this hypothesis, a pronounced activation and proliferation of microglia/macrophages was detected and their temporal/spacial expression pattern was quantified. Early phase neurogenesis, detected by doublecortin (DCX) labelling was restricted to the neurogenic zones at the early post-ischemic times investigated. Unlike published data about increased neurogenesis at later post-ischemic times, ischemia reduced the overall number of DCX-positive cells in the DG 3 days after global ischemia. Astrocytes were activated all over the forebrain 1 and 3 days post ischemia but did not proliferate. In addition nestin was used as a marker for pluripotent neural stem cells. After ischemia, nestin was co-localized almost completely with the astrocytic marker GFAP. The expression of nestin on astrocytes is in line with the assumption that activated astrocytes may be source of nascent neurons. However, it is also possible that cells with the neural precursor marker nestin migrate from adventitial progenitor cells or the ependymal layer of the ventricle walls to the damage and differentiate into astrocytes. Using the newly developed method of Thallium-autometallography it is possible to trace the cellular potassium metabolism. This provides the opportunity to visualize and further characterize the functional changes which had been detected by electrophysiological methods. In rats with complete loss of the CA1 pyramidal cells, the stratum moleculare and the granular neurons of the DG had changed from a slight staining (controls) to a very intensive labelling. This is the first morphological evidence of the functional impairments in the granular neurons after global ischemia and it suggests that a pronounced hyperpolarisation may be the underlying mechanism of the loss of function in the DG post ischemia. Additionally a vertical laminar periodicity of the selective vulnerability in the CA1 layer was observed. The results of this work show that the CA1 pyramidal cell layer is not the only impaired region at an early time points after global cerebral ischemia. Furthermore a decrease of DCX-positive cells and an impairment of the granular neurons in the DG were observed. Additionally a massive increase of microglia/macrophages and nestin/GFAP-positive cells all over the forebrain were also detected. These results demonstrate the complexity of the different processes during global ischemia which should be paid regard to in search of endogenous regenerative strategies. The imaging of neuronal activity at the cellular level revealed extraordinary new insights into post-ischemic brain function.
Thallium-autometallographie, electrophysiology in vivo ischemia, neurogenesis, microglia