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In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.

We investigated the temporal course of microglia activation in different brain regions after permanent middle cerebral artery (MCA) occlusion in mice and compared this microglia response with the appearance of apoptotic cells, Microglia activation and morphological changes of microglial cells were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse CR3 complement receptor. Cells showing morphological and biochemical features of apoptosis were identified using the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and light microscopy. As early as 30 min after onset of MCA occlusion activated microglia with hypertrophic cell bodies and stout processes were detected in the periphery of the ischemic lesion as identified by diffusion-weighted magnetic resonance imaging. A wider distribution and a progressive increase in the number of activated microglia was found with increasing time. Only few TUNEL-positive cells with apoptotic features were observed within the lesion area at 6 h after onset of cerebral ischemia. From 12 h after MCA occlusion onward a tremendous increase in the number of TUNEL-positive cells was found. Within the thalamus from 24 h onward microglia cells with few processes, irregular morphology and fragmented appearance were detected. Microglia activation in the thalamus progressed up to 4 weeks after MCA occlusion, but had declined after 90 days. Neuronal degeneration in the thalamus as determined by anti-neuronal nuclei immunohistochemistry progressed from 6 days after MCA occlusion onward. Only a few TUNEL-positive cells were found in the thalamus. In summary, microglia activation both in the primary cortical lesion area and in the secondarily affected thalamus preceded the manifestation of tissue injury. These observations encourage further studies on the role of microglia in focal cerebral ischemia.

We investigated the effects of 30-min heat shock on survival, DNA degradation, and nuclear morphology of primary rat cortical and hippocampal neurones. In cell cultures which were grown for 8 days in vitro (DIV), only a small portion of neurones showed apoptotic morphology after heat shock of 45 degrees C and typical DNA laddering was not detectable, despite the fact that nearly 50% of the neurones died within 24 h. The majority of the neurones presumably died by necrosis, as indicated by random DNA degradation. In neuronal cultures grown for 15 DIV, heat shock, however, resulted in DNA laddering, occurrence of apoptotic bodies and DNA strand breaks, typical of apoptosis. In these cultures, about 50% of the neurones showed apoptotic morphology following exposure to 45 degrees C in TUNEL and acridine orange staining, whereas glia were not affected in vitality. In addition we were interested whether the highly inducible member of the heat-shock protein family, HSP72, would be induced in apoptotic cells. Double staining for HSP72 and TUNEL revealed concomitant HSP72 induction and occurrence of DNA degradation only in very few neurones in 15-DIV cultures, which were growing adjacent to astrocytes. A clear association of the degenerative process and HSP72 expression, therefore, could not be established. These results demonstrate that environmental stress, such as heat shock, can induce apoptotic death in aged primary cultured neurones. The differentiation state and/or the presence of glial cell elements in the cultures appears to be an important factor for the occurrence of apoptotic features in cultured neurones.

Following transient forebrain ischemia selective and delayed neuronal degeneration occurs in the CA1 sector of the hippocampus. It is presently unclear whether this cell death is related to programmed cell death (PCD), which occurs in neurons during development of the CNS. Recently, the expression of various genes, such as c-fos, c-jun mkp-1, cyclin D1, and hsp70 was found to be associated with PCD in model systems. We and others have described that these genes are also upregulated in the hippocampus following ischemia. Most notably, c-fos, c-jun, and hsp70 are expressed specifically in CA1 neurons at survival times shortly preceding cell degeneration in rat models of global ischemia. In addition, the gene products could be detected by immunohistochemical methods, despite a general impairment of protein synthesis. These finding are especially relevant, since recent report suggests a functional role for Fos family proteins and c-jun in PCD in neurons of the superior cervical ganglion. These results could be indicative for the occurrence of a PCD-related program in CA1 neurons ad corroborate several other lines of evidences, such as occurrence of DNA fragmentation. Clearly, further studies are necessary to elucidate the functional role of the gene inductions following ischemia in vivo.