The dissertation is dedicated to investigate of regenerative effects at coculturing of stem cells with neural tissue after ischemic injury in vitro. Stem cells are a remarkable cell type, mainly because of the two key properties they possess: unlimited proliferation and unlimited differentiation. Stem cells can be maintained in culture for an extended amount of time, perhaps even indefinitely. It has been shown that the experimental oxygen-glucose deprivation (OGD) led to appearance of damaged pyramidal neurons and reactive gliosis in CA1 area of the hippocampus. The intensity of neurons and astrocytes immunoreactivity and their localization in the hippocampal CA1 area were depended significantly on the post-ischemia period. On the 3rd day after OGD and grafting, the GFP+ neural stem cells (NSCs) have been integrated into hippocampal neuropil, their shapes were changed - from roundish to ramified with radial processes. Double immunohistolabelling of slices against GFP (marker of transplanted stem cells) and NeuN (neurons) at that term after grafting didn't revealed colocalization of mentioned markers, indicating the absence specific proteins of mature neuron in the grafted cells. On the 7th day of the observation the shape of GFP+ NSCs has changed even more. We observed appearance colocalization of GFP and NeuN markers, which allowed to assume that the GFP+ NSCs didn't only integrated into the recipient tissue, but also differentiated to mature neurons. The number of GFP-positive neurons continue increased to the 14th and 21st days following NSCs grafting into organotypic slice cultures (OHCs). It should be noted, that GFP+ cells acquired the phenotype of mature neurons. In particular, these cells were characterized by well developed soma and branched dendrites. Also on the 14th and 21st days after OGD and transplantation the NSCs differentiated into glial cells - astrocytes and oligodendrocytes. Thus the GFP+ NSCs grafted into OHCs after OGD have been differentiated into neurons and glial cells. These results suggest that grafted cells could respond to signals of recipient tissue microenvironment, triggering cell differentiation and determining the direction of migration. Newly formed GFP+ neurons and glial cells were located exactly in the spaces between neurons of the injured hippocampal tissue. Thereby the grafted NSCs might likely replace the recipient tissue neurons being lost after experimental cerebral ischemic injury. In addition, we observed a decrease the level of glial cells activation: the vacuolization and the number of swollen astrocyte processes of hippocampal CA1 zone were reduced. In addition we have shown that bone marrow stem cells (BMSCs) also are able to confer beneficial effects after transplantation into neural tissue with ischemic injury. This effect is probably caused by the release of trophic factors, although the possibilities of replacement of dead neural cells by BMSCs are not excluded. The potential of differentiation of grafted BMSCs to neural direction was assessed for 14 days after the ischemic injury. At the 7th day after OGD and transplantation the BMSCs differentiated into microglial cells, and on the 14th day - as in microglial cells and in mature oligodendrocytes. These findings suggest that the transplanted stem cells respond to signals from the microenvironment of the injured tissue of the recipient, which in turn may trigger and regulate cell differentiation as well as to determine the direction of migration. Also we have investigate the effect of BMSCs at non-contact coculturing with ischemic neural tissue. Immunohistochemical analysis after 24 hours of BMSCs coculturing with OHCs after ischemia showed a significant reduction of caspase-3-positive nuclei of neural cells, as compared to those in ischemic damage without coculturing with BMSCs, and decrease of glial cells activation. After coculturing of hippocampal slices after OGD with BMSCs, cytoarchitectonic and types of neural tissue defined cells have been retained. Neurons were located tightly and compactly in the CA1 area. Thus coculturing of the OGD-treated slices with stem cells of different origin significantly improve the morphological status of the ischemic neural tissue. Stem cells in our model have shown neuroprotective effects. The direct mechanism of stem cells action in ischemic brain is still unknown. But we assume that such an improvement of morphological and functional state of the ischemic tissue takes place through activation of neurogenesis, synaptogenesis and neuroprotection due to growth factors. Key words: hippocampal organotypic slice culture (OHCs), oxygen-glucose deprivation (OGD), neural stem cells (NSC), bone marrow stem cells (BMSCs), cocultivation, immunohistochemical staining.
