The aims of the present work were to identify the neuronal form of nitric oxide synthase (nNOS type I) in brain structures in rats by immunocytochemistry, to compare the results with data from histochemical reactions for NADPH-diaphorase, and to develop the optimal conditions for fixation for detecting nNOS. The product of the histochemical reaction was found to be located strictly in the cytoplasm. Immunocytochemical detection of nNOS showed that along with the cytoplasmic reaction for nNOS, the nuclei of some neurons and gliocytes were immunopositive, though the cytoplasm of these cells gave negative reactions for nNOS. Selection of the optimal fixation conditions for specimens and the dilution of the primary antibody allowed reductions in the intensity of nuclear nNOS-type reactions without affecting the specific reaction of the cytoplasm for nNOS. These data provide evidence that the best detection of nNOS in paraffin sections is obtained using immersion fixation in Carnoy's fluid or post-fixation in this solution after perfusion with 4% paraformaldehyde.

Mesenchymal stem cells (MSCs)-based therapy is a promising modern attempt to improve the recovery after stroke. Experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in cultute and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO), followed by injection of 5 x 10(6) rat MSCs into the tail vein 3 days after MCAO. Control group animals received PBS injection (negative control). Therapy results were estimated by the following parameters: behavioral and neurological testing, the brain injure area, the state of damaged region "border" zone and the vessels quantity in the "borden" area. It was shown that control group animals (PBS injection) did not restore their initial behavioral and neurological state, while the experimental group animals (MSCs injection) showed the same parameters as intact rats at 2-3 weeks after MCAO. The size of the damaged region in the control group was approximately 1.5 as large as in the experimental group. The damage in the experimental group was limited to neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. Small vessels quantity in the "border" regions was twine higher compared to control group and was approximately equal to an intact brain vessel number. Moreover, it was shown for the first time that after MSCs transplantation the vessels quantity in the neocortex and caudate putamen of contralateral hemisphere was twice as much as in control. We demonstrated that the MSCs transplantation definitely exerted a positive influence upon the brain tissue reparation after stroke.

In experiments on male Wistar-Kyoto rats we studied the distribution of mesenchymal stem cells in intact body and in the presence of a focus of acute tissue inflammation. In healthy animals mesenchymal stem cells were transplanted intravenously. In the second case we used various routes of transplantation of mesenchymal stem cells: intravenous, into tissue adjacent to the inflammation focus, and into intact lobe of the damaged organ (prostate gland). Three weeks after transplantation, mesenchymal stem cells labeled with a fluorescent dye were detected in the bone marrow and intestine of intact animals. In case of inflammation focus, mesenchymal stem cells after transplantation migrated into the bone marrow, intestine, and prostate gland. After injection into the adjacent zone, these cells formed a compact agglomerate at the site of injection. After transplantation into the intact lobe of the prostate gland the cells migrated towards the inflammation focus. Thus, transplantation of mesenchymal stem cells into the venous blood is less traumatic and led to more uniform distribution of cells in the damaged tissue.

The aim of the present study was to demonstrate a neuronal form of NO-synthase (type I NOS) in rat brain using immunocytochemical technique in comparison with the histochemical reaction demonstrating NADPH-diaphorase activity, as well as finding of optimal fixation protocol for the NOS demonstration. The histochemical reaction product was found to have strict cytoplasmic distribution. Immunocytochemical visualization of nNOS revealed not only cytoplasmic, but also nuclear localization of nNOS in some part of neural and glial cells without cytoplasmic nNOS staining. Searching for an optimal protocol of the material fixation and the primary antibody dilution, we succeeded in diminishing the intensity of intranuclear nNOS-like reaction while the specific cytoplasmic nNOS reaction was retained. The results obtained show that the optimal demonstration of nNOS in paraffin sections requires immersion fixation in Carnoy solution or posfixation in this solution following a perfusion with 4% paraformaldehyde solution.

The possibility of mesenchymal stem cell differentiation in the cardiomyocyte direction was studied on Wistar-Kyoto rats with myocardial infarction induced by ligation of the left coronary artery. In vitro treatment of mesenchymal stem cells with 5-azacitidine led to spontaneous contractions of about 15% cells in culture. Analysis of the expression of matrix RNA showed expression of fetal and functional markers of the myocardium in this cell culture. In vivo on day 21 after myocardial infarction and intravenous transplantation of mesenchymal stem cells into the periinfarction area, myocardial cells carrying donor label were detected. Immunohistochemical analysis showed that these cells were cardiomyocytes integrated into the myocardium. These cells can be a result of differentiation of transplanted mesenchymal stem cells or fusion of endogenous cardiomyocytes with exogenous mesenchymal stem cells.

We studied the effects of mesenchymal stem cells on rejection of xenogenic bone transplant. Experiments were carried out on Wistar-Kyoto rats transplanted chicken demineralized bone matrix populated or not populated with mesenchymal stem cells to the site of parietal bone defect. Histological analysis showed complete resorption of the xenogenic transplant not populated with mesenchymal stem cells by day 119. In experimental animals chicken matrix without signs of tissue inflammation was in fact completely retained over the entire period of observation. Numerous new vessels, mineralization fields, and areas of bone tissue formation were seen in the transplant.

The effect of intravenous transplantation of mesenchymal stem cells on the recovery of cognitive functions was studied in Wistar-Kyoto rats after brain stroke induced by occlusion of the middle cerebral artery in the left hemisphere. Analysis 2 and 5 weeks after stroke showed that transplantation of mesenchymal stem cells 3 days after middle cerebral artery occlusion reduced the area of cerebral injury, preserved cognitive functions, and decreased mortality in experimental animals.

The aim of the present work was to develop optimal protocols for immunocytochemical reactions for nuclear protein NeuN for light and laser confocal microscopy which avoid the thermal antigen demasking procedure, which degrades the state of the tissue and requires use of expensive adhesive-coated slices. Maximal antigen retention was obtained after fixation in zinc-formalin and Bouin’s fluid (maximum one day). Two protocols are proposed allowing the thermal demasking procedure to be avoided during detection of neuron marker NeuN on paraffin sections examined by light and confocal microscopy.