E. G. Gilerovich

Mesenchymal stem cells transplantation could be beneficial for treatment of experimental ischemic stroke in rats

Cell therapy is prospective, modern attempt to ischemic stroke treatment. It has been being widely worked out recently. We suggest mesenchymal stem cells (MSC) as a cell therapy agent in the therapy of this disease. Experiments were carried out in inbred male Wistar-Kyoto rats. Animals were subjected to middle cerebral artery occlusion (MCAO). MSCs were isolated from rat bone marrow, expanded in culture and labelled with vital fluorescent dye PKH-26. Then 5 x 10(6) cells were injected into the tail vein on the day of MCAO and three days later. Control group animals received PBS injection (negative control). Cognitive function restoration was estimated by Morris Water Maze testing during 6 weeks after MCAO. Animals were sacrificed 1, 2, 3, 5 days and 1, 2, 4 and 6 weeks after operation. Intravenous MSC transplantation decreased post-operation mortality and benefited behavioural and neurological recovery. Experimental groups animals revealed changes in aseptic inflammation processes which were completed faster comparing to control group. That effect correlated with accelerated glial scar formation. Reduction of the infarct volumes and such post-stroke after-effects as border zone gliosis and liquor cysts formation accompanied by increased angiogenesis and subventricular zone cells proliferation were shown after cell therapy. The obtained results referred to both cell therapy groups. Thus, MSC injection benefited post-stroke rehabilitation irrespective of transplantation time. However, further investigation should be carried out in order to find out the mechanism of their action.

Immunocytochemical detection of brain neurons using the selective marker NeuN.

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.

Advantages and Disadvantages of Zinc-Ethanol-Formaldehyde as a Fixative for Immunocytochemical Studies and Confocal Laser Microscopy

We present here an analysis of our own results obtained by fixing various tissues in zinc-ethanol-formaldehyde (ZEF). As compared with other fixation methods, fixation in ZEF was found to provide greater sensitivity for immunocytochemical reactions for many study antigens, in many cases also allowing thermal demasking of antigens to be avoided and giving higher image quality using fluorescence and confocal laser microscopy. However, studies of low molecular weight antigens showed diffusion of antigens from their initial locations. These data lead to the conclusion that fixation of specimens in ZEF has potential for use both in immunocytochemical studies, including use of fluorescence and confocal laser microscopy, and in general histological practice.

Immunocytochemical detection of neuronal NO synthase in rat brain cells.

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.

Distribution of Neuroglobin in the Human Cerebellar Cortex (an immunohistochemical study)

Neuroglobin is a recently discovered heme-containing protein located mainly in the brain in mammals. We report here the first data on the distribution of neuroglobin in the human cerebellum, obtained from immunohistochemical studies. Reactions for neuroglobin were seen in all the cases studied (n = 7), though its intensity varied. Clear reactions were seen in Purkinje cells and in the cerebellar glomeruli.

Effect of transplantation of mesenchymal stem cells on neuronal survival and formation of a glial scar in the brain of rats with severe traumatic brain injury

We studied the effect of various methods of transplantation of mesenchymal stem cells on neuronal survival in rat brain 1 and 6 weeks after severe traumatic brain injury. It was found that intracerebral and systemic transplantation of mesenchymal stem cells improves neuronal survival in the piriform cortex of the contralateral hemisphere without affecting neuronal survival in the marginal zone of the traumatic cavity and amygdaloid nuclei. Intracerebral transplantation of mesenchymal stem cells increases the content of the astroglial component of the scar in the borderline zone of the traumatic cavity.

Gaba- and Serotonin-Immunoreactive Structures and Ca2+-Binding Protein in the Neocortex of the Reeler Mouse Mutant

The transmitter organization of anomalously formed neocortex was studied in reeler mutant mice by immunohistochemical studies of GABA- and serotoninergic structures and Ca2+-binding protein. GABAergic structures were identified in terms of the localization of glutamate decarboxylase (GDC) within them, this being an enzyme involved in GABA synthesis. The neocortex of reeler mutant mice was found to contain an unusual distribution of cells morphologically and chemically identical to Cajal—Retzius cells — beneath layer I rather than in its upper third, as seen in normal animals. GDC-immunoreactive label accumulated in the neuropil of the intermediate and deep layers, layer I containing only occasional granules. Serotonin-immunoreactive fibers did not form superficial or deep plexuses, as seen in normal animals, though they did reach their innervation targets. Thus, the anomalously formed neocortex which lacks the typical cytoarchitectonic organization, showed abnormalities in the structure of both intrinsic and projectional transmitter systems.

Modification of histogenetic processes in rat nervous tissue after administration of dexamethasone during prenatal development.

Experiments on rats using confocal microscopy and immunocytochemistry showed that single doses of the synthetic corticosteroid hormone dexamethasone to pregnant females during the period of elevated sensitivity of the developing fetal nervous system (day 13 of intrauterine development) led to modification of histogenetic processes in the brain, reflected as changes in its structural-functional characteristics during postnatal ontogenesis.

Immunohistochemical studies of the structural bases of inhibition in the central cerebellar nuclei in mice.

The distribution of glutamate decarboxylase-immunoreactive structures in the central nuclei of the cerebellum, its first afferent component, was studied at the light and electron microscope levels. Axosomatic, axodendritic, and axospinous synapses were detected, in which the presynaptic parts contained glutamate decarboxylase (GDC); this enzyme is involved in GABA synthesis. Additionally, these investigations revealed axoaxonal synapses in which both poles were GDC-reactive. The central nuclei of the cerebellum were found to have an intrinsic GABAergic system.

Detection of Glomeruli in the Human Cerebellum Using an Immunocytochemical Reaction for Synaptophysin and Confocal Laser Microscopy

The aim of the present work was to develop methods for the detection of complex synaptic groups (glomeruli) in the human cerebellum using an immunocytochemical reaction for synaptophysin. The protocols presented here yield high-quality preparations for light and confocal laser microscopy on which individual glomeruli and intraglomerular axon terminals can be clearly identified. These preparations are suitable for quantitative analysis.