[MORPHOLOGICAL BASES OF REORGANIZATION OF THE RAT CEREBELLAR CORTEX IN SENESCENCE].

A histological and immunohistochemical study of the cerebellar cortex of senescent Wistar rats (36-month-old) was performed in comparison to young rats (36-month-old).The cerebellar cortex of senescent animals typically showed degeneration of the Purkinje cells accompanied by immunochemically determined loss of the calcium-binding protein calbindin. These results suggest that presence of calbindin in the Purkinje cells is a criterion of functional activity of these neurons. The Purkinje cells degeneration is accompanied by lesion of the synaptophysin-containing basket cell networks, which is an indication of impaired function of inhibitory (GABAergic) axo-axonal synapses. During senescence significant rear-rangement is observed in the glomeruli of the granular layer of the cerebellum, which are the structures where primary afferent animals disintegrate suggesting of alteration in the transmission of information from the cerebrum to the cerebellum.

[Efficacy of mesenchymal stem cells intracerebral transplantation for the correction of age-related cerebral microcirculation alterations in rats].

Using a television-based vital microscopy method and immunohystochemical analysis, we have assessed the effect of intracerebral transplantation of syngeneic mesenchymal stem cells (MSC) on the brain cortex structure and the microcirculation in the pia mater of old rats. Using "open field" system, we have studied the effect of MSC transplantation on position-finding and discovery behavior of older animals. We have found that density of microvascular network of the pia mater increased ca. 1.9-fold in MSC recipients, compared to age-matched intact animals. Density of the arteriolar area of microvascular network of the pia mater increased ca. 2-fold. Reactivity of the newly formed arterioles was nearly equal to that of native microvessels. Intracerebral transplantation procedure itself was traumatic for brain cortex of rats, but it had no effect on the microcirculation in the contralateral hemisphere. Intracerebral transplantation of MSC did not improve locomotor behavi- or and emotional stage of old rats, did not increase their position-finding and discovery activity.

[Neuroglobin distribution in the human cerebellar cortex (an immunohistochemical study)].

Neuroglobin is a recently discovered heme-containing protein located predominantly in the mammalian brain. This paper for the first time presents the data on neuroglobin distribution in human cerbellum using immunohistochemistry. Neuroglobin immunoreactivity in the cerebellum was found in all the cases studied (n = 7), although its intensity varied. Distinct reaction was found in Purkinje cells and the areas of cerebellar glomeruli.

[The effect of intracerebral transplantation of mesenchymal stem cells on orientation and exploratory behavior of aged animals].

Male Wistar-Kyoto rats (12 and 22-24 months old) were intracerebrally transplantated with syngeneic mesenchymal stem cells (MSC). Three weeks or one year later, orientation and exploratory behavior of the same animals (not 2 years old) was assessed using an «open field¼ test. Older rats (22-24 months old) were inhibited in the basic behavioral acts in comparison to younger animals (2-3 months old). Transplantation of MSC did not cause improvement of an orientation and exploratory behavior of aged rats. Morphological and immunohistochemical analysis suggests intracerebral MSC transplantation led to the injury of the cortex of the ipsilateral hemisphere.

[Postnatal neurogenesis: cells identification and nomenclature].

Elaboration of the concept of neural stem cells at the end of the twentieth century resulted in the appearance of the multitude of new terms used for the designation of cells localized in the proliferative zones of the brain. Concurrently, a problem arose concerning the correlation of newly named cells, generally known elements of the nervous tissue, and classic views on its development. In the present study, an attempt is made on the basis of the literature data review and authors' own experience in the exploration of proliferative zones, to classify cell populations involved in the neurogenesis considering, primarily, the possibilities of their morphological and immunocytochemical in situ identification.

[Distribution of alpha-tubulin in the structures of rat forebrain].

The distribution of alpha-tubulin, the major protein of the microtubules, was studied using immunohistochemistry in the rat forebrain structures. The differential distribution of alpha-tubulin immunoreactivity was detected: high immunoreactivity was found in cingulate and pyriform cortex, olfactory tubercles and optic chiasm, while the weakest immunohistochemical staining was observed in caudatoputamen, superficial layers of septum, cingulum and areas around the third cerebral ventricle. Immunoreactivity in the nervous cells was distributed along perikaryon periphery and in the apical dendrite. It is suggested that the intensity of alpha-tubulin immunohistochemical reaction could reflect the functional state of the neurons.

[Advantages and disadvantages of zink-ethanol-formaldehyde as a fixative for immunocytochemistry and confocal laser microscopy].

The paper presents an analysis of authors' results obtained using fixation of various tissues in zink-ethanol-formaldehyde (ZEF). It was found that fixation in ZEF, in comparison with other methods of fixation, allowed to achieve higher sensitivity of immunocytochemical reaction for a large number of antigens studied and, in many cases, to avoid heat unmasking of antigens. It also provided high resolution of the images obtained using the fluorescent and confocal laser microscopy. However, the studies of antigens with small molecular mass revealed the antigen diffusion from the site of original localization. The data obtained suggest that fixation of a material in ZEF fixative is promising for both immunocytochemical studies, including those using the fluorescent and confocal laser microscopy, and general histological practice.

Morphological changes in rat hippocampus after brain injury.

We performed morphological analysis of the structure of rat hippocampus after ablation of the left sensorimotor cortex. Four experimental groups were formed: two control groups (intravenous and intracerebral injections of the culture medium) and two experimental groups (intravenous and intracerebral transplantation of MSC). Ten weeks after surgery, disturbed cytoarchitectonics and great number of dead neurons were found in all zones of the hippocampus in animals of the control groups. In animals receiving cell therapy, no pathological changes in the structure of the hippocampus were found: hyperchromatic neurons were absent and the cells had regular shape and closely adjoined to each other.

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.

[Analysis of morphological manifestations of the inflammatory reaction in wistar rat spinal cord in experimental model].

Morphological changes in the spinal cord of rats with different intensity of pathological symptoms were studied at the peak of the experimental encephalomyelitis development. Light-microscopical and immunohistochemical methods were used. Distribution of proliferating cell nuclear antigen (PCNA), astrocyte marker - glial fibrillar acidic protein (GFAP), and microglia and macrophage marker Iba-1, was studied. Heterogeneity in morphological manifestations of the experimental allergic encephalomyelitis was shown. Four typical patterns of morphological manifestations of the disease were demonstrated depending on the preferential involvement of pia mater, vessels, spinal cell nuclei or conductive tracts in the pathological process.

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.

Morphofunctional characteristics of the lumbar enlargement of the spinal cord in rats.

The topography of the lumbar enlargement of the spinal cord in rats was studied; an immunohistochemical method was used to determine the distribution of synaptophysin--a membrane protein of synaptic vesicles. Synaptophysin-immunoreactive structures were detected in the gray matter of all Rexed laminae, around most neurons and in the neuropil. Previously undescribed subpial synaptic contacts were detected immunohistochemically in the white matter and confirmed by electron microscopy. A non-myelinated component of the corticospinal tract, including axonal varicosities and synaptic contacts, was observed in the dorsal part of the white matter of the lumbar enlargement of the spinal cord.

[Morphofunctional characteristic of the rat distal spinal cord after its complete experimental section with subsequent treadbun training of the animal].

Motor activity of rats has been studied after complete experimental section of spinal cord at the lower thoracic level. A treadbun training performed one day after the operation has been shown to lead to the appearance of movement of hindlimbs and to restoration of function of support of the body weight. In our opinion, the key moment in initiation of locomotor movements is stimulation of foot. Morphoimmunohistochemical study (detection of nuclear protein of proliferation cells, synaptophysin, and glial fibrillary acid protein) of the lumbar enlargement has allowed revealing reorganization of motoneurons, interneurons, and afferent chain in the distal part of the sectioned spinal cord. In trained animals there are observed the normal structure of motoneurons and the appearance of aggregates of synaptophysin-immunoreactive structures lost after the operation.

[Mesenchymal stem cells-based therapy of brain ischemic stroke in rat].

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.

[Mesenchymal stem cells transplantation influences upon dynamics of morphological changes in rat brain after stroke].

Study of dynamic morphological changes if the brain after ischemic stroke is an important phase of pre-clinical trial of mesenchymal stem cell (MSC) therapy for this widespread disease. Experoments were carried out in inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture and labeled with vital fluorescent dye PKH26. Animals were subjected to middle cerebral artery occlusion (MCAO) followed by injection of 5 x 10(6) rat MSCs into the tail vein on the day of MCAO. Control group of animals received PBS injection (negative control). Animals were sacrificed in 1, 2, 3 and 5 days and in 1, 2, 4 and 6 weeks after operation. MSCs were revealed in the brain on the third day transplantation. They distributed around brain vessels in both the ipsilateral and contralateral hemispheres. This pattern of distribution remained unchanged during 6 weeks of observation. It was demonstrated that inflammation process and scar formation in the experimental group progressed 25-30 % faster than in the control group. MSC transplantation stimulated endogenous stem cell proliferation on the subependimal zone of lateral ventricles (subventrecular zone). What is more, MSC injection showed neuroprotective effect: almost all penumbra neurons in animals treated with cell therapy retained their normal structure, whereas in animals of control group penumbra neurons died or had signs of serious damage.

[Immunocytochemical demonstration of neuronal NO-synthase in rat brain cells].

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.

[Morpho-functional characterictic of rat lumbar spinal cord enlargement].

Topography and the distribution of synaptophysin-immunoreactive structures were studied in the rat lumbar spinal cord enlargement. Synaptophysin (synaptic vesicle marker) was found in the gray matter of all Rexed laminae around most neurons and in neuropil. Subpial synaptic contacts, that were not described previously, were found in the white matter by immunohistochemistry and their presence was confirmed by electron microscopy. They were localized directly beneath the astrocyte processes, that cover the spinal cord externally. In the dorsal portion of the white matter within the lumbar spinal cord enlargement, the unmyelinated synaptophysin-immunoreactive component of pyramidal tract was detected which included axonal varicosities and synaptic contacts.

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.

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.

Gaba- and serotonin-immunoreactive structures and ca(2+)-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 Ca(2+)-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.