Neural progenitor and hemopoietic stem cells inhibit the growth of low-differentiated glioma.

The effects of neural progenitor and hemopoietic stem cells on C6 glioma cells were studied in in vivo and in vitro experiments. Considerable inhibition of proliferation during co-culturing of glioma cells with neural progenitor cells was revealed by quantitative MTT test and bromodeoxyuridine incorporation test. Labeled neural progenitor and hemopoietic stem cells implanted into the focus of experimental cerebral glioma C6 survive in the brain of experimental animals for at least 7 days, migrate with glioma cells, and accumulate in the peritumoral space. Under these conditions, neural progenitor cells differentiate with the formation of long processes. Morphometric analysis of glioma cells showed that implantation of neural progenitor and hemopoietic stem cells is accompanied by considerable inhibition of the growth of experimental glioma C6 in comparison with the control. The mechanisms of tumor-suppressive effects of neural and hemopoietic stem cells require further investigation.

Reaction of oligoglia to spinal cord injury in rats and transplantation of human olfactory ensheathing cells.

In experiments on rats with lateral TVIII hemisection of the spinal cord and transplantation of ensheating olfactory cells, we studied structural changes at the lesion site and adjacent rostral and dorsal regions of the spinal cord. The state of oligodendrocytes and myelin fibers and motor function in experimental animal were analyzed. Open field testing (BBB test) showed that motor functions steadily increased (by 13% on average) within the interval from day 21 to day 53 after transplantation. Histological examination showed that groups of transplanted cells carrying human nuclear marker (HNu(+)cells) were still present at the lesion site 30 days after surgery. Some of these cells migrated in the rostral and caudal directions from the injection site to a distance up to 6 mm. At the initial period after hemisection, the number of oligodendrocytes (O4(+)-cells) in the immediate vicinity to the lesion site decreased 2-fold, but no significant changes in the number of neurons were found in the rostral and dorsal fragments of the spinal cord compared to the corresponding parameter in controls. Sixty days after transplantation, the cross-section area in the rostral part of the spinal cord at a distance of 3 mm from damage site increased by 15.3% compared to the control. The number of O4(+)-cells at the lesion site and in adjacent rostral and caudal parts of the spinal cord by 22.8% surpassed that in the control group. The number of remyelinated axons also increased. These findings suggest that the absence of pronounced structural changes in the rostral and caudal parts of the spinal cord compared to lesion site at early stages after damage and cell transplantation. At the same time, pronounced activation of oligodendrocytes in this region suggests their involvement together with Schwann cells into remyelination of regenerating axons, which can serve as a factor of partial restoration of motor functions after spinal cord injury.

Prevention of neurodegenerative damage to the brain in rats in experimental Alzheimer's disease by adaptation to hypoxia.

We report here studies addressing the possibility of preventing neurodegenerative changes in the brain using adaptation to periodic hypoxia in rats with experimental Alzheimer's disease induced by administration of the neurotoxic peptide fragment of beta-amyloid (Ab) into the basal magnocellular nucleus. Adaptation to periodic hypoxia was performed in a barochamber (4000 m, 4 h per day, 14 days). The following results were obtained 15 days after administration of Ab. 1. Adaptation to periodic hypoxia significantly blocked Ab-induced memory degradation in rats, as assessed by testing a conditioned passive avoidance reflex. 2. Adaptation to periodic hypoxia significantly restricted increases in oxidative stress, measured spectrophotometrically in the hippocampus in terms of the content of thiobarbituric acid-reactive secondary lipid peroxidation products. 3. Adaptation to periodic hypoxia completely prevented the overproduction of NO in the brains of rats with experimental Alzheimer's disease, as measured in terms of increases in tissue levels of stable NO metabolites, i.e., nitrites and nitrates. 4. The cerebral cortex of rats given Ab injections after adaptation to periodic hypoxia did not contain neurons with pathomorphological changes or dead neurons (Nissl staining), which were typical in animals with experimental Alzheimer's disease. Thus, adaptation to periodic hypoxia effectively prevented oxidative and nitrosative stress, protecting against neurodegenerative changes and protecting cognitive functions in experimental Alzheimer's disease.

Study of the efficiency of transplantation of human neural stem cells to rats with spinal trauma: the use of functional load tests and BBB test.

Human ensheating neural stem cells of the olfactory epithelium were transplanted to adult male rats immediately after contusion trauma of the spinal cord at T9 level rostrally and caudally to the injury. Voluntary movements (by a 21-point BBB scale), rota-rod performance, and walking along a narrowing beam were monitored weekly over 60 days. In rats receiving cell transplantation, the mean BBB score significantly increased by 11% by the end of the experiment. The mean parameters of load tests also regularly surpassed the corresponding parameters in controls. The efficiency of transplantation (percent of animals with motor function recovery parameters surpassing the corresponding mean values in the control groups) was 62% by the state of voluntary motions, 37% by the rota-rod test, and 32% by the narrowing beam test. Morphometry revealed considerable shrinking of the zone of traumatic damage in the spinal cord and activation of posttraumatic remyelination in animals receiving transplantation of human neural stem cells.

[Influence of the neural stem cell transplantation on the restoration of CNS functions in rats with cortical stroke].

Ischemic stroke was modeled in white pedigreeless rats by the superficial blood vessel devascularization in the sensorimotor cortex. The preparations of neural progenitors--rat embryonic neural stem cells (rENSC) and human olfactory epithelium-derived neural stem cells (hOENSC) and differentiated fibroblasts ("cell control") were transplanted at the perimeter of the devascularized region. These cells marked with vital tracer stayed alive in the brain parenchyma for at least 16 days. The monitoring of contralateral forepaw motor deficit during 8 weeks demonstrated that only rats with rENSC transplantation had the stable and significant improvement of performance in cylinder test and swimming test (forepaw inhibition test) in comparison to "cell controls" and rats without cell transplantation. The maximal difference in the relative values (the efficacy) was 25% to the end of the experiment. There was no difference in the indicators of vibrissae-elicited forelimb placing test between experimental groups. The methodological approach used makes it possible to broaden the study of mechanisms of neural stem cells' therapeutic effect in stroke.

[Prevention of the brain neurodegeneration in rats with experimental Alzheimer's disease by adaptation to hypoxia].

The study focused on a possibility of preventing brain neurodegeneration by adaptation to intermittent hypoxia (AH) in rats with experimental Alzheimer's disease (AD) modeled by injection of a neurotoxic bert-amyloid peptide fragment (Ab) into n. basalis magnocellularis. AH was produ- ced in an altitude chamber (4.000 m; 4 hours daily; 14 days). The following results were obtained after fifteen days of the Ab injection: (1) AH substantially prevented the memory impairment induced by Ab, which was determined using the conditioned avoidance reflex test; (2) the AH significantly restricted the enhanced oxidative stress, which was determined spectrophotometrically by thiobarbituric acid-reactive substance level in the hippocampus; (3) the AH completely prevented Ab-induced nitric oxide (NO) overproduction in brain, which was measured by tissue level of nitrite and nitrate; (4) pathologically changed and dead neurons (Niessle staining) were absent in the brain cortex of rats exposed to AH before the Ab injection. Therefore AH seems to effectively prevent oxidative and nitrosative stress thereby providing protection of brain against neurodegeneration and preservation of cognitive function in experimental AD.

Morphofunctional changes in the rat spinal cord after focal photothrombosis.

The aim of the present work was to study pathomorphological and functional changes after induced focal photothrombosis of blood vessels in the thoracic part of the spinal cord in rats. Neuron abnormalities characteristic of ischemia were seen at the focus of experimental photothrombosis and in the transitional zone, along with symptoms of impaired motor and pelvic organ function. The focal photothrombosis method can be used to model spinal cord ischemia for the development of pharmacological correction methods and the recovery of impaired sensorimotor functions.

[Morpho-functional changes in the spinal cord of rats after focal photothrombosis].

The aim of this study was to determine the pathomorphological and functional changes after the induced focal photothrombosis of blood vessels in the thoracic region of rat spinal cord. Neuronal damage, characteristic of ischemia, both in the focus of an experimental photothrombosis and in transitional zone, were detected together with the symptoms of motor function and pelvic organ failure. The investigated method of focal photothrombosis induction may be used for modeling of spinal cord ischemia in the development of methods for pharmacological correction and partial restoration of damaged sensomotor functions.

Spontaneous neural differentiation of stem cells in culture of human olfactory epithelium.

We studied differentiation of stem cells in dissociated cultures of olfactory epithelium. Staining with anti-nestin antibodies revealed stem cells in the primary monolayer culture of the olfactory epithelium from adult human. Proliferation of these cells during culturing in serum-containing medium in the presence of nerve growth factors FGF2 and NGF led to the formation of neurospheres freely floating in the medium or attached to the substrate. Further long-term culturing and cloning of dissociated cells from these neurospheres in media not containing nerve growth factors led to spontaneous neural differentiation of the olfactory epithelium stem cells. The cells with phenotypic signs of differentiated neurons were stained with antibodies against beta-tubulin and neurospecific enolase. Differentiated neurons formed diffuse and spatially organized neuronal networks. We hypothesized that factors triggering neural differentiation of olfactory epithelium stem cells are produced byastrocytes present in these cultures.

Homocysteine toxicity in organotypic cultures of rat retina.

Effects of homocysteine in toxic concentrations on retinal neurons were studied in vitro. In organotypic roller cultures of postnatal (8-12-day-old) and adult rat retina homocysteine caused multiple damage to neurons in the outer nuclear layer, in deep compartments of the inner nuclear layer, and ganglion cell layer.

Neuroprotective and antiamnesic effects of Semax during experimental ischemic infarction of the cerebral cortex.

Semax had a pronounced neuroprotective and antiamnesic effect during focal photoinduced ischemia of the prefrontal cortex. Intranasal administration of Semax for 6 days decreased the volume of cortical infarction and improved retention and performance of conditioned passive avoidance response.

Roller organ cultures of the retina from postnatal rats.

Whole retinas of 2-14-day-old rats were cultured in a roller device for 2-14 days. Floating retinas of 7-14-day-old rats formed hole spheroid structures (spheroids) with the wall completely retaining the linear structure and layer-by-layer cellular and fibrous architecture, including the outer nuclear, outer plexiform, inner nuclear, inner plexiform layers, layers of ganglion cells and nerve fibers. The retina obtained at earlier terms of development often formed folds, with pyknotic nuclei of dead neurons in their deep compartments. In organ cultures of the retina isolated from rats at early postnatal periods, rosettes were formed in sites of local injury to the outer nuclear layer and pigmented epithelium. Roller organ cultures can be used for in vitro studies of the development and experimental diseases of the retina.

Multipotent stem and progenitor cells of the olfactory epithelium.

In recent decades, a wide spectrum of fetal and embryonic stem and progenitor cells were used for cell therapy of diseases of the central nervous system, but the olfactory glial ensheathing cells exhibited certain advantages due to their biological properties and capacity to stimulate regeneratory processes in spinal injury. The therapeutic effect of a heterogeneous complex of olfactory epithelial cells is more pronounced; apart from glial ensheathing cells, this complex includes fibroblasts, Schwann cells, stem and progenitor cells of this structure. The use of minimally invasive methods for isolation of human olfactory epithelial tissue is important for clinical practice, because they provide cells for autologous transplantation and rule out graft rejection immune reaction and the risk of transmission viral infection and transfer of genetic defects, which can be associated with allotransplantation.

Culturing of specialized glial cells (olfactory ensheathing cells) of human olfactory epithelium.

A monolayer of dissociated glial cells of human olfactory epithelium was cultured in Petri dishes and 12-well plates using a polylysine-laminin substrate. Primary cultures were subcultured after 10-15 days. The cell cultures were analyzed by phase contrast microscopy at all stages of culturing. A cytological study involved histological methods (trypan blue staining) and immunocytochemical visualization of GFAP, nestin, and low-affinity nerve growth factor receptors. At the final stage of culturing (5 passages) the monolayer cultures included 2 types of cells: GFAP- and p75-positive glial cells and nestin-positive fibroblasts.

Characteristics of the development of the sensorimotor cortex in rats aged 7-8 days in roller cultures of free-floating brain slices.

The development and formation of the neuronal architectonics of organotypic structures of the sensorimotor cortex in rats aged 7-8 days were studied in roller cultures. Free-floating slices were cultured for 2-3 weeks. Serial paraffin sections of cultured tissue were stained with fast cresyl violet as described by Nissl. Initially planar sections of the cortex were found to change their configuration during the process of cultivation and were transformed into spherical structures, retaining the major histotypic features of cortical formations. Radially orientated pyramidal cells and fusiform neurons formed a cortical rudiment, not discriminated into layers, over the whole surface of the spherical tissue structures. In free-floating slices of the sensorimotor cortex of rats aged 7-8 days in roller cultivation, histogenetic processes continued, leading to the formation of histotypic cortical structures similar to the phylogenetically more ancient allocortical formations of the forebrain.

Organotypic cultures of free-floating slices of human embryo medulla oblongata.

The aim of the present work was to produce organotypic cultures of human embryo brain tissues, as needed for investigation of the effects of serum factors on mental diseases and their pharmacological treatment. Cultures were made using slices of medulla oblongata from human embryos aged 9-10 weeks, obtained from medical abortions. Free-floating slices were cultured using a modified roller method for four weeks. Light and electron microscopy, along with immunocytochemistry, demonstrated that cultured slices showed differentiation and growth of neurons, astro- and microgliocytes, with formation of the synaptic contacts and glioneuronal interactions typical of organotypic cultures. However, along with differentiated neurons and glial cells, there was some persistence of undifferentiated cellular elements with signs of stem cells and neuron and gliocyte precursor cells. These experiments showed that organotypic roller cultivation of free-floating slices of human embryo brain tissue can be used to study the processes of neuron and gliocyte development, the mechanisms of neurotoxicity and neuroprotection using a variety of morphological (including stereological) and biochemical study methods.

Roller organotypic cultures of postnatal rat retina.

Floating retinal sections from 7-12-day-old rats form ball-shaped retinal bodies during roller culturing. Histological studies of serial sections of retinal bodies showed that their outer surface is formed by the retina completely retaining organotypic cytoarchitectonics. Some retinal bodies have laminar structure consisting of several layers of the retina. At the initial stages of culturing some retinal bodies contain a cavity, which later is completely obliterated due to the growth of axons of ganglion cells and migration of glial cells and fibroblasts. This study demonstrated the possibility of long-term survival, differentiation, and in vitro axonal regeneration of ganglion cells, the main retinal efferent neurons, which can provide the basis for investigation of pathology and drug correction of injuries and stimulation of regeneration of these cells in experimental glaucoma models.

[Developmental characteristics of the sensorimotor cortex of 7-8 days old rats in the roller cultures of free-floating brain slices]. / Osobennosti razvitiia sensomotornoi kory 7-8-dnevnykh krys v rollernykh kul'turakh svobodnoplavaiushchikh srezov mozga.

Development and formation of neuronal architectonic of organotypic structures of sensomotor cortex of 7-8-day-old rats was studied in roller cultures. Free-floating cortical slices were cultured for 2-3 weeks. Serial paraffin sections of cultured tissue were stained with cresyl violet fast using Nissl's method. It was shown that during cultivation cortical slices changed their initial flat configuration and transformed into spherical bodies that retained main histiotypic features of cortical formations. Radially oriented pyramidal and fusiform neurons formed cortical structure that was not subdivided into individual layers and covered the whole surface of spherical tissue bodies. It is concluded that histogenetical processes were continuing in free-floating slices of sensomotor cortex of 7-8-day-old rats during roller cultivation. They result in formation of histiotypic cortical structure similar to phylogenetically more ancient allocortical formations of the forebrain.

Stimulatory effect of green tea extract on the growth of neurites in the rat spinal ganglion culture.

Addition of green tea extract in concentrations of 0.004-0.006% to the nutrient medium markedly stimulated the growth of spinal ganglion neurites of 1-2-day-old rats.