V. A. Otellin

Glial Fibrillary Acidic Protein in Astrocytes in the Human Neocortex

Glial fibrillary acidic protein (GFAP), expressed in the brain by astrocytes, is one of the major immunocytochemical markers of these cells. The aim of the present work was to study the structure of GFAP-positive astrocytes in the human neocortex. Immunocytochemical confocal laser microscopy was used to characterize the main types of GFAP-immunoreactive astrocytes in the human neocortex. These were astrocytes of layer I, forming the superficial glial delimiting membrane, along with transmembrane astrocytes of layer I, with very long processes penetrating several layers of the cortex, astrocytes of the middle layers of the neocortex, mostly of the protoplasmic type and involved in forming perivascular delimiting membranes, and typical white matter fibrous astrocytes. These data may help unify assessments of histopathological processes seen in various types of gliosis in the CNS.

Formation of the neocortex in mice developing in conditions of prenatal serotonin deficiency.

We report here detailed studies of structural changes occurring in the mouse neocortex formed and stratified in conditions of prenatal experimental blockade of serotonin synthesis. Studies were performed using F1(C57BL/CBA) hybrid mice. Endogenous serotonin levels were decreased by exposure of the mice to parachlorophenylalanine, which inhibits the key serotonin synthesis enzyme tryptophan hydroxylase. Offspring brains were studied at 1, 5 and 10 days (n = 10–15 for each time point) of postnatal development. Controls consisted of intact animals at the same periods of development. These experiments showed that prenatal blockade of serotonin synthesis leads to impaired formation of all neocortical layers, impaired growth, development, and differentiation of neurons, and alterations in neuron shape and size. As postnatal development proceeded, significant numbers of neurons died in the brain structures of these animals.

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.

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.

Structural organization of astrocytes in the rat hippocampus in the post-ischemic period

The aim of the present work was to study the location and structural organization of astrocytes in the rat hippocampus, which contain immunoreactive glial fibrillary acid protein (GFAP) after ischemic damage to the brain after intracerebroventricular administration of the neuroprotective agent creatine and without treatment. Light microscopy and immunocytochemical methods were used to study the brains of 26 adult male Sprague-Dawley (Koltushi) rats, some of which were subjected to total cerebral ischemia (12 min) under anesthesia with subsequent reperfusion (seven days). Creatine was given to 11 animals intracerebroventricularly using an osmotic pump (Alzet Osmotic Mini-Pump). The results showed that GFAP-immunoreactive hippocampal astrocytes were concentrated in two main zones (the stratum lacunosummoleculare of field CA1 and the stratum polymorphae of the dentate fascia). The neuroprotective effect of creatine had the result that moderate ischemic damage to the hippocampus did not lead to changes in the zones containing activated astrocytes. The redistribution of GFAP-positive astrocytes in the post-ischemic period was associated with loss of pyramidal neurons in cytoarchitectonic field CA1. Complete loss of pyramidal neurons in this area of the hippocampus leads to a qualitatively new level of astrocyte activation - proliferation.

Transplantation of human embryonal nervous tissue into the spinal cord of adult rats.

human embryos, containing an anlage of the neocortex can be used as the initial material for transplantation [3, 4]. It has been placed into the cerebral cortex of adult rats, and the taking of the graft has been observed [5]. The time course of the proliferative processes and the differentiation of the cellular elements have been studied in this model, and it has been demonslrated that the prolonged existence, for six months and longer, of the trap. splant without signs of a cellular immune reaction is possible in a number of instances. The purpose of the present investigation was the elucidation of the characteristics of the development of a transplant of human embryonal nervous tissue in the rat spinal cord. Taking the great potentials for growth of segments of the telencephalic vesicles of human embryos, and data regarding the fact that they take well in homografts of the spinal cord of mammals [3, 4, 8, 7], into account, this division of the embryonal brain was specifically selected as the initial material for transplantation. Material and Methods. The investigation was carried out on 68 adult Wistar rats, weighing 200-250 g. Laminectomy of the VI thoracic vertebra was carried out in the animals under anesthesia (Calypsol in a dose of 200 mg/kg in combination with the inhalation of ether vapors). With monitoring by means of an operative microscope, the dura mater was opened, and a segment of donor brain measuring 2 mm 3 was introduced by a means of a glass cannula with an internal diameter of 0.5 mm into the spinal cord. Segments of the wall of the telencephalic vesicle of human embryos at the 8-10 week period of development, obtained after artificial interruption of pregnancy, were transplanted. The initial material for the transplantation was kept for several hours in Hanks solution at t ~ = 4~ After the operation the animals were maintained in the usual vivarium conditions. Morphological investigation using light and electron microscopic methods was carried out 2, 3, 4, 8, and 12 weeks later. For light microscopy the material was fixed in Bouins fluid. Paraffin sections, 5-7 Inn were stained with hematoxylin-eosin and toluidine blue after Nissl. For the electron microscopy, fixation of the brain was carried Out by perfusion of a 2.5% solution of glutaraldehyde in a 0.01 M phosphate buffer at pH 7.3-7.4, with subsequent treatment with a 2% solution of OsO4 in a 0.05 M cacodylate buffer, The material dehydrated in a series of alcohols of increasing concentration and in propylene oxide, and mounted in Epon. After the semithin sections were studied, collimated trimming of the blocks was carried out. The ultrathin sections, 50 nm in thickness, were obtained on an LKB ultratome, and studied under a JEM 100-B electron microscope. Results and Discussion. The wall of the telencephalic vesicle of the donor embryo at the 8-10 week period of development consists of cells of various degrees of maturity. Undifferentiated neuroepithelial cells, capable of mitotic division, are located in the ventricular zone. They are the forerunners of both nerve and glial cells. These cells, which are capable of migration, have an elongated form when stained with hematoxylin-eosin. A large number of mitotically dividing cells are observed particularly in the ventricular zone. The anlage of the ncocortex, the primary gray matter of the cortex, properly appears in embryogenesis from the eighth week of intrauterine development as a result of the migration of cells from the ventricular zone [1, 6]. It consists of cells of a high degree of maturity. As the formation of the primary gray matter of the cortex and the increase in the number of cells

Serotonin is involved in the regulation of histogenetic processes in rat embryonic neocortex.

We compared the dynamics of the development of ectopic transplants of embryonic (day 14) primordial neocortex from rats injected with serotonin inhibitor (para-chlorophenylalanine; 400 mg/kg) on day 11 of pregnancy and transplants of similar primordial neocortex incubated before transplantation in a medium with serotonin (3 µg/ml). The study of mitotic activity and differentiation of transplanted cells showed that serotonin promoted survival of the transplanted neuroepithelial cells and their differentiation into nerve cells, and is involved in the regulation of their proliferation. We hypothesized that serotonin accelerated the cell cycle of transplanted cells, thus accelerating the neuron differentiation.

Immunocytochemical Detection of Astrocytes in Brain Slices in Combination with Nissl Staining

The present study was performed to develop a simple and reliable method for the combined staining of specimens to allow the advantages of immunocytochemical detection of astrocytes and assessment of the functional state of neurons by the Nissl method to be assessed simultaneously. The protocol suggested for processing paraffin sections allows preservation of tissue structure at high quality and allows the selective identification of astrocytes with counterstaining of neurons by the Nissl method. The protocol can be used without modification for processing brain specimens from humans and various mammals-except mice and rabbits.

Formation of the neocortex in rats after prenatal hypoxia.

Exposure of pregnant rats to the atmosphere containing 7.56% of oxygen for one hour was found to cause the disturbances in the nerve system development in their offspring. The exposure to acute hypoxia at days 13 or 16 of embryonic development induced an attenuation and underdevelopment of brain cortical layers, disturbances in cell orientation and differentiation, i.e. caused the modifications of basic histogenetic processes that were active during this period, including proliferation, migration and differentiation, thus resulting in the changes of structural characteristics of neocortical layers. The effect of hypoxia during the later fetal period--at day 19 of embryonic development, when cell proliferation in the brain ceases while the processes of differentiation are enhanced,--is less damaging. The cortical layers formed in experimental animals are not significantly different from those in control rats, however the death of individual neurons and gliocytes takes place.

Initial stage of vascular bed development in telencephalon of human embryo.

It was found that intracerebral blood vessels in human embryo telencephalon first appear on the 7th week of prenatal development as capillaries with poorly differentiated walls and signs of functional immaturity. The formation of the basal capillary membrane consisting of laminin and type IV collagen starts immediately after the formation of primary capillary network.