I. P. Grigor’ev

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.

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.

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.

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.

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.

Иммуногистохимическая характеристика нейронов черного вещества головного мозга человека

AIM To determine the cytochemical characteristics of unchanged neurons of the human substantia nigra using a wide range of immunocytochemical markers some of which (glutamate decarboxylase-65, PGP 9.5, non-phosphorylated neurofilament proteins, alpa-tubulin) have never been used for study of human dopaminergic neurons. MATERIAL AND METHODS Fragments of human midbrain (17 men and women, aged from 28 to 78 years) from the archives of the Department of General and Specific Morphology of the Institute of Experimental Medicine were used. The study was performed using classical neurohistological techniques and immunocytochemistry using antibodies to 15 different proteins. RESULTS Most neurons in substantia nigra exhibited a reduced expression of common neuronal markers such as neuronal nuclear protein NeuN, PGP 9.5 protein, and neuron-specific enolase. GABAergic (GAD65-immunopositive) neurons were not found in the substantia nigra. Single cholinergic neurons without neuromelanin were identified in the dorsal part of the substantia nigra. Calcium-binding proteins calbindin and calretinin were not found in the majority of nigral cells although calbindin was rarely seen in some neurons of the dorsal part and calretinin in the ventral one. Nitric oxide synthase (NOS) was present in the substantia nigra both in neuropil and neuronal bodies. CONCLUSION The results suggest the unique cytochemical properties of the nigral neurons, which may be related to their increased susceptibility to lesion and degeneration.

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.

Distribution of Alpha-Tubulin in Rat Forebrain Structures

Immunohistochemical studies addressed the distribution of α-tubulin microtubules in rat forebrain structures. Non-uniform staining was seen with the reaction for α-tubulin. High immunoreactivity was detected in the cingulate and piriform cortex, olfactory bulbs, and the optic chiasma. The weakest immunohistochemical reactions were seen in the corpus striatum, the superficial layers of the septum, the cingulum, and structures around the third ventricle. Immunoreactivity in neurons was clearly apparent at the periphery of the perikaryon and in the apical dendrite. It is suggested that the intensity of the immunohistochemical reaction for α-tubulin may reflect the functional state of neurons.