Morphofunctional Features of Microglial Cells during the Administration of Orexin A.

The neurotransmitter orexin A (1 µl of a solution with a concentration of 0.3 mM) was injected into the second brain ventricle of wild-type C57BL/6 mice; 1 h later, LPS was injected intraperitoneally at a dose of 2 mg/kg to activate microglial cells. Administration of orexin A attenuated activation of microglial cells assessed by changes in the length of filopodia. Injection of orexin was followed by an increase in the length of the processes, which indicates a decrease in activity of microglial cells. Using double immunohistochemical staining, we found that the number of microglial cells and orexin A receptors on microglial cells increased after LPS injection. Our findings confirm the participation of orexin A in the modulation of the functional activity of microglial cells.

The Blood-Brain Barrier in Neuroimmune Interactions and Pathological Processes.

The blood-brain barrier (BBB) is a kind of filter, highly selective in relation to various types of substances. The BBB supports the immune status of the brain and is an important regulator of neuroimmune interactions. Some of the molecular and cellular features of the BBB, as well as the five main pathways of neuroimmune communication mediated by the BBB, are analyzed in this article. The functions of the BBB in neuroimmune interactions in various diseases are discussed: multiple sclerosis and Alzheimer's and Parkinson's diseases. The latest data on BBB dysfunction in COVID-19 coronavirus infection caused by the SARS-CoV-2 virus are considered.

Effect of Skin Application of Dorogov's Antiseptic-Stimulant on Behavioral Reactions in Rats.

Local application of the Dorogov's antiseptic-stimulant onto the skin of 2-month-old male Sprague-Dawley rats in a low dose (0.5 g/kg/day) for 1 week increased motor and ultrasonic activity of experimental animals and the content of activated c-Fos+ neurons on the cingular, motor, insular, and pyriform cortex and in the striatum.

[CELL-MOLECULAR BASIS OFNEUROIMMUNE INTERACTIONS DURING STRESS].

The review represents a modern concept about cells-molecular basis of mechanisms of neuro-immune interactions, the data on the effects of destabilizing factors (electric pain stimulation, rotation, cold and psychoemotional stress) on the functioning of neurons and immune cells. It must be underlined, that under the stress conditions take place the alterations of ligand-receptors interactions on the membrane of lymphocyte. In particular the reaction of these cells to regulating signal - application of Interleikin-1 grow up after mild stress, but it falls down after an influence of severe stress factors. Special attention is paid to the role of the orexinergic system in mechanism of realization of CNS reactions to application of antigens. In the present work the possible methods of correction of imbalance in functional interactions between nervous and immune systems, caused by different destabilizing factors, are reviewed.

[Receptors for oreksins: structure, localisation and activation mechanisms].

The review concerns morpho-functional characteristics of orexin receptors. The data on their structure, signal transduction pathways, biological effects of orexin receptors 1 or 2 depending on association with different G-proteins are presented. Localisation of orexin receptors in various CNS structures as well as in peripheral organs mediates regulation of different physiological functions by orexins. Low concentration of orexins in peripheral blood and orexin-containing cells in ganglions and internal organs suggests a possibility to activate orexin-sensitive cells distantly, paracrinely or autocrinely. The data on effects of selective or non-selective orexin receptor antagonists is analysed.

[Immunoreactivity of hypothalamic orexin neurons and expression level of preproorexin gene in them after lipopolysaccharide injection].

Hypothalamic orexin neurons are involved in the regulation of many physiological functions. The immunoreactivity of these neurons is shown to be altered after LPS injection. This phenomenon is characterized by definite time-space pattern and depends on dose of antigen applied. The expression level ofpreproorexin gene in rat hypothalamus was investigated in 2, 4 and 6 hours after injection of 25 and 500 mkg/kg b. w. LPS. Both injections of higher and lower doses resulted in the increase of expression level of preproorexin gene after 2 hours that could suggest an enhancement of orexin synthesis in neurons. There were no significant changes in 4 and 6 hours after injection. The comparative analysis of the data obtained earlier with immunohistochemistry, and the data shown in the present study suggest the mechanisms of orexin neurons reaction to injection of LPS in different doses, i. e. the more considerable prevalence of orexin utilization over its synthesis in hypothalamic cells after injection of subseptic (500 mkg/kg) dose of LPS.

[Response of hypothalamic orexin-containing neurons to stimuli of antigen and non-antigen nature].

Orexin is a hypothalamic peptide, a neurotransmitter described in 1998. Orexinergic neurons are localized in hypothalamic structures and play a significant role in regulation of various physiological functions. The localization oforexin-containing neurons and their projections in hypothalamus of Wistar rats and other structures of CNS are presented. The participation of orexinergic neurons in the regulation of feeding behavior and in the sleep/wake cycle as well as their involvement in the regulation of immune functions is discussed. There are experimental data, containing comparative analysis oforexin-containing neurons responses to stimuli of antigenic and non-antigenic nature, which suggest functional heterogeneity of orexin-containing neurons of hypothalamus that leads, particularly, to involvement of different neurons in the realization of brain reaction to antigen and non-antigen stimuli. Both analyses ofpreproorexin gene expression level and morphofunctional characteristics of orecxin-containing neurons of hypothalamus after antigen challenge suggest the possibility of their participation in the mechanisms of realization of brain reaction to antigen challenge.

[Cellular and molecular mechanisms of interaction between the neuroendocrine and immune systems under chronic fatigue syndrome in experiment].

One of the main mechanisms of chronic fatigue syndrome development involves disturbances of interaction between the immune and neuroendocrine systems. The adequate experimental model for the search of these mechanisms is induction of fatigue in animals via the single intraperitoneal administration of synthetic double-stranded RNA - Poly I : C. Investigation of alterations in cytotoxic and proliferation activities of splenocytcs, the intensity of immunomodulatory cytokines signaling via the sphingomyelin pathways in membrane P2 fraction of the brain cortex, as well as the activity of hypothalamic-pituitary adrenal (HP A) axis in the dynamics of chronic fatigue syndrome in rats has performed. Inhibition of both cytotoxic and proliferative activities of splenocytes during the period of fatigue development has been shown. Priority data concerning the suppression of the activity of neutral sphingomyelinase (nSMase) - the key enzyme of the sphingomyelin cascade - in membranes ofthe cells from the brain cortex on the 3d day after Poly I : C administration to rats have been obtained. It was found that Poly I : C injection to rats led to disturbed HPA axis functions which was manifested by decreased corticosterone concentration in standard functional assays with ACTH and hydrocortisone administration. It is suggested that disturbances in interaction between the immune and neuroendocrine systems during development of chronic fatigue syndrome, including alterations in HPA axis activity, are realized both on the level of changes in the activity of immune-competent cells and immediately on membranes of the brain cells.

[In search of the guilty: analysis of encephalitogenic T cell migration pathways on preclinical phase of EAE after their intravenous or intraperitoneal injection].

Adaptive-transfer experimental autoimmune encephalomyelitis is an inflammatory neurodegenerative disease which is induced by injection of activated encephalitogenic T cells. Adaptive-transfer experimental autoimmune encephalomyelitis is a major experimental tool for investigation ofT cell function in multiple sclerosis development. Activated myelin basic protein specific T cells are able to invade and inflame the central nervous system which is followed by axonal injury and paralysis on the third day after injection. In the prodromal phase of EAE encephalitic T cells migrate through different organs which alter their phenotype before invading the CNS. We compared migratory patterns of encephalitic T cells after intravenous and intraperitoneal injection to elucidate which organs may play an important role in the formation of "migratory" phenotype. We found that encephalitogenic T cells ultimately migrate through spleen and parathymic lymph nodes regardless of the start point of cells migration after i.p. and i.v. injection. We hypothesise that cellular and extracellular components of these organs could be involved in the formation of T cells "migratory" phenotype which is necessary for penetration via blood-brain barrier.

Expression of the c-Fos gene in the rat hypothalamus in electrical pain stimulation and UHF stimulation of the skin.

Studies of the pattern of activation of hypothalamic cells in conditions of electrical pain stimulation demonstrated an increase in the proportion of c-Fos-positive cells (to 72.02-98.95%), predominantly affecting cells of size 10-50 microm2. UHF irradiation of the skin activated hypothalamic cells of size 10-70 microm2 in the paraventricular and dorsomedial nuclei and in the periforniceal zone of the lateral hypothalamic field. Combined action of UHF irradiation of the skin and electrical pain stimulation led to decreases in the proportions of cells activated as a result of pain stimulation in the anterior hypothalamic field, the ventromedial nucleus, and the basal part of the lateral hypothalamic field. Changes in the distribution of activated cells by size (mainly a reduction in the proportion of cells of area 10-30 microm2 and an increase in the proportion of larger cells) were detected in the ventromedial nucleus and the basal part of the lateral field. UHF irradiation of the skin was found to have a modifying effect, consisting of a reduction in the degree of activation of hypothalamic cells evoked by electrical pain stimulation.

Morphometric analysis of hypothalamic cells showing c-Fos proteins after movement restriction and EHF-irradiation.

A morphometric analysis of hypothalamic cells containing c-Fos-like protein detected by an indirect immunoperoxidase method was carried out to clarify the responses to movement restriction and skin electromagnetic high frequency (EHF) irradiation (7.1mm, 42.2GHz, 20mW output power) simultaneously to three acupuncture projection areas. The morphometry of c-Fos-protein positive neurons by their number and type was analyzed. Movement restriction (40min) induced c-Fos protein expression primarily in cells with 10-50mum(2) sizes (associative type neurons) only in anterior hypothalamic nucleus and lateral hypothalamic area; while additional EHF-irradiation of acupuncture projection areas (under movement restriction) induced c-Fos expression in all hypothalamic structures and mostly in cells with 70-150mum(2) sizes (relay type neurons), i.e. changed the pattern of activated cell type distribution. In conclusion, the findings show that modest stress under which experimental animals often are exposed increase c-Fos protein expression in hypothalamic centers and skin EHF-irradiation of acupuncture projection areas seem to increase that.

Comparative analysis of the expression of c-Fos and interleukin-2 proteins in hypothalamus cells during various treatments.

The aim of the present work was to perform a combined analysis of the degree of activation of the anterior hypothalamus of the rat and expression of the interleukin-2 gene during treatments of different types: mild stress ("handling") and adaption to it, as well as intranasal administration of physiological saline and the peptides Vilon (Lys-Glu) and Epitalon (Ala-Glu-Asp-Gly). Changes in the numbers of c-Fos-and IL-2-positive cells in structures of the lateral area (LHA) and anterior (AHN), supraoptic (SON), and paraventricular (PVN) nuclei of the hypothalamus in Wistar rats. Ratios of the quantities of c-Fos-and IL-2-positive cells were determined in intact animals and after activation of brain cells initiated by different treatments; the influences of adaptation to handling on the nature of changes in the expression of these proteins was also studied. Combined analysis of the intensity of expression of these two proteins - c-Fos, a marker of neuron activation and a trans-factor for the IL-2 cytokine gene and other inducible genes, and IL-2 - in intact animals and after various treatments showed that the process of cell activation in most of the hypothalamic structures studied correlated with decreases in the quantity of IL-2-positive cells in these structures; different patterns of changes in the numbers of c-Fos-and IL-2-positive cells were seen in response to different treatments in conditions of stress and adaptation to it.

Stress-induced changes in cellular responses in hypothalamic structures to administration of an antigen (lipopolysaccharide) (in terms of c-Fos protein expression).

Stress is known to affect the intensity of the immune response. The involvement of central regulatory structures in mediating these changes was addressed by analyzing the extent of activation of neurons in the hypothalamus (in terms of the number of c-Fos-positive cells) in rats 2 h after i.v. administration of lipopolysaccharide alone and on the background of electrical pain stimulation. Studies were performed using 52 male Wistar rats weighing 200-250 g. c-Fos protein expression was studied by immunohistochemical analysis. Increases in the quantity of c-Fos-positive cells 2 h after administration of lipopolysaccharide were seen in the following hypothalamic structures: AHN, PVH, LHA, VMH, DMH, and PH. After electrical pain stimulation, the number of c-Fos-positive cells increased in these same hypothalamic structures (AHN, PVH, LHA, VMH, DMH, and PH). The combination of electrical pain stimulation and lipopolysaccharide administration led to a decrease in the extent of activation in hypothalamic structures AHN, PVH, LHA, and VMH as compared with the characteristic reaction to lipopolysaccharide without electrical pain stimulation. Electrical pain stimulation suppressed the intensity of the immune response induced by lipopolysaccharide (as assessed by local hemolysis and counts of the numbers of spleen antibody-forming cells). Thus, changes in the extent of activation of hypothalamic structures (AHN, PVH, LHA, VMH) correlated with the development of stress-induced immunosuppression, i.e., morphofunctional mapping of the extent of activation of hypothalamic structures allowed identification of which changes in hypothalamic cell activity occurred with stress-induced changes in immune system responses to antigen administration.

Responses of hypothalamic orexin-containing neurons to cyclophosphamide, EHF-irradiation of the skin, and their combination in rats.

Orexins are neuromediators that participate in the regulation of feeding behavior, energy metabolism, circadian rhythms and perception of pain. The aim of the present study was to clarify the responses of the hypothalamic orexin-containing neurons to an intraperitoneal injection of cyclophosphamide (CPA), extremely high frequency (EHF)-electromagnetic stimulation of skin, which is used to modulate side effects of cytostatics and their combination. The activation of orexin-containing neurons was determined by recording of the intensity of c-Fos protein expression. Injection of cyclophosphamide (40mg/kg) or EHF-irradiation of the skin decreased the staining of orexin-containing neurons, which was most pronounced in the subfornical region of the lateral hypothalamic area (LHAs). A redistribution of orexin from the perinuclear space to the processes of these cells took place, which occurs after the activation and the expression of the c-fos-gene. c-Fos protein was expressed in most neurons with minimum content of orexin, i.e. activation of these neurons correlated with the redistribution of orexins caused by skin EHF-irradiation and injection of cyclophosphamide (CPA). EHF-irradiation of the skin before and after injection of CPA increased the staining of orexin-containing neurons, i.e. it prevented the redistribution of orexin.

[Expression of c-Fos gene in the rat hypothalamus upon electric painful stimulation and EHF irradiation of the skin].

Studies of the activation pattern of hypothalamic cells upon electric painful stimulation have revealed an increase in the counts of c-Fos-positive cells (72.02-98.95%) predominantly of a size ranging from 10 to 50 microm2. EHF irradiation of the skin activates hypothalamic cells of the size ranging from 10 to 70 microm2 in the paraventricular, dorsomedial nuclei, and in perifonical zone of the lateral hypothalamic area. The combined action of EHF skin irradiation and electric painful stimulation leads to a decrease in the counts of cells activated in result of the painful stimulation in the anterior hypothalamic area, ventromedial nucleus, and the basal part of the lateral area. Changes in size distributions of activated cells are found in the ventromedial nucleus and the basal part of the lateral area, occur mainly through decreased counts of smaller cells (10-30 microm2) and increased counts of lager cells. The modulatory effect of EHF irradiation of the skin was found to be expressed as a reduction in hypothalamic cell activation caused by electric painful stimulation.

[Parallel analysis of c-Fos protein and interleukin-2 expression in hypothalamic cells under different influence].

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.

Interleukin-2 concentration in hypothalamic structures of rats receiving peptides during mild stress.

The number of hypothalamic IL-2-containing cells changed in rats receiving Vilon and Epithalon during mild stress (handling). The number of IL-2-positive cells in hypothalamic structures decreased 24 h after intramuscular injection of Epithalon and 2 h after intranasal administration of the test peptides. Adaptation of animals to experimental conditions prevented the decrease in the number of IL-2-positive cells in the supraoptic nucleus after intranasal administration of Epithalon.

Expression of the c-Fos gene in hypothalamic cells and cytotoxic activity of natural killer cells in the spleen of rats after treatment with Cytoxan.

In experiments on rats we studied the effect of cyclophosphamide-containing drug Cytoxan on activation of neurons in hypothalamic structures involved in the regulation of natural killer cell activity in the spleen and changes in cytotoxicity of these cells. Administration of Cytoxan in a dose of 60 mg/kg increased the number of c-Fos-positive cells in the ventromedial hypothalamus and lateral hypothalamic area and reduced interferon-alpha-induced cytotoxic activity of natural killer cells. Our findings attest to the involvement of central mechanisms of regulation of splenic natural killer cells into side effects of Cytoxan.

[Structure and functions of the brain orexin-containing neurons].

The results of investigations of the new discovered brain orexin neurons, their chemical structure, localization and functions are reviewed. The following data are described: the specifics of orexins mRNA, orexins A and B and their receptors; connections between orexin neurons and neurons from different structures of the brain and spinal cord and the participation of the orexin neuron system in the functional regulation.

[Stress-induced changes of hypothalamic structure cell responses to antigen injection (LPS) (revealed by c-Fos protein expression)].

Stress stimuli are known to influence the intensity if immune response. To elucidate the role of central regulating structures in this changes, analysis of activation level of hypothalamic neurons (revealed by quantity of c-Fos-positive cells) was carried out in rats within 2 hours after intravenous LPS injection and after this--impact associated with electric pain stimulation (EPS). The investigation was carried out in 52 male Wistar rats, 200-250 g. The c-Fos protein expression was analyzed with immunohistochemical method. The increase of c-Fos-positive cells number in 2 hours after LPS injection was observed in AFTN, PVH, LHA, VMH, DMH and PH. After electrical pain stimulation, the quantity of c-Fos-positive cells increased in the same structures. Combined application of electric pain stimulation and LPS injection results in diminished activation level in AHN, PVH, LHA and VMH as compared with typical response to single LPS injection without EPS. The EPS suppresses intensity of the immune response induced by injection of LPS (revealed by local hemolysis method with calculation of antibody-forming cells quantity (%) in the rat spleen). Thus the activation level changes of hypothalamic structures (AHN, PVH, LHA, PH) correlate with development of stress-induced immunosuppression, i. e. morphofunctional description of hypothalamic structures activation as revealed by pattern of activated cell alterations in hypothalamic structures during realization of stress-induced changes of immune system responses to antigen injection.