HIF-1α- and HIF-2α-Immunopositive Neurons and Capillaries in the Prefrontal Cerebral Cortex of Rats with Experimental Myocardial Infarction.

The quantitative content of HIF-1α- and HIF-2α-immunopositive brain neurons in Wistar rats was studied 1, 15, and 30 days after modeling of myocardial infarction. In rats of the control group, the immunohistochemical markers HIF-1α and HIF-2α in the prefrontal cortex of the brain were determined in few pale-colored neurons and capillaries. One day after myocardial infarction simulation, the number of HIF-1α+ neurons increased, and on day 15 it reached the maximum level: the concentration of immunopositive neurons and capillaries increased by 24.7 and 18.4%, respectively, in comparison with the control. After 30 days, the number of HIF-1α+ structures decreased, but remained above the control values. The number of neurons and capillaries positively stained for HIF-2α peaked only on day 30 of the postinfarction period.

The Role of Carbon Monoxide in the Reactions of Arterial Branches of the Uterine Artery of Rats to Low-Intensity Irradiation with a Helium-Neon Laser.

Biomicroscopy was used to study order I-IV branches of the uterine artery in sexually mature female Wistar rats after a single 5- or 10-min direct exposure of the right broad ligament of the uterus to a helium-neon laser beam in the red band of the spectrum (λ=632.8 nm, output power 2 mW, intensity 20 mW/cm2). Under the influence of laser exposure, there was a significant increase in the diameter of small uterine branches and a weaker increase in large vessels with a well-developed muscular membrane. Preliminary administration of zinc IX protoporphyrin hemoxygenase blocker (30 mmol/kg) reduced reactivity of the vascular bed to laser irradiation, mainly of order I-II branches. It was concluded that carbon monoxide can act as an intermediary between the laser exposure and uterine vessels.

Reaction of Arterial and Venous Branches of the Broad Ligament of the Rat Uterus to Low-Intensity Red Laser Irradiation.

Using the biomicroscopy method, we studied the reaction of arterial and venous vessels of the broad ligament of the uterus in outbred female rats to irradiation with helium-neon laser (λ=632.8 nm; power output 2 mW). Small arteries were found to be most sensitive to laser irradiation. The veins of the broad ligament of the uterus demonstrated lower reactivity to laser irradiation of the same duration than arterial vessels, which can be explained by morphological, functional, and hemodynamic differences.

Dynamics of Organization of the Microcirculatory Bed and Mast Cells of the Uterus in Rats at Different Times of the Day.

Structural organization of the microcirculatory bed and mast cells in the uterus of mature female Wistar rats (n=60) were examined every 2 h over 24 h. During night hours, many vessels characterized by intensive reaction to NADPH-diaphorase and endothelial NO synthase were detected in the endometrium, and the density of these vessels and mast cells was quite high. During the day and evening hours, the capillaries with high levels of enzymes involved in NO synthesis and mast cells were less numerous. NO directly regulating the diameter of arteries and the intensity of transcapillary exchange can act as a signal molecule in the temporal dynamics of the vasoceptive action of estrogens and their receptors in the uterus.

Effect of the Hemoxygenase-Carbon Monoxide (HO-CO) System on the Reactivity of Uterine Artery Branches in Rats.

First to fourth-order branches of the uterine artery in sexually mature female Wistar rats were studied by biomicroscopy. After administration of a CO donor hemin (60 mM), the diameters of large uterine branches with a well-developed muscle layer markedly increased, while the increase in diameter of small vessels with one often interrupted layer of smooth muscle cells increased insignificantly. Zinc protoporphyrin IX (30 mM) in all cases blocked this effect. However, zinc protoporphyrin IX does not affect NO-mediated reaction of the branches of the uterine artery caused by administration of L-arginine (60 mM), and L-NAME did not significantly affect reactivity of uterine artery branches associated with the hemoxygenase-CO system. In contrast to NO, CO produced less potent and rapid, but more sustained effect. The target for the hemoxygenase-CO system is mainly arteries with developed muscular layer, while the target for the NO synthase-NO is small vessels where endothelium plays a Rdecisive role in the regulation of vasomotor reactions.

Comparative Study of HIF-1α- and HIF-2α-Immunopositive Neurons and Capillaries in Rat Cortex under Conditions of Tissue Hypoxia.

We measured the content of HIF-1α and HIF-2α-immunopositive neurons and microvessels in the brain of Wistar rats during the first 24 h of tissue hypoxia induced by subcutaneous injection of cobalt dichloride (50 mg/kg). In control rats (without hypoxia), immunohistochemical marker HIF-2α in cortex of parietal lobe was not detected, and HIF-1α was detected only in few weakly stained pale neurons and capillaries. In 30 min after injection of the cobalt salt, the number of HIF-1α+ neurons increased by 25.6% (in capillaries by 12.3%), many of these were characterized by intensive reaction; the quantitative parameters reached their maximum level within 1-3 h. However, the concentration of immunopositive neurons returned to the control values in 6 h after hypoxia modeling (capillaries in 9 h). In contrast to HIF-1α, the number of neurons and capillaries containing HIF-2α reached a maximum level in 6-12 h of hypoxia. The relative density of HIF-2α+ capillaries increased most pronouncedly (by 23.6%); the relative density of neurons increased by 18.9%. The relative density of HIF-2α+ cells did not change significantly to the end of the experiment. Thus, HIF-1α is more essential for regulation of adaptation to hypoxia in neurons and HIF-2α is more important for the endothelium of microvessels.

Dynamics of Distribution of Capillaries with Matrix Metalloproteinase-2 and Its Tissue Inhibitor in Rat Brain during Development of Experimental Hypertension.

The capillaries containing MMP-2 and its tissue inhibitor TIMP-2 were examined in cerebral cortex and white matter obtained from intact Wistar rats (n=5) and the rats with progressing experimental renovascular hypertension (n=35). In hypertensive rats, the changes in intensity of the immunohistochemical reaction and in the density of capillaries expressing TIMP-2 significantly differed from the corresponding values in MMP-2-positive capillaries, which resulted in pronounced deviation of MMP-2/TIMP-2 index from the control level (especially in cerebral cortex) probably attesting to enhanced risk of complications in cases with arterial hypertension.

Immunolocation of Heme Oxygenases in the Walls of Cerebral Arteries of Various Diameters in Rats.

The distribution of two enzymes involved in the formation of carbon monoxide, heme oxygenases 1 and 2, in the pial branches of orders I-V of the middle cerebral artery basin and in intracerebral vessels was studied in adult Wistar rats. Immunohistochemical studies detected hemeoxygenase-2 in the endothelium of the small pial and intracerebral arterioles and in myocytes of pial branches I-III. Heme oxygenase 1, an inducible form of the enzyme, is normally not expressed in the cerebral vessels, but the enzyme is expressed in response to sodium metaarsenite. In this case, heme oxygenase markers are detected in myocytes of pial arteries I-II and in the endothelium of small pial and intracerebral vessels. Sodium meta-arsenite is inessential for immunolocation and quantitative distribution of heme oxygenase 2 in the vessels.

Age-Specific Characteristics of CO-Mediated Reaction of the Pial Arteries of Various Diameters in Rats.

First-fourth order pial branches of the median cerebral artery were studied by biomicroscopy in male Wistar rats aged 1 and 12 months. Irrespective of age, CO-mediated mechanisms are involved in the regulation of the basal tone of pial vessels of various diameters (more so of arteries with well-developed muscular tunic). Injection of hemin confirmed that endogenous production of CO maintained vasodilatation and this effect was most pronounced in large pial branches of young animals, while zinc protoporphyrin IX blocked this effect in all cases. On the other hand, zinc IX protoporphyrin did not modify NO-mediated reaction caused by injection of L-arginine, while hemin compensated (though not completely) vasoconstriction induced by NO synthase blocker L-NAME. In contrast to NO, the effect of CO on blood vessels was not so rapid and potent, but more lasting. Other targets for CO were arteries with well-developed muscular tunic, while targets for NO were small vessels. The vasomotor effects of both gas transmitters were more pronounced in young animals.

ENDOTELIAL-DEPENDENT OF THE REGULATION OF ANGIOGENESIS.

The review presents cellular and molecular mechanisms regulating angiogenesis. In response to the angiogenesis inducers impact the activated endothelial cells and their precursors (progenitor cells) synthesize and produce angiogenic molecule that differ by chemical origin and biological functions but all of them enable these cells to influence both directly and indirectly on new vessels growth. Among the great number of angiogenic molecules the scientists hold interests in the following: the set of vascular endothelial growth factors, the set of the fibroblast growth, transforming growth factor, tumor necrosis factor and some other soluble polypeptides which occurred to be an effective regulators of angiogenesis. However, despite the evident achievements in studies of cellular and molecular mechanisms of angiogenesis it is still difficult to control this process. Therefore the main goal of the study was to review endothelial-dependent factors and mechanisms of capillary vessels growth regulation.

Effects of Chronic Tobacco Smoking on the Distribution of Tachykinin Receptors in Rat Pial Arteries.

Pial arteries of different diameter were studied in intact rats and after 6-month modeling of chronic tobacco smoking in rats. Expression of tachykinin NK1 receptors in pial arteries was studied by biomicroscopy and immunohistochemical methods. Chronic tobacco smoking induced considerable reorganizations of the arterial bed. The intensity of changes depended on the diameter of vessels. In small pial vessels that directly participate in the blood supply to the brain, pronounced vasodilatation and enhanced expression of NK1 receptors in the endothelium mediating the effects of substance P were observed; the number of these vessels also increased. The intensity of the response to tobacco smoke components decreased with increasing vessel diameter.

Topochemistry of Internuclear and Intranuclear Interneurons of the Vasomotor Area in the Medulla Oblongata of Hypertensive Rats.

Immunohistochemical examination with the antiserum against neuronal NO synthase and cystathionine ß-synthase was used to study the following two pools of interneurons in Wistar rats at various periods after the development of renovascular hypertension: intranuclear interneurons (lying in the projection of the solitary nucleus, reticular gigantocellular nucleus, and parvocellular nucleus) and 2 groups of internuclear interneurons (small interneurons, area 50-300 µ(2); and large interneurons, area above 350 µ(2)). Intranuclear and internuclear interneurons probably play a role in the central mechanisms of hemodynamics regulation. These interneurons differ by not only in topochemical parameters, but also functional properties (different resistances to BP changes). Intranuclear interneurons are characterized by high sensitivity of the gas transmitter systems to a continuous increase in BP, which results in remodeling and dysfunction of the bulbar part of the cardiovascular center. Large internuclear interneurons demonstrate a strong reaction to BP rise, which confirms their involvement into hemodynamics regulation. By contrast, small internuclear interneurons retain their characteristics in arterial hypertension and probably perform an integrative function.

[LOCALIZATION AND QUANTITATIVE ASSESSMENT OF OXYGEN-DEPENDENT HYPOXIA-INDUCIBLE FACTOR 1α IN THE BRAIN OF MITTEN CRAB ERIOCHEIR JAPONICA IN NORM AND IN ACUTE ANOXIA (AN IMMUNOHISTOCHEMICAL STUDY)].

Using immunoblotting and immunocytochemistry, the expression of hypoxia-inducible factor 1α (HIF-1α) was studied in the brain of the mitten crab Eriocheir japonica in norm and at 2, 4, 6 and 12 hours of anoxia on the model of water deprivation. In intact crabs, the number of immunopositive neurons was small, but it increased with anoxia duration. Particularly pronounced increase in the proportion of neurons with the HIF-1α expression was found in cell group 6. In group 9/11. the highest expression index was observed between 2-6 hours of anoxia. In group 17, significant changes in the proportion of immunopositive cells was observed only after 2 hours of anoxia. After 6 hours of anoxia, proportion of neurons with HIF-1α expression within all cell groups was reduced, but the reactions appeared in the blood cells. It is assumed that the increase in the proportion of immunopositive neurons and the appearance of the expression of HIF-1α in blood cells in the anoxic brain play an important role in providing compensatory and protective processes, enhancing adaptive capacity of mitten crab under the conditions of hypoxic stress.

[COMPARATIVE CHARACTERISTICS OF uNOS-POSITIVE STRUCTURES IN THE CNS OF SOME SPECIES OF CRUSTACEANS].

We conducted a comparative study of NO-ergic system in the CNS of 10 species of crustaceans subclass Malacostraca, belonging to orders Stomatopoda and Decapoda, with a common habitat in Ussuri Bay (Sea of Japan). Both similar characteristics and differences in content and distribution of universal NO-synthase (uNOS) were revealed in homologous parts of the brain and ventral nerve cord of the investigated species of crustaceans. We discuss the involvement of nitric oxide in the regulation of physiological functions of decapod crustaceans and its role in the processes of adaptation to the environmental conditions.

Norepinephrinergic and nitroxidergic neurons of vasomotor nuclei in hypertensive rats.

The distributions of norepinephrinergic and nitroxidergic neurons in solitary tract nucleus, locus coeruleus, and in the small-celled and gigantocellular reticular nuclei were examined in sham-operated and experimental Wistar rats with induced renovascular hypertension. In control rats, the greatest population of norepinephrinergic cells was revealed in the projections of locus coeruleus and reticular gigantocellular nuclei, while the larger number of nitroxidergic neurons were observed in the solitary tract and the small-celled nuclei. During renovascular hypertension, the earliest and the most pronounced changes in luminescence intensity and the number of nitroxidergic neurons were observed in the solitary tract nucleus, while the changes in locus coeruleus were minimal. The significant changes in similar parameters of norepinephrinergic neurons were documented only in reticular gigantocellular nucleus and locus coeruleus, but they were delayed and less pronounced in comparison with the changes of nitroxidergic neurons.

[Interneurons Brainstem of the Human].

OBJECTIVE: To conduct a comparative study of the structural organization of internuclear interneurons involved in the metabolism of nitrogen monoxide, hydrogen sulphide and carbon monoxide in the caudal brain stem humans. METHODS: The cross-sectional study was performed. We used histochemical and immunohistochemical methods to study the internuclear interneurons expressing neuronal nitric oxide synthase, cystathionine ß-synthase and heme oxygenase-2 which are located between the giant and small cell reticular nuclei (cell group 1), small cell reticular nucleus and the nucleus of the solitary tract (cell group 2) or is surrounded by reticular lateral nucleus (cell group 3). RESULTS: The work was carried out on the corpses of 6 men 18-44 years old who died from causes unrelated to the damage of the central nervous system. We revealed the differences between internuclear organization and intranuclear interneurons and local structural features of internuclear interneurons that are more clearly visible between the cell group 1 including a relatively large number of large neurons and cell groups 2 and 3 (p < 0.001). In the latter two groups small cells with high concentrations of the neuronal nitric oxide synthase and heme oxygenase-2 dominated. CONCLUSION: Despite the fact that the number of internuclear interneurons significantly is less than the number of intranuclear cells, they have structural prerequisites to control integration processes in the brain. Internuclear interneurons produce gasotransmitters and classical mediators of nerve impulse, form a local chain of interneurons between vasomotor nuclei, share many links with the above and the lower parts of the brain.

Two pools of interneurons in the bulbar region of the cardiovascular center of rats.

Wistar rats were subjected to renovascular hypertension. Then, we studied two pools of neurons: intranuclear neurons located in projection area of the solitary tract nucleus and reticular, magnocellular, and parvicellular nuclei; and two groups of internuclear neurons, which included both small neurons 50-300 µm(2) in size and large neurons more than 350 µm(2) in size. Immunohistochemical methods with antisera against neuronal nitric oxide synthase, cystathionine-ß-synthase, and heme oxygenase-2 were used. The studies were performed 8, 16, and 24 weeks after the development of renovascular hypertension. We have found that at least two pools of interneurons, intra- and internuclear cells, may be involved in the mechanisms of regulation of hemodynamics. These pools of interneurons differ in some topochemical and functional features, including different resistances to an increased blood pressure.

[Regulation of Ovarian Function: Part of the Gas Transmitters NO, CO and H2S].

This review discusses the management of ovarian function and participation in the process of a new class of cellular regulators--Gasotransmitters (NO, CO and H2S). According to modern concepts in regulatory processes, in addition to hypothalamic-pituitary mechanisms involved a large and diverse group of ovary cells (interstitial, internal theca and atretic bodies, follicular epithelium, teka-luteal and granulosa-luteal cells of the corpus luteum), which not only mediate the gonadotropic stimulation, but satisfy the for the production of a wide range of biologically active substances. These materials are expected to participate in all these processes gaseous mediators. However, our knowledge of topochemistry, mechanisms and the role of these substances in the important organs of the reproductive system-ovary, fragmentary and do not allow to form a holistic understanding of the cellular mechanisms by which Gasotransmitters have a regulating effect on the individual structures of the ovary in normal functioning of the body and disease. Creation of an experimental model for studying the mechanisms of gaseous mediators in different cell types of ovarian, including endothelial and smooth muscle cells of blood vessels, in which the functional significance of these substances may be particularly significant, will provide fundamental knowledge for the development of new approaches to the treatment of diseases of the reproductive system.

[A COMPARATIVE STUDY OF CATECHOLAMINERGIC AND NITROXIDERGIC VASOMOTOR NEURONS IN THE NUCLEI OF THE CAUDAL PART OF THE BRAINSTEM OF THE RAT].

Immunohistochemical methods for the demonstration of tyrosine hydrolase (TH) and neuronal form of nitric oxide synthase (nNOS) were used to study the distribution of catecholaminergic and nitroxidergic vasomotor neurons respectively, in the nuclei of the medulla oblongata and the pons of 12 Wistar rats. Most often the expression of TG was found in neurons located in the nucleus and several reticular nuclei (gigantocellular, paragigantocellular, caudal pons nucleus), but the proportion of immunoreactive neurons did not exceed 8-14%. In the other nuclei (reticular parvocellular nucleus and oral pons nucleus, spinal nucleus of the trigeminal nerve) the value of this parameter ranged from 1 to 3%. In a large group of nuclei with proven vasomotor function such neurons were constantly not detected. In the structures with high content of catecholaminergic neurons, nNOS-positive cells were found, as a rule, in fewer numbers than in the nuclei with a limited number of TH-positive neurons.

[Histochemical and immunohistochemical localization of choline acetyltransferase in the nuclei oblongata rat brain].

ChAT-positive neurons in the nuclei of the medulla oblongata of Wistar rats have been studied with the use of histochemical and immunohistochemical methods. We have found that the topography and the number of cholinergic neurons at the projection nuclei studied largely depend on the method of detection of these neurons. Histochemical method always revealed more neurons than immunohistochemistry. Such a feature of ChAT-positive neurons detection was clearly seen among the majority of nuclei in the medial region and in some of the nuclei in the lateral region of the medulla oblongata. The number of immunoreactive cells in the nuclei varied from 17 to 26%, whereas the histochemical reaction determined 1.5-3 times more neurons in the same nuclei. ChAT-positive cells in the nuclei of the back seam were detected mainly by a histochemical method.