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.

[The distribution of matrix metalloproteinases and their tissue inhibitors in the brain vascular bed exposed to chronic tobacco smoke]. / Ékspressiia matriksnykh metalloproteinaz i ikh tkanevykh ingibitorov v sosudistom rusle golovnogo mozga pri khronicheskoi intoksikatsii tabachnym dymom v éksperimente.

AIM: To study the distribution of MMP-2 and MMP-9 and their inhibitors (TIMP-2 and TIMP-1 respectively) in the brain vascular bed of rats exposed to chronic tobacco smoke. MATERIAL AND METHODS: Localization and expression of MMP-2, MMP-9, TIMP-2 and TIMP-1 in the pial branches (I-V order vessels), intracerebral arteries and capillaries of rats exposed to tobacco smoke were studied for 36 weeks. The level of enzymatic activity was assessed by the relative quantity of enzymopositive arteries and amount of fragments per 1 mm2 and rate of immunohistochemical reaction. Specific capillary density per mm2 of brain tissue and optical density of the immunohistochemical product were calculated. RESULTS: MMP-2 and TIMP-2 were found in all segments of the arterial course in control animals. In rats exposed to tobacco smoking, the expression of MMP-2 increased only in intracerebral arteries and capillaries while TIMP-2 level decreased. MMP-9 and TIMP-1 were noted only in single vessels, mainly small pial and intracerebral arteries, in intact animals. In rats exposed to tobacco smoke, MMP-9 expression significantly increased in all segments of the arterial course whereas the increase in TIMP-1 was observed mainly in large pial arteries. CONCLUSION: In physiological conditions, the dynamic balance between MMP-2 and TIMP-2 maintains basic tissue metabolism. Products of tobacco combustion are inductors of the inducible MMP-9 which promotes morphofunctional changes. The imbalance in MMP-9 - TIMP-1 system causes the degradation of extracellular matrix in different segments of the brain arterial course promoting the development of cerebral dysfunction.

[Changes in spatial relationships between catecholamine- and nitroxidergic neurons in the nuclei of the caudal brain stem in the development of hypertension]. / Izmenenie prostranstvennykh vzaimootnoshenii katekholamin- i nitroksidergicheskikh neironov v iadrakh kaudal'noi chasti stvola mozga pri razvitii arterial'noi gipertenzii.

AIM: To obtain the data on the spatial relationships between catecholamine (TH-positive) and nitroxidergic (nNOS-positive) neurons in vasomotor nuclei of the medulla in different periods of hypertension development. MATERIAL AND METHODS: The experiment was performed on male Wistar rats (n=45) with induced renovascular hypertension (RVH). TH and nNOS in neurons of solitary tract nuclei, reticular small-and giant cell nuclei were detected using immunohistochemical methods. RESULTS AND CONCLUSION: The most early and severe changes in the intensity of reaction and amount of nNOS-positive neurons were noted in the solitary tract nucleus. Significant changes in the quantitative parameters of TH-positive neurons in RVH were identified only in the reticular giant cell nucleus but they appeared later and were less expressed compared to nNOS-positive cells. This resulted in the changes of spatial relationships between two types of neurons and remodeling of the bulbar region of the cardiovascular center.

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.

[The cellular and molecular mechanisms of angiogenesis regulation in the brain]. / Kletochno-molekuliarnye mekhanizmy reguliatsii angiogeneza v golovnom mozge.

This review presents the data on cellular and molecular mechanisms of angiogenesis regulation linked to the vascular epithelium. According to current conceptions, activated endothelial cells and their predecessors (progenitor cells) are involved in the regulation of angiogenesis. These cells synthesize angiogenic molecules differing by the chemical structure and mechanism of biological effect and allowing a direct or indirect control over each stage of angiogenesis. Both the excess and insufficient angiogenesis can lead to fast and irreversible changes in nervous tissue under certain conditions. For this reason, the balance in the system of molecular stimulators and inhibitors of angiogenesis is especially important for brain function. Without adequate reperfusion of an affected brain area the post-stroke neuroreparation, which can be provided with timely stimulation of angiogenesis, is unattainable and the intensification of this process in tumors, on the contrary, has adverse consequences. Growth of a tumor and its metastatic spread are substantially associated with an increase in the level of tumor tissue vascularization, and blocking angiogenesis is often the only productive way to limit the growth of a tumor. However our knowledge of mechanisms of angiogenesis regulation in the brain on the cellular and molecular level in physiological and pathological conditions is still insufficient, and, therefore, the influence of angiogenic factors on tissue targets do not always cause the expected effects.

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.

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.

[Effects of chronic tabacco smoking on the cerebral blood flow]. / Vliyanie khronicheskogo tabakokureniya na tserebral'nuyu gemodinamiku.

AIM: To study an effect of chronic smoking on cerebral hemodynamics and cerebrovascular reactivity in different segments of the arterial system of the brain in men. MATERIAL AND METHODS: Male patients enrolled in the study were divided into two groups: controls (n=11) and smokers (n=24) with index 30.7 pack-year smoking history. Haemorheological parameters: viscosity of blood, aggregation of erythrocytes, hematocrit, fibrinogen were evaluated in both groups. Using carotid duplex ultrasound (MyLab 50 Esaote, Italy), intima-media thickness (IMT) of common carotid arteries (CCA) was measured. Parameters of cerebral hemodynamics were evaluated using transcranial Doppler ultrasound (Nicolet Companion Biomedikal, USA). Tests of the cerebrovascular reactivity were used to study arteries of three levels: common carotid arteries, middle cerebral arteries (MCA) and pial arteries. RESULTS: An increase in erythrocytes, hemoglobin, hematocrit, viscosity of blood, aggregation of erythrocytes and fibrinogen was found in the group of smokers compared to the control group. There were an increase in IMT of CCA by more than 50% and a decrease in the mean flow velocity in CCA in smokers. Also smoking decreased MCA mean flow velocity, but this decrease was 2-3 times smaller than in the common carotid arteries. Vasoconstrictor reactions prevailed in MCA, as indicated by the increase in the peripheral vascular resistance index. An increase in vasospastic reactions in pial arteries in smokers was discovered. At the same time, the vasodilatation reaction was maintained or not significantly reduced in pial arteries. CONCLUSION: Chronic smoking is one of the significant factors causing changes in haemorheological parameters, damage of vascular wall, initiation of atherogenesis and disturbance of cerebral hemodynamics. The study of velocity characteristics and peripheral vascular resistance indices in the group of smokers revealed different reactions of different segments of the brain arterial system.

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.

[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.

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.

[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.

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.

[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.