Hypercapnia Modulates the Activity of Adenosine A1 Receptors and mitoK+ATP-Channels in Rat Brain When Exposed to Intermittent Hypoxia.

The mechanisms and signaling pathways of the neuroprotective effects of hypercapnia and its combination with hypoxia are not studied sufficiently. The study aims to test the hypothesis of the potentiating effect of hypercapnia on the systems of adaptation to hypoxia, directly associated with A1-adenosine receptors and mitochondrial ATP-dependent K+ -channels (mitoK+ATP-channels). We evaluated the relative number of A1-adenosine receptors and mitoK+ATP-channels in astrocytes obtained from male Wistar rats exposed to various respiratory conditions (15 times of hypoxia and/or hypercapnia). In addition, the relative number of these molecules in astrocytes was evaluated on an in vitro model of chemical hypoxia, as well as in the cerebral cortex after photothrombotic damage. This study indicates an increase in the relative number of A1-adenosine receptors in astrocytes and in cells next to the stroke region of the cerebral cortex in rats exposed to hypoxia and hypercapnic hypoxia, but not hypercapnia alone. Hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels in astrocytes and in cells of the peri-infarct region of the cerebral cortex in rats. In an in vitro study, hypercapnia mitigates the effects of acute chemical hypoxia observed in astrocytes for A1-adenosine receptors and mitoK+ATP-channels. Hypercapnia, unlike hypoxia, does not affect the relative number of A1 receptors to adenosine. At the same time, both hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels, which can potentiate their protective effects with combined exposure.

[Plasma acid reproduces oxidative and nitrosative stress in bladder tissue in vitro: experimental study].

AIM: To analyze some effects of plasma acid in vitro on the bladder tissue obtained from laboratory animals and to evaluate the possibility of its application for in vitro modeling of IC/BPS. MATERIALS AND METHODS: The tissue samples of the bladder wall were obtained from female Wistar rats aged 3 months (n=16, weighing 180-200 g). The tissues were processed for 1 hour in the plasma acid prepared by spark discharge of water for injection in air. The immunohistochemical study of obtained samples was performed. RESULTS: The changes in the expression profile of bladder epithelial cells under the action of plasma acid in vitro were found indicating the development of oxidative, nitrosative and dicarbonyl stress, impaired expression of NADPH oxidase DUOX2 and VEGF, and a decrease in cell proliferative activity, which, in general, corresponds to the main mechanisms of urothelial alterations specific for the IC/BPS. CONCLUSION: The revealed effects of plasma acid on bladder epithelial cells confirm the possibility of using it as an inducer of urothelial cell damage typical for IC/BPS in the in vitro models.

Neurogenic Potential of Implanted Neurospheres Is Regulated by Optogenetic Stimulation of Hippocampal Astrocytes Ex Vivo.

The protocol of optogenetic ChR2-mediated activation of astrocytes was used in a model of artificial neurogenic niche, neurospheres implanted into ex vivo organotypic cultures of mouse hippocampus. The electrophysiological characteristics of the hippocampus and expression of molecules involved in the mechanisms of activation of astrocytes and microglia (GFAP, CD38, C3/C3b, Cx43, CD11b, and CD18) were evaluated. Photoactivation of astrocytes led to activation of neurogenesis and changes in the expression of molecules (Cx43 and CD38) that determine bioavailability of NAD+ to ensure proliferative activity of cells in the neurogenic niche. Implantation of neurospheres into organotypic slices of the hippocampus caused an increase in C3/C3b expression and suppression of the synaptic plasticity of hippocampal neurons.

[Effect of sliding discharge on proliferation and death of brain microvessel endothelial cells in vitro]. / Vliianie skol'ziashchego razriada na proliferativnuiu aktivnost' i gibel' kletok éndoteliia tserebral'nykh mikrososudov in vitro.

The dose-dependent effects of plasma exposure to a unipolar nanosecond sliding discharge over the surface of the culture medium in a closed plate on the cells of cerebral endothelium in vitro were studied. Using a 24-well plate, the surface plasma energy density of one pulse was 360 µJ/cm2 at a pulse frequency of 100 Hz. It has been shown that in the creeping discharge plasma there is an active excitation of air molecules, the formation of positive nitrogen and oxygen ions, and the formation of carbon monoxide. In the dose density range of 0-32 J/cm2, the dose-dependent effects were assessed in the 4-12 h post-radiation period. Cell death was analyzed with an assessment of the total number of cells, necrotic cells, cells in apoptosis (phosphatidylserine externalization, internucleosomal DNA fragmentation) and their proliferative activity (Ki67-immunopositive cells). A preliminary assessment of subtle dose-dependent effects indicates the peculiarities of the effect of small doses.

[Features of the in vitro expression profile of hippocampal neurogenic niche cells during optogenetic stimulation]. / Osobennosti ékspressionnogo profilia kletok v modeli neirogennoi nishi gippokampa in vitro pri optogeneticheskoi stimuliatsii.

In the central nervous system of mammals, there are specialized areas in which neurogenesis - neurogenic niches - is observed in the postnatal period. It is believed that astrocytes in the composition of neurogenic niches play a significant role in the regulation of neurogenesis, and therefore they are considered as a promising "target" for the possible control of neurogenesis, including the use of optogenetics. In the framework of this work, we formed an in vitro model of a neurogenic niche, consisting of cerebral endothelial cells, astrocytes and neurospheres. Astrocytes in the neurogenic niche model expressed canalorodopsin ChR2 and underwent photoactivation. The effect of photoactivated astrocytes on the expression profile of neurogenic niche cells was evaluated using immunocytochemical analysis methods. It was found that intact astrocytes in the composition of the neurogenic niche contribute to neuronal differentiation of stem cells, as well as the activation of astroglia expressing photosensitive proteins, changes the expression of molecules characterized by intercellular interactions of pools of resting and proliferating cells in the composition of the neurogenic niche with the participation of NAD+ (Cx43, CD38, CD157), lactate (MCT1). In particular, the registered changes reflect a violation of the paracrine intercellular interactions of two subpopulations of cells, one of which acts as a source of NAD+, and the second as a consumer of NAD+ to ensure the processes of intracellular signal transduction; a change in the mechanisms of lactate transport due to aberrant expression of the lactate transporter MCT1 in cells forming a pool of cells developing along the neuronal path of differentiation. In general, with photostimulation of niche astrocytes, the total proliferative activity increases mainly due to neural progenitor cells, but not neural stem cells. Thus, optogenetic activation of astrocytes can become a promising tool for controlling the activity of neurogenesis processes and the formation of a local proneurogenic microenvironment in an in vitro model of a neurogenic niche.

Current Methods for the Isolation and Cultivation of Microglia (Review).

The role and morphological features of microglia (M1 and M2 microglia, "stellate", "amoeboid", giant, round-shaped, rod-shaped, dysfunctional, etc.) in vivo under physiological conditions and during the development of neurodegenerative diseases have been described. Various methods and techniques of microglia isolation from adult (density gradient isolation, use of "magnetic beads", from mesenchymal bone marrow progenitor cells) and newborn (obtaining from a mixed glial culture, density gradient isolation) animals have been considered, including microglia isolation from the cerebral cortex or hippocampus. Various methods of cell cultivation have been shown, including obtaining two-dimensional and three-dimensional cell cultures (on scaffolds, hydrogels, nanofibers), co-cultures on slice cultures of the hippocampus, as well as changes in microglia during cultivation.

Hypercapnia and Hypoxia Stimulate Proliferation of Astrocytes and Neurons In Vitro.

We compared proliferative activity and hypoxic tolerance in a co-culture of neurons and astrocytes in vitro after preliminary exposure to normobaric hypoxia and/or permissive hypercapnia in vivo. Preliminary hypoxic exposure increased the cell index throughout the 72-h period of observation, the effect of hypercapnia was observed on days 1 and 3 of the experiment, and the effect of hypercapnic hypoxia was noted only on day 1. Preliminary hypoxic exposure has a protective effect on nerve cells under conditions of chemical hypoxia. This suggests that hypercapnia and hypoxia activate proliferative activity of nerve cells, which can be viewed as a mechanism of their neuroprotective effectiveness.

Inhibition of Apoptosis is a Potential Way to Improving Ischemic Brain Tolerance in Combined Exposure to Hypercapnia and Hypoxia.

We compared the intensity of apoptosis in the peri-infarction area of the brain after isolated and combined exposure to hypoxia and hypercapnia prior to focal ischemic stroke modeling. Hypoxia and hypercapnia reduced the number of TUNEL-positive cells in the peri-infarction area, and their combination was most effective in comparison with effects of isolated exposures. The maximum neuroprotective effect of combined exposure to hypoxia and hypercapnia in comparison with isolated exposures was determined by inhibition of apoptosis in the peri-infarction zone.

Perinatal Brain Injury is Accompanied by Disturbances in Expression of SLC Protein Superfamily in Endotheliocytes of Hippocampal Microvessels.

The peculiarities in expression of transport proteins and the proteins implicated in the control of glycolysis by the cellular components of neurovascular units were examined in animals of different age under normal conditions and after modeled perinatal stress or hypoxic brain injury. In both cases, the specialties in expression of transport proteins in ontogenesis were revealed. The perinatal hypoxic brain injury resulted in up-regulation of MCT1, MCT4, and GLUT4 expression in endotheliocytes of hippocampal microvessels accompanied by transient elevation of HIF-1α and GSK3 expression.

[CLINICAL AND PATHOGENETIC PECULIARITIES OF DEVELOPMENT OF ENDOTHELIAL DYSFUNCTION].

The purpose of the study was to examine the relationship between functional parameters, arterial rigidity, lipid profile, markers of systemic inflammation and endothelial dysfunction in patients with COPD and COPD + coronary heart disease. We examined 110 subjects divided into 3 groups. G group 1 included 40 patients with severe and very severe COPD, group 2 consisted of 40 patients with severe and very severe COPD + coronary artery disease, the control group was comprised of 30 healthy volunteers. We studied parameters of respiratory function, the level of blood oxygenation, the main characteristics of arterial rigidity plasma lipid, TNF-α, CRP, fibrinogen, sPECAM-1 levels and the expression of CD38/ADP-ribosylcyclase in peripheral blood lymphocytes. The study revealed increased rigidity of the central arteries in the patients of groups 1and 2 regardless of the duration of observation and the presence of coronary artery disease, as evidenced by the increase of the pulse wave velocity in the aorta. Patients of both groups had elevated levels of TNF-α, CRP and fibrinogen indicating systemic inflammatory response. Taken together, the enhanced expression of CD38 in peripheral blood lymphocytes, elevated concentration of soluble CD31 (sPECAM-1) in both groups related to bronchial obstruction and neutrophil elastase activity as well as increased rigidity of the vascular bed gives evidence that the CD38 and sCD31 relationship plays a role in the formation of endothelial dysfunction, and dysregulation of vascular tone in COPD patients. Disorders of lipid metabolism combined with increased rigidity of the vascular wall, elevated levels of markers of systemic inflammation and endothelia dysfunction, suggest that patients with COPD are at risk of cardiovascular events.

TIGHT JUNCTIONS PROTEINS IN CEREBRAL ENDOTHELIAL CELLS DURING EARLY POSTNATAL DEVELOPMENT.

Formation and functional plasticity of the blood-brain barrier is associated with the molecular events that occur in the brain neurovascular unit in the embryonic and early postnatal development. To study the characteristics of barriergenesis under physiological conditions, as well as recovering from perinatal hypoxia and early life stress, we examined the expression of proteins of cerebral endothelial tight junctions (JAM, ZO1, CLDN5) in rats aged 7, 28 and 70 days of postnatal development (P7­P70). Under physiological conditions, we have found that the number of endothelial cells expressing JAM, ZO1, CLDN5 slightly increases in the cortex, hippocampus and amygdala of the brain in the period from P7 to P70. Perinatal hypoxia significantly increased the number of cells expressing proteins of tight junction proteins (JAM, CLDN5) up to the age P28­P70, whereas the number of cells expressing ZO1 was reduced in the same period of time. Early life stress led to an imbalance between the number of cells expressing ZO1 proteins and that expressing tight junctions proteins, but these changes were in opposite direction to that observed in perinatal hypoxia

[MODULATION OF LACTATE PRODUCTION, TRANSPORT AND RECEPTION BY CELLS IN THE MODEL OF BRAIN NEUROVASCUL. UNIT I.]

Metabolic activity of cells within a neurovascular unit is among the factors determining structural and functional integritY of the blood-brain barrier and the an- giogenesis process. in order to verify the hypothesis about the role Of g1YcolYtic activity in the perivascula astroglialcells associated with lactate release in the development of functioning of cerebral microvessel endothelial cells, we have used a three-component model of the brain neurovascular unit in vitro. The cells o f n o n -en d o th elia l o rig in w ere in c u b a te d in th e p rese n ce o f m o d u la to rs o f la c ta te pro d u c n ago ni glu c ose ta a G ly c o s o) , bas t h e oe t a n t a at- blocker of monocarboxylate transporters MCTlprCT and recepltiors of3Ctate0produasan (2-donisyoflactate G e8 breceptor) Iasa estbishe vthat that te suppression of lactate production and transport, prdc o1,adrcpin(C-O-Aa n (2gdoxysgflucoase as a glycolysis inhibitor), transport (phloretin as a sukr of lacaroduto transport , aswellasastimultionof3lactate receptors in astroglial cells, lead to aberrant development of endothelial layer, ther by u g g e tin t h efor atio o f anti ngi gencmi roen ircm ent for cerebral endothelium due to inappropriate lactate-m ediated effects. KeYw.ords:-n-eur-ovascular unit; metabolism; glYcolysis; lactate.

[Inflammation and brain aging].

The review covers current concepts on cell and molecular mechanisms of neuroinflammation and aging with the special focus on the regulation of cytokine-producing activity of astroglial cells and intercellular communication. The review reflects that a key component of the aging phenomenon as a result of ineffective implementation of anti-inflammatory response are processes of the dysregulated cytokine production, in particular, an increase in the secretion of proinflammatory cytokines and an imbalance in the expression of the receptors and receptor associated proteins. Interpretation of the molecular mechanisms of cell conjugating neuroinflammation and aging cells can give rise to new therapeutic strategies that are relevant to the treatment of a wide range of central nervous system diseases and the development of new experimental models of diseases of the central nervous system.

Perinatal hypoxic-ischemic brain injury affects the glutamatergic signal transduction coupled with neuronal ADP-ribosyl cyclase activity.

Agonists of glutamate ionotropic (NMDA) and metabotropic receptors (mGluRI and mGluRIII) had the regulatory effect on ADP-ribosyl cyclase/CD38 activity in cerebellar granular cells of newborn rats. Perinatal hypoxic-ischemic brain injury was followed by dysregulation of this mechanism.

Perinatal hypoxic and ischemic damage to the central nervous system causes changes in the expression of connexin 43 and CD38 and ADP-ribosyl cyclase activity in brain cells.

The expression of connexin 43 and CD38 and ADP ribosyl cyclase activity in brain cells were studied in rats with experimental hypoxic and ischemic damage to the CNS. Changes in the expression and activity of the enzyme were detected over the course of ischemic injury indicating a possible contribution of NAD(+)-converting activity and NAD(+)-transporting processes to the pathogenesis of acute cerebral ischemic injury.