[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.

[Development of blood-brain barrier under the modulation of HIF activity in astroglialand neuronal cells in vitro]. / Osobennosti formirovaniia gematoéntsefalicheskogo bar'era pri moduliatsii aktivnosti HIF v kletkakh astroglial'noi i neironal'noi prirody in vitro.

Barriergenesis is the process of maturation of the primary vascular network of the brain responsible for the establishment of the blood-brain barrier. It represents a combination of factors that, on the one hand, contribute to the process of migration and tubulogenesis of endothelial cells (angiogenesis), on the other hand, contribute to the formation of new connections between endothelial cells and other elements of the neurovascular unit. Astrocytes play a key role in barriergenesis, however, mechanisms of their action are still poorly examined. We have studied the effects of HIF-1 modulators acting on the cells of non-endothelial origin (neurons and astrocytes) on the development of the blood-brain barrier in vitro. Application of FM19G11 regulating expression of HIF-1 activity and GSI-1 suppressing gamma-secretase and/or proteasomal activity resulted in the elevated expression of thrombospondins and matrix metalloproteinases in the developing blood-brain barrier. However, it caused the opposite effect on VEGF expression thus promoting barrier maturation in vitro.

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.

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.

Expression of Glutamate and Glutamine Transporter Proteins in Neurovascular Unit Cells In Vitro.

Glutamine transporter protein SLC1A5 and glutamate transporter protein EAAT2 responsible for cell-cell communication and energetic coupling were studied using in vitro model of multicellular neurovascular unit consisting of astrocytes, neurons, and endotheliocytes under standard conditions and during chemical hypoxia in vitro. Hypoxic damage to the neurovascular unit cells increased the number of SLC1A5-expressing cells and reduced the number of EAAT2-expressing astrocytes. Metabolic uncoupling in the neurovascular unit cells under hypoxic conditions resulted from abnormal expression of glutamine and glutamate transporter proteins, which is indicative of impaired glutamine and glutamate transport.

In Vitro Modeling of Brain Progenitor Cell Development under the Effect of Environmental Factors.

We studied in vitro development of brain progenitor cells isolated from healthy 7-9-month-old Wistar rats and rats with experimental Alzheimer's disease kept under standard conditions and in enriched (multistimulus) environment in vivo. Progenitor cells from healthy animals more rapidly formed neurospheres. Considerable changes at the early stages of in vitro development of brain progenitor cells were observed in both groups kept in enriched environment.

Elimination of iron-containing magnetic nanoparticles from the site of injection in mice: a magnetic-resonance imaging study.

Suspension of magnetic nanoparticles (0.7 g/liter) obtained from Klebsiella oxytoca culture was injected intraperitoneally (1 ml), intramuscularly (in the hip; 100 µl), and subcutaneously (200 µl) or administered orally instead of drinking water for 2 days. The presence of magnetic nanoparticles was evaluated detected by MRI in 15 min and 2 h after injections and in 1 and 2 days after the beginning of oral consumption of the suspension. Magnetic nanoparticles were eliminated from the site of intramuscular and intraperitoneal injections and after oral consumption. The period of elimination after intramuscular and intraperitoneal injections did not exceed 2 h, while after oral consumption it corresponded to the time of gastrointestinal tract contents evacuation.

[THE MODEL OF NEUROVASCULAR UNIT IN VITRO CONSISTING OF THREE CELLS TYPES].

There are many ways to model blood brain barrier and neurovascular unit in vitro. All existing models have their disadvantages, advantages and some peculiarities of preparation and usage. We obtained the three-cells neurovascular unit model in vitro using progenitor cells isolated from the rat embryos brain (Wistar, 14-16 d). After withdrawal of the progenitor cells the neurospheres were cultured with subsequent differentiation into astrocytes and neurons. Endothelial cells were isolated from embryonic brain too. During the differentiation of progenitor cells the astrocytes monolayer formation occurs after 7-9 d, neurons monolayer--after 10-14 d, endothelial cells monolayer--after 7 d. Our protocol for simultaneous isolation and cultivation of neurons, astrocytes and endothelial cells reduces the time needed to obtain neurovascular unit model in vitro, consisting of three cells types and reduce the number of animals used. It is also important to note the cerebral origin of all cell types, which is also an advantage of our model in vitro.

[Current concepts of perinatal ischemic injury in the brain neurovascular unit: molecular targets for neuroprotection].

Perinatal hypoxic-ischemic brain injury is a relevant medical and social problem. Among many pathological processes in the neonatal period perinatal hypoxic-ischemic injury is a major cause of further hemorrhage, necrotic and atrophic changes in the brain. This review presents recent data on the basic mechanisms of the hypoxic-ischemic brain injury along the concept of neurovascular unit (neurons, astrocytes, endothelial cells, pericytes) with the focus on alterations in cell-to-cell communication. Pathological changes caused by ischemia-hypoxia are considered within two phases of injury (ischemic phase and reperfusion phase). The review highlights changes in each individual component of the neurovascular unit and their interactions. Molecular targets for pharmacological improvement of intercellular communication within neurovascular unit as a therapeutic strategy in perinatal brain injury are discussed.