[LONG-TERM EFFECTS OF EXPOSURE TO PERINATAL HYPOXIA ON MICROVASCULAR ENDOTHELIUM OF NEOCORTEX IN RATS].

Morphological characteristics of the elements of vessel walls in the microvasculature of rat neocortex were studied after perinatal hypoxia and subsequent introduction of salifen, a derivative of GABA, at long-term postnatal periods. It is shown that salifen at a therapeutic dose has a protective effect on the endothelium of blood vessels in the microvasculature of neocortex. It is found that after exposure to hypoxia followed by application of salifen no endothelial hypertrophy, appearance of numerous processes of endotheliocytes in the vascular lumen, and narrowing of the capillary lumen occur. The formation of la- mina densa is almost identical to that in the control. Swelling of perivascular astrocytes and reactive chan- ges of pericytes are also absent. In all layers of the neocortex the density of vessel distribution in the mic- rovasculature as well as their cross-sectional area in adult animals of the control group and after exposure to hypoxia and salifen application were approximately identical. It is shown that changes and restructu- ring of the capillary bed after exposure to hypoxia and application of salifen take place at earlier terms of development whereas by the maturity period stabilization of the structural parameters of the microvas- culature occurs. The protective effect of salifen on the elements of capillary walls determines the clinical effectiveness of its use and serves as a basis for further research in this direction.

[EXPRESSION OF SEROTONIN TRANSPORTER IN THE DORSAL RAPHE NUCLEUS DURING THE EARLY POSTNATAL PERIOD IN NORMAL STATE AND UNDER PRENATAL DEFICIENCY OF THE SEROTONERGIC SYSTEM IN RATS].

The expression of the serotonin transport membrane protein (5-NTT) in the dorsal raphe nucleus (DNR) was investigated in laboratory Wistar rats during the early postnatal period. The results of the immunocytochemical study using primary antibodies--anti-Serotonin transporter antibody (AbCam, UK)--showed that during the first 3 postnatal weeks the intensity of 5-NTT expression in DNR of control animals changes. At the earliest postnatal times the main part of subnuclear neurons (dorsal, ventral and lateral ones) of the dorsal raphe nucleus (DNR-d, DNR-v, DNR-lat) was shown to intensely express 5-NTT. Sites of 5-NTT localization are found on the membrane surface of neuron bodies and processes in neuropile. The reduction in the number of neurons expressing 5-NTT and of its binding sites was observed on P10. At this time a redistribution of 5-NTT localization sites occurs: they are very few on neuron bodies and dendrites but are located rather densely on the plasma membrane of axons. The number of neurons expressing 5-NTT gradually increases with age and in neuropile the density of 5-NTT localization sites rises. It is shown that during the prenatal development the reduction of serotonin level in all parts of the DNR leads to a reduction in both the number of neurons expressing 5-NTT and sites of its localization in the early postnatal period, this trend continuing with age.

[FORMATION OF GABA-ERGIC NEURAL NETWORK IN BÖTZINGER COMPLEX IN RATS DURING EARLY POSTNATAL PERIOD IN NORM AND IN PRENATAL DEFICIENCY OF ENDOGENOUS SEROTONIN].

The dynamics of the distribution of GABAergic neurons and neurons expressing different types of GABA receptors (GABAAα1 and GABAB1) was studied in Bötzingercomplex (BötC) in the early postnatal period (the period of functional maturation of the respiratory system in mammals) in norm and prenatal reduction of serotonin content in Wistar rats. The brain was studied on postnatal Days 5, 9 and 20 in two groups of rat pups: control (n=9), born by intact females, and experimental (n=13), born from mothers that received parachlorophenylalanine, causing the depression of endogenous serotonin level. Imunocytochemical methods were used to detect the neurons producing GABA and expressing GABAAα1 and GABAB1 receptors. It was shown that the maturation of the inhibitory GABAergic network in BötC occurred in the early postnatal period (by Day 9). Simultaneously with GABA, the expression of GABAAα1 and GABAB1 receptors took place, however their maturation has the distinctive features. The formation of GABAAα1 receptors occurred earlier (by Day 9) and coincided in time with the expression of GABA. The maturation of GABAB1 receptors happened later ­ only by the third week. Prenatal serotonin deficiency caused a delay in the expression of GABA and GABAAα1 receptors by the neurons of BötC, as well as the disruption of the formation of a network of terminals and synapses containing GABA, GABAAα1 and GABAB1 receptors.

[EXPRESSION OF SEROTONIN TRANSPORT PROTEIN IN THE DORSAL RAPHE NUCLEUS IN THE EARLY POSTNATAL PERIOD IN RATS].

In this work an expression of serotonin transport protein (5-HTT) was studied in the dorsal raphe nucleus (DRN)--in its dorsal, ventral and lateral subnuclei in Wistar rats (n = 15) during the early postnatal period. Histological methods and immunocytochemical staining demonstrating 5-HTT were used in the investigation. It was shown that at postnatal Day 5 major part of neurons of the subnuclei studied intensively expressed 5-HTT. However, by Day 10, the level of its expression decreased dramatically, but with age (by Day 20) the expression was increased once again. This was manifested by the augmentation of size of 5-HTT-positive neuronal population, the increase in the density of plexus network of their processes. The detected changes of 5-HTT expression indicate the varying degrees of functional activity of serotonin in the DRN in the early postnatal period.

[Distribution of GABAergic neurons in pneumotaxic center nuclei in the early postnatal period in norm and in prenatal deficiency of serotoninergic system in rats].

The aim of this study was to determine the distribution of GABAergic neurons in pneumotaxic center structures (parabrachial complex medial subnucleus and Kölliker-Fuse nucleus) in norm and in deficiency of serotoninergic system during the prenatal period of development in Wistar rats. Reduction of endogenous serotonin levels in fetal rats was achieved by tryptophan hydroxylase inhibition with para-chlorophenylalanine (PCPA), which was administered to female rats on Day 16 of gestation. Material was obtained from the area of the pons from experimental and control (intact) rat pups at early postnatal (Days 5, 10 and 12) and juvenile (Day 20) periods. At each time point, 5-6 animals were studied from both experimental and control groups. To demonstrate GABAergic neurons, antibodies against glutamate decarboxylase (GAD-67), the enzyme involved in its synthesis, were used. The results have shown that Kölliker-Fuse nucleus contained a population of GABAergic neurons at early postnatal period, the size of which was preserved until juvenile age. In parabrachial complex medial subnucleus during the early postnatal period, a small number of GABAergic neurons was detected, which was somewhat increased by juvenile age. Serotonin deficiency in pneumotaxic center structures lead to a reduction of the numbers of GABAergic neurons, GABAergic synapses and their clusters. A reduction of serotonin levels during the prenatal period may cause the disturbances in the inhibitory afferent signaling of the pneumotaxic center nuclei and lead to the changes of local inhibitory GABAergic networks in its nuclei, resulting in the disturbances of the inhibitory processes in the center structures.

[THE EFFECT OF PERINATAL HYPOXIA ON THE STRUCTURE OF BLOOD-BRAIN BARRIER IN RATS TREATED WITH SALIFEN].

The work was performed on Wistar rats, which were dividedinto 3 groups: 1st group--experimental rats subjected to hypoxia and treated with salifen (15 mg/kg for 14 days), 2nd group--control rats exposed to hypoxia only without treatment, and 3rdgroup--intact animals (8-10 animals in each group). Using themethods of light and electron microscopy, the effect of salifen onthe structural characteristics of the elements of the blood-brainbarrier (BBB) in the neocortex was studied in rats after exposureto hypoxia in the early postnatal period--on postnatal Day 2(model of human preterm pregnancy). The results showed thatsalifen had a positive effect on the state of the microvasculatureafter perinatal hypoxia, in particular, on the state of endothelialcells. Its active participation in the compensatory-adaptive reactions of the BBB in response to hypoxia exposure was detected,and the prospects of further studies of the protective properties ofsalifen are emphasized.

[EXPRESSION OF SEROTONIN TRANSPORTER IN THE VENTROLATERAL PART OF THE NUCLEUS OF THE SOLITARY TRACT IN RATS IN THE EARLY POSTNATAL PERIOD IN NORM AND FOLLOWING SEROTONERGIC SYSTEM DEFICIENCY IN THE PRENATAL PERIOD OF DEVELOPMENT].

The expression of serotonin transporter (5-HTT) was examined using the immunocytochemical method in the ventrolateral part of the nucleus of the solitary tract in Wistar rats in the early postnatal period (Days 5 and 10) in norm (n=10) and after prenatal serotonin deficiency (n=12). Temporary expression of the 5-HTT was demonstrated during the early postnatal period in the ventrolateral division of the nucleus of the solitary tract, that was more pronounced in the most caudal part of the ventral submucleus. It was shown that in the rostral part of ventral and lateral subnuclei, the number of neurons synthesizing 5-HTT was low and remained unchanged with age. In the caudal region of the ventral subnucleus, a large number of neurons synthesizing 5-HTT was found on Day 5 of postnatal period, that was significantly reduced with age. In the caudal region of the lateral subnucleus, a small number of neurons expressing 5-HTT was detected, that also decreased with age. It was found that in the caudal region of both ventral and lateral subnuclei, the level of 5-HTT expression was significantly higher than in the rostral region. Prenatal deficiency of serotonin resulted in a reduction of the number of neurons synthesizing 5-NTT in all the subnuclei investigated.

[REACTION OF STRUCTURAL ELEMENTS OF HEMATOENCEPHALIC BARRIER IN NEWBORN RATS TO NORMOBARIC HYPOXIA].

For the last time a particular interest of investigators has been attracted to the period of early newborn state when active process of adaptation of the organism to new life conditions occur defining its increased sensitivity to the effect of unfavorable environmental factors. An important place among these processes belongs to formation of homeostasis mechanisms and, primarily, the barrier mechanisms. The purpose of the present study was to investigate reactions of the hematoencephalic barrier (HEB) to action of perinatal normobaric hypoxia (a model of incomplete human pregnancy). Using light and electron microscopy, our investigation showed that after action of hypoxia all wall elements of neocortex capillaries revealed structural alterations which may cause impairment of microcirculation and increased permeability of HEB. It is established that differentiation of the basal membrane of a capillary wall takes place during the early perinatal period and the indicator of its differentiation is the formation of its plates - laminae rara et densa. After action of hypoxia, besides a delay of formation of the basal membrane, a vesicular type of its degeneration occurs. Key words: perinatal hypoxia, hematoencephalic barrier, capillary, endothelial cells, basal membrane.

[Reactions of neural elements of neocortex on action of hypoxia at the early neonatal period in rats].

In this work we studied reactions of neural elements of various neocortex areas (sensomotor, visual, auditory) on action of acute normobaric hypoxia. The study is performed on the model of human premature pregnancy (action of normobaric hypoxia on rat pups at the 2nd postnatal day). There are revealed monotypical and monodirected structural reconstructions in all studied neocortex parts. The chosen parameters of hypoxic action initiate several direct reactions as early as at the next day: a decrease in sizes of cell bodies and in volume of the cytoplasm, as well as an enhancement, as compared with control, of the apoptotic cell death. By the end of the neonatal period (5 days), several ultrastructural alterations indicating deceleration of processes of differentiation of nerve cells are revealed: arrest of processes of complication of smooth and rough endoplasmic reticulum and of Golgi apparatus, a small number of single ribosomes and polysomes in the cytoplasm, a decrease of the number of growth cones of axons and dendrites in neuropil, delay and disturbance of myelination processes in nerve fibers. The detected morphofunctional reconstructions can serve the structural ground for development of neonatal encephalopathies.

[Reactions of the interneuronal synapses of rat brain to hypoxia during the early postnatal period].

The reactions of forming synapses in rat neocortex to the effect of hypoxia in the early postnatal period (day 2) were studied. Using immunocytochemistry for synaptophysin demonstratoion and electron microscopic methods, the sensorimotor cortex was studied in rats at days 3, 4 and 10 of postnatal development (6 to 10 animals of each age) in both experimental and control groups (intact animals). Immunocytochemical study of the control animals demonstrated significant differences in the quantitative distribution of synaptophysin-positive structures in the different layers of the neocortex in the early postnatal period of development (day 5). It is shown that after exposure to perinatal hypoxia, more than 2-fold decrease of the optical density of the immunocytochemical reaction product took place together with the reduction of synaptophysin-positive granules distribution density in all cortical layers of. At the same time, electron-dense terminals demonstrating early degenerative processes were found. In the neuropil of the neocortex, a sharp decline in the number of growth cones, small processes and forming synapses was accompanied by significant changes of the electron density of synaptic, especially post-synaptic, membranes and densities. In the experimental animals, the number of growth cones and emerging synaptic structures were shown to increase only by postnatal day 10. Thus, the effects of hypoxia in the early postnatal period, causing disturbances of synaptogenesis, persist throughout the whole neonatal period examined.

[The distribution of GABA-ergic neurons in rat neocortex in the postnatal period after the perinatal hypoxia].

The distribution of GABA-ergic neurons in different areas of the neocortex (frontal, sensorimotor, visual cortex) was studied in Wistar rats at different time periods of postnatal development after their exposure to perinatal hypoxia. To identify these neurons, the antibodies against GAD-67, the marker of GABA-ergic neurons, were used. It was found that the exposure to perinatal hypoxia caused a significant reduction in the number of GAD-67-expressing neurons in both upper and deep layers of the cortex in juvenile age (day 20 of postnatal period), that persisted until the prepubertal period (day 40). In experimental animals at postnatal day 40, the numbers of neurons that synthesized GAD-67, were two times lower in each of the layers of the neocortex than those in control animals. It is suggested that a drastic reduction in the number of GABA-ergic neurons in the neocortex could be a result of the damaging effects of acute perinatal hypoxia on the processes of progenitor cell migration from the subventricular zone, or on the synthesis of the factors controlling these migration processes as well as on GABA-ergic neuron maturation, leading to a delay of GAD-67 expression.

[Changes of the structural organization of the nucleus raphe pallidus after the decrease of endogenous serotonin level in the prenatal period of development in rats].

The role of serotonin in the nucleus raphe pallidus (NRP) development and the dynamics of its serotonin-producing neurons were studied during various time points of the postnatal period in normal Wistar rats and in animals developing prenatally under the conditions of serotonin deficiency. It was shown that NRP contained 2 populations of serotoninergic neurons with different morphological characteristics. At the initial stages of postnatal development (Day 5) serotonin-producing neurons included only large neurons, while the synthetic activity of small neurons appeared later (by Day 10). With age, under normal conditions,the size of large neurons and their number were increased which is indicative of continuing process of differentiation and/or functional load augmentation. The size and number of small neurons were practically unchanged with age. Serotonin deficiency during prenatal development lead to the disturbance of NRP structural organization. In comparison with the control animals, the size and the number of serotonin-producing neurons of both populations was decreased, their size remained unchanged with the age. Part of the neurons underwent degeneration, resulting in the reduction of their numbers. The damage observed may change the serotoninergic innervation of the medullary nuclei, responsible for the cardiorespiratory the control, thus causing the disturbances of cardio-vascular and respiratory systems.

[Reactive microglial changes in rat neocortex and hippocampus after exposure to acute perinatal hypoxia].

The dynamics of reactive changes of a population density of microglial cells and the reversibility of their phenotypic forms were studied in the brain of neonatal rats at different time intervals after 1 hr-long exposure to acute normobaric hypoxia in the pressure chamber at the second postnatal day. Different areas of the neocortex (frontal, motor, somatosensory and visual) and of the hippocampus (CAI, CA3, CA4 and fascia dentata) were examined 1 hr, 3 hrs, 1 and 5 days after exposure to hypoxia. Microglial cells were demonstrated using an immunocytochemical staining with the monoclonal antibodies against Iba- 1 antigen. The results have shown that the reaction of microglia to acute hypoxia in both the neocortex and the hippocampus of the new-borns developed simultaneously and synchronously with the augmentation of cell death. The increase of a population density of amoeboid form of microglial cells in the brain areas studied was recorded already after 1 hour as a result of their migration from the subventricular region and the areas adjacent to large vessels from where they practically disappeared. The number of amoeboid microglial cells in this area has recovered rather quickly (in 3 hrs). The population densify of microglial cells, especially of amoeboid forms, sharply increased with the augmentation of cell death and remained unchanged for about 5 days.

[Structural changes in the subnuclei of parabrachial complex in the deficiency of serotoninergic system in the prenatal period of rat development].

The study was performed on the offspring, obtained from 8 female Wistar rats, which were administered parachlorophenylalanine on day 16 of gestation, to deplete the endogenous serotonin in their offspring. Dorsal and ventral subnuclei of the parabrachial complex lateral part were studied in rats on postnatal days 10 and 14 days (5-6 animals for each time point) using histological, immunocytochemical and morphometric methods. It was shown that serotonin deficiency changed the structure of the subnuclei. In the ventral subnucleus, the population of large multipolar neurons was more sensitive to low levels of serotonin. In experimental animals, the size of neuron cell bodies was reduced 1.6 times in comparison with the control, while cell numbers in the population remained practically unchanged. Morphological characteristics of smaller neurons corresponded to those in the control group, but their numbers were reduced, which probably can explain some rarefaction of neurons in the nucleus. In dorsal subnucleus of the experimental animals, the neurons of both populations had more pronounced changes. Sizes of the cell bodies in both large and small neurons was significantly decreased 2.0 and 1.8 times, respectively; the volume of their cytoplasm was significantly reduced, compared to that in control animals, while the number of cells in both populations remained practically unchanged.The reaction demonstrating glial fibrillary acidic protein has shown that the astrocytic glial population was increased in both subnuclei in the experimental animals in the early postnatal period.

[Characteristic of serotoninergic neurons of medullary nucleus raphe obscurus in norm and in serotoninergic system deficiency during the prenatal period of development in rats].

Morphological characteristics of the serotoninergic neurons forming nucleus raphe obscurus (NRO), were studied in rats at the early stages (days 5, 10, 12 and 14) of the postnatal period in normal rats and in animals whose prenatal development took place under the conditions of serotonin deficiency. NRO was found to contain three subpopulations serotonin-producing neurons (large, medium and small), which had different sensitivity to serotonin level during development. The results have shown that serotoninergic system deficiency during the prenatal period resulted in the changes of NRO structural organization and in the decrease of the rate of this nucleus formation, serotonin-producing neurons differentiation and the reduction of their total number by approximately a factor of 1.6. At the same time, the significant changes of the dimensions of serotoninergic neurons of all types took place. In control animals, the size of large, medium and small neurons was 1.8, 1.4 and 1.5 times greater than that in experimental animals, respectively. Reduction of the neuron dimensions was associated with the changes of a nucleo-cytoplasmic ratio. The volume of the cytoplasm and of Nissl bodies was significantly decreased. Along with it, the cell destruction was noted that increased with age. Synchronously with it, the marked astrocytic reaction developed, which could further lead to gliosis.

[Effect of hypoxia in early perinatal ontogenesis on behavior and structural characteristics of the rat brain].

The study has shown the acute hypoxia in newborn rat pups to lead to disturbances of processes of formation of brain structures, behavior reactions, and learning in the subsequent ontogenesis. The single normobaric hypoxia at the 2nd day of life causes retardation of such integrative parameter or genera development and growth as body mass at the period of feeding. In such animals, essential disturbances of the sensorimotor development were revealed in forms of delay of reflex reactions of turning on a plane, negative geotaxis, and avoidance of edge. Also detected was action of hypoxia on hanging on a rope by using front legs (a symptom of muscle weakness). Morphological study has shown stereotypic reaction to the early postnatal action of hypoxia in all studies of all studied functional zones of neocortex - motor, sensomotor, auditory, visual. The death of nerve cells is predominant in the II-III associative layers, the sizes and number of pyramidal neurons are sharply decreased. Different hippocampus fields maturing in mammals show a characteristic response to hypoxia. In individual hippocampus fields there was detected different degree of death of neurons, predominant in the CA3 and CA4 fields. A possibility of modeling of perinatal encephalopathy with minimal brain dysfunctions in children is discussed.

[Participation of serotonin in the mechanisms of the formation of trigeminal motor nucleus].

The formation of trigeminal motor nucleus (TMN) was studied in the early postnatal period in 21 female Wistar rats which received the serotonin biosynthesis inhibitor para-chloro-phenylalanine at prenatal Day 16 (the period of serotoninergic system formation). It was shown that the serotonin deficit during the prenatal period in rats resulted in the changes of TMN structural organization. In the early postnatal period, the delay of neuropil development, the reduction of cell body size with the partial loss of Nissl substance in some of the neurons, the presence of degenerating neurons with the signs of hyperchromatosis in all the parts of the nucleus, especially in TMN ventromedial part, were detected. At later stages, the destruction of motoneurons became slower, though some of them had morphological abnormalities. With the increase of the postnatal age (by Day 20) the number of motor neurons decreased, apparently, as a result of the gradual intensification of cell death. Simultaneously with the motor neuron degeneration in TMN parts studied, the astrocytic gliosis was observed.

[Synaptogenesis in the dorsal raphe nucleus of rat medulla oblongata in serotonin deficiency].

The purpose of the present study was to examine the synaptogenesis in the dorsal raphe nucleus (DRN) in Wistar rats (n = 8-10 for each time point) in the end of prenatal (days 19 and 20) and early postnatal (days 5 and 20) periods, and to determine the role of serotonin in the formation of synaptic contacts during this period of development. It is shown that at prenatal dayl9 neuropil started to develop in DRN -d, DRN -v and DRN -1 in control (intact) animals, while the synaptic contacts appeared on cell processes. At prenatal day20 synaptic contacts were detected for the first time on neuronal cell body surface. The density of synaptophysin-positive granules on both the cell processes in the neuropil, and neuronal cell bodies was sharply and considerably increased by postnatal day 5. Subsequently, until postnatal day 20, the augmentation of their density was insignificant. Serotonin deficiency in a second half of prenatal development, induced by a single injection of parachlorophenylalanine to female rats at gestational day 16 resulted in a significant delay of synaptogenesis in DRN of their offspring in both prenatal and early postnatal periods. With the increase of postnatal age the density synaptic contacts was unequally augmented in various parts of DRN: in DNR-d it approached the control level, while in DRN-v and DRN-1 it remained significantly reduced. The results received suggest serotonin participation in synaptogenesis in DRN.