Study of distribution of the spine apparatus protein synaptopodin in cortical brain parts of rats exposed to hypoxia at different periods of embryogenesis

A comparative study of the nervous tissue and distribution of the spine apparatus protein synaptopodin was performed in all layers of the brain sensorimotor cortex and hippocampal CAl area in control rats and in the rats exposed to hypoxia at E14 and E18. It was found that beginning from the 20th day of postnatal development, a statistically significant decrease of the mean number of labile synaptopodin-positive spines in the stratum radiatum moleculare of the hippocampal area CAl was observed in rats exposed to hypoxia both at E14 and E18. The decrease of the number of labile spines in the sensorimotor brain cortex was revealed only in the I layer beginning from the 20th day after birth in the rats exposed to hypoxia at E14. Maximal differences in the studied brain areas were observed in adult rats exposed to hypoxia at E14 in the neocortex—a decrease by 23 ± 10%, in hippocampus—by 24 ± 8%, respectively. However, no increased degeneration of neurons was detected in adult animals. It is suggested that disturbances in cognitive functions and in the capability for learning observed in rats after prenatal hypoxia can be due to a decrease of the amount of the labile synaptopodin-positive spines, which leads to a change of the structural-functional properties of neuronal networks and to a decrease of their plasticity.     

Changes in the number of nitrergic neurons following kainic acid administration and repeated long-term hypoxia

Using histochemical analysis (NADPH-diaphorase) we have been investigating the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 h a day). At the age of 22 or 90 days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the hippocampus, in the dentate gyrus and in the PAC. In 22-day-old animals both hypoxia and KA increased the number of NADPH-diaphorase positive neurons in the hilus, CA1, CA3 areas of the hippocampus and in the PAC. On the contrary, KA given to hypoxic animals lowered the number of NADPH-diaphorase positive neurons in the dentate gyrus. In 90-day-old animals, hypoxia and KA given to both normoxic and hypoxic animals lowered the number of NADPH-diaphorase positive neurons in some areas of the central nervous system.     

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.     

Possibility of shortening an aerosol immunization session with dry live plague vaccine]

A comparative study of the 10- and 30-minute seances of the animal vaccination with the dry living plague aerozol EB vaccine demonstrated that the biological and the weight aspiration doses for a 10-minute vaccination seance constituted 96 and 89% of the dose, respectively, during a 30-minute exposure. According to the results of the passive hemagglutination test immunological shifts developing in guinea pigs after the 10- and 30-minute exposure proved to be equal. On this basis it is recommended to decrease the seance of the animal vaccination from 30 to 10 minutes.     

Long-lasting changes in the density of nitrergic neurons following kainic acid administration and chronic hypoxia

Using histochemical analysis (NADPH-diaphorase) we have investigated the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 hours a day). At the age of 1 year, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in CA1 and CA3 areas of the hippocampus, in the dentate gyrus and in the PAC. Both, hypoxia and KA lowered the number of NADPH-diaphorase positive neurons in the hilus, dorsal and ventral blades of the dentate gyrus, CA1 and CA3 areas of the hippocampus. On the contrary, KA given to the hypoxic animals increased the number of NADPH-diaphorase positive neurons in the dorsal blade of the dentate gyrus and PAC.     

Effects of Hypoxia on the Activities of Noradrenergic and Dopaminergic Neurons in the Rat Brain

The effects of hypoxia (10% O2, 90% N2) on the content, biosynthesis, and turnover of noradrenaline (NA) and 3,4-dihydroxyphenylethylamine (dopamine, DA) in the rat brain were examined. Up to 24 h following exposure to hypoxia, NA content in the whole brain was decreased, whereas DA content remained unchanged. The accumulation of 3,4-dihydroxyphenylalanine (DOPA) after central decarboxylase inhibition was decreased. The turnover rate of DA after synthesis inhibition was markedly decreased up to 8 h and returned to the control level within 24 h. In contrast, the turnover rate of NA was all but unchanged, except for a 4-h exposure. The 2-h exposure to the hypoxic environment resulted in a significant decrease in NA content and DOPA accumulation in all brain regions tested, but no significant change was observed in DA content. The turnover rate of DA was remarkably decreased in all brain regions tested, whereas the rate of NA was slightly decreased only in the cerebral cortex and hippocampus. These results suggest that although hypoxia decreases the biosynthesis of both NA and DA, the effects of oxygen depletion on the functional activities of NA neurons differ considerably from those of DA neurons: Only in the cerebral cortex and hippocampus are the NA neurons slightly sensitive to hypoxia, whereas the DA neurons are most sensitive in all brain regions.     

Change of the neocortex nervous tissue in rat ontogenesis after hypoxia at various terms of embryogenesis

The performed study has shown that in rats submitted to hypoxia (3 h, 7% O2) at the 14th day of embryogenesis (E14) as compared with control animals, density of disposition of cells in the brain cortex decreased for the first month of postnatal ontogenesis (maximally by 40.8% by P20). In dying neurons, swelling of the cell body, lysis of organoids, and disturbance of the cytoplasmic membrane intactness were observed. Two waved of neuronal death by the mechanism of caspase-dependent apoptosis were revealed; the first involved large pyramidal neurons of the V layer (P10-20), the second--small pyramidal and non-pyramidal neurons of the II--III layers (P20-30). In neuropil of molecular layer, a decrease of the mean amount of labile synaptopodin-positive dendrite spines was observed, as compared with control. In rats exposed to hypoxia at E18, no changes of cell composition and structure of the nervous tissue were found in the studied brain cortex areas. Thus, formation of the cortex nervous tissue in postnatal ontogenesis of rats submitted to hypoxia at the period of neuroblast proliferation-migration is accompanied not only by a change of the cell composition of various cortex layers in early ontogenesis, but also by a decrease of the number of the synaptopodin-positive spines in molecular layer, the decrease being preserved in adult animals.     

Structural changes in the neocortex nervous tissue in rat ontogenesis after hypoxia at various terms of embryogenesis

The performed study has shown that in rats submitted to hypoxia (3 h, 7% O₂) at the 14th day of embryogenesis (E14) as compared with control animals, density of distribution of cells in the brain cortex decreased for the first month of postnatal ontogenesis (maximally by 40.8% by P20). In dying neurons, swelling of the cell body, lyses of or ganoids, and disturbance of the cytoplasm membrane intactness were observed. Two waves of neuronal death by the mechanism of capsize-dependent apoptosis were revealed; the first involved large pyramidal neurons of the layer V (P10–20), the second-small pyramidal and non-pyramidal neurons of the layers II–III (P20–30). In neurosis of molecular layer, a decrease of the mean amount of labile synaptopodin-positive dendrite spines was observed, as compared with control. In rats exposed to hypoxia at E18, no changes of cell composition and structure of the nervous tissue were found in the studied brain cortex areas. Thus, formation of the cortex nervous tissue in postnatal ontogenesis of rats submitted to hypoxia at the period of neuroblast proliferation-migration is accompanied not only by a change of the cell composition of various cortex layers in early ontogenesis, but also by a decrease of the number of the synaptopodin-positive spines in the molecular layer, the decrease being preserved in adult animals.     

Study of distribution of protein of the spine apparatus synaptopodin in cortical brain parts of rats submitted to hypoxia at different periods of embryogenesis

A comparative study of the nervous tissue and distribution of the spine apparatus protein synaptopodin was performed in all layers of the brain sensorymotor cortex and hippocampal CA1 area in control rats and in the rats submitted to hypoxia at E14 and E18. It was found that beginning from the 20th day of postnatal development, in rats submitted to hypoxia both at E14 and E18 there was observed a statistically significant decrease of the mean number of labile synaptopodin-positive spines in the stratum radiatum molecular of the hippocampus area CA1. The decrease of the number of labile spines in the sensorymotor brain cortex was revealed only in the I layer beginning from the 20th day after birth in the rats submitted to hypoxia at E14. Maximal differences in the studied brain areas were observed in adult rats (exposed to hypoxia at E14: in the neocortex--a decrease by 23 +/- 10%, in hippocampus--by 24 +/- 8%, respectively). In adult animals, the increased degeneration of neuzons was not detected. It is suggested that disturbances in cognitive functions and in the capability for learning observed in rats after prenatal hypoxia can be due to a decrease of the amount of the labile synaptopodin-positive spines, which leads to a change of the structural-functional properties of neuronal networks and to a decrease of their plasticity.     

The long-term effect of acute hypoxic hypoxia on shuttlebox avoidance learning in rats

Long-term influence was studied of the acute hypoxic hypoxia seance on rats behaviour in situation of elaboration of the conditioned reaction of active avoidance of electric shocks in shuttle chamber. It was found that in 2.5-3 months after the hypoxia seance, the experimental animals significantly differed from the intact controls by dynamics of CR elaboration (rats which had hypoxia were ahead of the control ones) and by distribution of the conditioned reactions latencies (for experimental animals this distribution was shifted to minor values). The character of these behavioural shifts coincided with that observed in the group of rats with local unilateral hippocampus lesion. The obtained results and numerous data presented in literature on the influence of the hippocampus lesion on animals shuttle avoidance learning, allow to conclude that the seance of hypoxic hypoxia leads to the disturbance of the hippocampus function. This conclusion conforms to the data on diffusive death of the hippocampal and neocortical neurones as a result of hypoxia action.     

Effect of maternal hypoxia on the neurogenesis of the cerebral cortex of rat progeny (autoradiographic study)]

Rats with 15-day pregnancy were exposed to two-hour hypoxia corresponding to 8,000 m altitude. On the 18th day of pregnancy they were administered thymidine-3H three times. Quantitative autoradiographic studies were performed on brain cortex neurons of 30-day rat progeny. The animals who had sustained intrauterine hypoxia were shown to have obviously higher number of labeled neurons in IId, IIId and Vth layers of the sensomotor area than controls. Differences in the label intensity were also revealed. It is suggested that maternal hypoxia may delay differentiation and maturation of the brain cortex neurons in the progeny.     

Disturbances in Formation of the New and Old Cortex at Changes of Conditions of Embryonic Development

Using a model of acute hypoxia during pregnancy of rats, changes in the development of old (hippocampus) and new (sensorimotor) cortex associated with disturbance of neuronogenesis have been revealed in the studied brain structures at the period of action of a pathological factor. It was found that in rats submitted to hypoxia at the 13–14th days of embryogenesis, the number of degenerating neurons (including the pyramidal ones) at various levels of chromatolysis increased since the 5th day after birth; the increase was present for the entire first month of postnatal development. In the cortex of rat pups submitted to prenatal hypoxia there were observed deformation of neuronal bodies, vacuoles in the cytoplasm, shrinkage of apical dendrites of pyramidal neurons and delayed development of the structure (time of the appearance of spikes, formation of structural elements and the size of the cells) of the nervous tissue of the brain of the rat pups exposed to prenatal hypoxia. The columnar structure of the cortex was disturbed. In hippocampus, the process of degeneration of neurons started by 2–3 days later than in the cortex; by two weeks of postnatal development a massive degeneration and death of a part of neurons were also revealed. The morphometrical analysis showed a decrease in the number of neurons and their total area in the sensorimotor cortex (the layer V) and an increase in the number of glial elements at the 10–17th days after birth. In the hippocampus a decrease in the area occupied by neurons and in their size was detected in adult animals. The adult rats submitted to prenatal hypoxia were found to have disturbances of memory and learning. A correlation was shown between the disturbances of the conditions of embryonic development and the changes in the ability of learning and storage of new skills in the offspring.     

The ultrastructure of the neurons of a graft developing in the brain of rats experiencing hypoxia

Fragments of the brain cortex of 17- or 18-day-old rat embryos were allotransplanted into the brain cortex of rats subjected to hypoxia. Four days later the graft consisted of mixed differentiating neuroblasts. By the 100th to 130th day after transplantation the graft contained mature neurons, differentiating neurons and neuroblasts. Hypochromic neurons showing the signs of intracellular reparation were also detected. A well-developed neuropile was localized inside the graft. In contrast to the normal brain, neurons in the graft were not organized in layers.     

大鼠液压冲击脑损伤热休克蛋白70基因表达的研究

目的：观察大鼠侧位液压冲击脑损伤时ＨＳＰ７０的表达分布特点及时序性变化。方法：雄性ＳＤ大鼠,给以０．２ＭＰａ液压冲击,造成脑损伤,应用免疫组织化学技术观察冲击后不同时间ＨＳＰ７０在脑组织内的表达特点。结果：冲击侧大脑皮层和脑干ＳＨＰ７０阳性神经辊冲击后２ｈ和４ｈ出现,７并逐渐增强直至１２ｈ;冲击后４ｈ,冲击侧海马ＨＳＰ７０免疫阳性细胞开始出现,４￣１２ｈ,海马ＨＳＰ７０免疫阳性细胞数无明显改变。结     

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.     

The short-term and long-lasting changes in DNA and RNA synthesis in the cerebral cortex cells of animals subjected to hypoxia and nerve tissue transplantation]

The DNA and RNA synthesis in the cells of the brain cortex of intact rats and animals subjected to hypoxia, hypoxia with subsequent transplantation or by the local brain injury has been investigated. The DNA synthesis changes insignificantly in the case of hypoxia, it enhances slightly in the area of the injury and increases much more after transplantation. The RNA synthesis decreases considerably immediately after hypoxia and decreases much more 120 days later. Using the ultracentrifuge method it has been found that under the effect of hypoxia the number of nervous cells decreases, the number of glial cells does not change. The local injury in the nervous tissue enhances abruptly the synthesis in neurons and glial cells in the hypoxia-exposed animals, the embryonic nervous tissue transplantation normalizes the number of neurons in the specimens under study and the RNA synthesis in the neurons and glial cells.     

Heat shock protein modulation of KATP and KCa channel cerebrovasodilation after brain injury

Fluid percussion brain injury (FPI) impairs pial artery dilation to activators of the ATP-sensitive (K(ATP)) and calcium-activated (K(Ca)) K(+) channels. This study investigated the role of heat shock protein (HSP) in the modulation of K(+) channel-induced pial artery dilation after FPI in newborn pigs equipped with a closed cranial window. Under nonbrain injury conditions, topical coadministration of exogenous HSP-27 (1 mug/ml) blunted dilation to cromakalim, CGRP, and NS-1619 (10(-8) and 10(-6) M; cromakalim and CGRP are K(ATP) agonists and NS-1619 is a K(Ca) agonist). In contrast, coadministration of exogenous HSP-70 (1 mug/ml) potentiated dilation to cromakalim, CGRP, and NS-1619. FPI increased the cerebrospinal fluid (CSF) concentration of HSP-27 from 0.051 +/- 0.012 to 0.113 +/- 0.035 ng/ml but decreased the CSF concentration of HSP-70 from 50.42 +/- 8.96 to 30.9 +/- 9.9 ng/ml at 1 h postinsult. Pretreatment with topical exogenous HSP-70 (1 mug/ml) before FPI fully blocked injury-induced impairment of cromakalim and CGRP dilation and partially blocked injury-induced impairment of dilation to NS-1619. These data indicate that HSP-27 and HSP-70 contribute to modulation of K(+) channel-induced pial artery dilation. These data suggest that HSP-70 is an endogenous protectant of which its actions may be unmasked and/or potentiated with exogenous administration before brain injury.     

Gene expression analysis following hypoxia–reoxygenation in rat gastric epithelial cells using a high-density oligonucleotide array

Abstract

Recent investigations have demonstrated that the signaling of hypoxia–re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia–reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N₂ and 5% CO₂ at 37°C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia–reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia–reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.     

Histone alteration in the nuclei of different types of neurons in a "mirror" epileptiform focus in rat brain]

Total histones estimated after M. Alfred and I. Geschwind were studied cytophotometrically in nuclei of neurons of different types (layers III and V in the frontal-parietal cortex and in the thalamic lateral nucleus) of the "mirror" epileptiform focus in the rat brain on the 11th and 63d day after cobalt implantation. On the 11th day of the experiment, integral optic density of the histones decreased by 33%, 25% and 37%, and the area of the nuclei -- by 27%, 24% and 35% in the neurons of layers II and V in the frontal-parietal cortex and in the thalamic lateral neucleus, respectively. On the 63d, integral optic density of the histones in the neurons mentioned increased respectively by 42%, 52% and 42%, and the area of the nuclei -- by 21%, 43% and 27%. Concentration of the histones in the nuclei decreased by 3--9% on the 11th day and increased, except layer V, by 9--18% on the 63d of the experiment. Interconnection between changes in histone content and total proteins in cytoplasm of functionally different neurons in the "mirror" epileptiform focus is disscussed, with special reference to histones as a regular of genetic activity for protein synthesis.