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.     

Synaptosomal Uptake and Release of Dopamine in Rat Striatum After Hypoxia

Hypoxia induces alterations of central monoaminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long-lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short-term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+-evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2-11) caused long-lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+-stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.     

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.     

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.     

Effect of 5 alpha-pregnan-3 alpha-ol-20-one on nitric oxide biosynthesis and plasma volume in rats

Plasma 5 alpha-pregnan-3 alpha-ol-20-one (pregnan) levels and nitric oxide (NO) biosynthesis increase during pregnancy. These factors have independently been implicated in the control of blood pressure and volume. We wished to determine whether pregnan might be responsible both for the increase in NO biosynthesis and for the increase in plasma volume observed during pregnancy. Virgin female Long-Evans rats were implanted with indwelling cannulas and maintained on a low nitrate/nitrite diet. After the rats recovered from surgery, 500 microg of pregnan or vehicle were given daily for 2 days. NO biosynthesis and plasma volume were measured in conscious animals before and after treatment. Pregnan caused a significant increase in NO biosynthesis (1.9 +/- 0.8 micromol/24 h, n = 10) compared with the vehicle-treated control group (0.3 +/- 0.4 micromol/24 h, n = 10, P < 0.05). Similarly, there was a significant increase in plasma volume in the pregnan-treated group (0.7 +/- 0.2 ml/100 g, n = 11) compared with the vehicle-treated control group (0.2 +/- 0.1 ml/100 g, n = 11, P < 0.05). These results confirm that pregnan can mimic pregnancy by its ability to increase both NO biosynthesis and plasma volume.     

The effect of cellular glucoprivation on skin temperature regulation in the rat

Studies were undertaken to determine the effects of cellular glucoprivation on temperature responses in morphine-addicted and placebo-treated rats and to compare these responses to those observed during naloxone-precipitated morphine withdrawal. Naloxone caused a tail skin temperature (TST) response of 5.7 +/- 0.5 degrees C in morphine-dependent rats. Intraperitoneal administration 2-deoxyglucose (2DG) caused TST responses in placebo-treated and morphine-dependent rats of 4.8 +/- 0.6 and 6.2 +/- 0.5 degrees C, respectively. These data indicate that the activation of the sympathetic nervous system by cellular glucoprivation causes a TST response which is equivalent in magnitude to that induced by precipitating withdrawal with naloxone. This effect of 2DG appears to be mediated by the brain, since icy administration of 2DG caused a TST response, similar to that induced by naloxone treatment of morphine-dependent rats. Collectively, these data suggest that a TST increase is a component of the response of rats to local brain glucoprivation induced by 2DG.     

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.     

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.     

Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system

Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.     

Effect of Acute Hypoxia on Ascorbate Content of Plasma, Cerebral Cortex, and Adrenal Gland

Levels of ascorbic acid (AA) in the plasma, brain, and adrenal gland of rats were determined after 15 min of hypoxia (PaO2 less than 25 mm Hg) and following asphyxia. In rabbits, AA plasma levels were followed up to 75 min of reoxygenation following 15 min of hypoxia of the same severity. A significant increase (approximately 70%) in AA levels was found in plasma of rats and rabbits after hypoxia and asphyxia. This increase was found to be transient, with a return to normal levels within 1 h after resumption of normal oxygenation. Pretreatment with dexamethasone reduced the increase in AA level in both rabbits and rats. Adrenalectomy in rats, performed 24 h before the experiment, abolished the response to hypoxia. Ascorbate levels in the cerebral cortex, hypothalamus, and adrenal gland of awake rats subjected to hypoxia or asphyxia were found to be the same as in normoxic rats. Our results suggest that the observed changes in plasma AA levels are probably mediated through adrenocorticotropic hormone and that the adrenal gland is the major source of ascorbate efflux into the circulation during oxygen deprivation.     

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.     

Effects of short-term and prolonged aerogenic hypoxia on gamma-glutamyl transpeptidase activity in the brain,liver, and biological fluids of young rats

Posthypoxic fluctuations in the levels of two excitatory amino acids, glutamate and aspartate, may be related to changes in mechanisms(s) which are responsible for their reuptake. As gamma-glutamyl transpeptidase (GGT) plays a role in mediating the uptake of glutamate and aspartate into various compartments of the brain, we studied changes in the activity of this enzyme in main regions of the brain in young and adult rats. We found a posthypoxic increase in bound GGT activity in some brain regions of 18-day-old animals after acute exposure, but no changes were observed after prolonged altitude hypoxia, with the exception of a decrease in cortical GGT activity. In contrast, acute hypoxia decreased GGT activity in the cortical capillaries to 59%, but prolonged hypoxic exposure was ineffective. However, the activity of soluble GGT in the cerebrospinal fluid of both groups of rats was several-times elevated in comparison with controls. At the same time, bound GGT activity was increased in the liver after acute or prolonged altitude hypoxia. The soluble GGT activity in plasma was only increased after prolonged exposure. Ninety days after prolonged hypoxic exposure the bound GGT activity was reduced in all brain regions to about 60–70% of controls (significantly higher in females than in males) as long-term developmental sequel from early postnatal hypoxia.     

胚胎大鼠宫内缺氧动物模型的建立

目的-建立适合不同胎龄大鼠宫内缺氧的动物模型。方法-将孕15、19天的孕鼠随机分为对照组和缺氧组,缺氧组孕鼠用低张性缺氧原理致鼠胚宫内缺氧,将鼠胚大脑组织作c-Fos蛋白和一氧化氮合酶(nitricoxide synthase,NOS)免疫组化双标染色后,进行观察分析。结果-缺氧组鼠胚大脑神经元c-Fos蛋白和NOS阳性细胞均较对照组增多(P<0.05)。结论-可采用低张性缺氧方法使孕鼠缺氧来制造胚胎大鼠宫内缺氧的动物模型。     

Inducible nitric oxide synthase expression and plasma bilirubin changes in rats under intermittent hypoxia treatment

It has been reported that intermittent hypoxia treatment prevents oxidative injuries to the brain and protects the heart against ischemia-reperfusion injury. Both anti-oxidative defensive systems and prevention of free intracellular calcium overload might be the result of intermittent hypoxia. Thus, the purpose of this study was to explore the effects of intermittent hypoxia (8 h at 12 % O2 per day) for 0, 7 or 14 days on inducible nitric oxide synthase (iNOS) expression in the spleen and on splenic calcium response to the mitogen phytohemagglutinin (PHA). The results demonstrated that administration of intermittent hypoxia for 7 days caused severe hemolysis of erythrocytes in the spleen and the hemolytic condition was ameliorated by intermittent hypoxia for 14 days. However, a significant decline in splenic weight and an increase in plasma total bilirubin levels appeared in rats after hypoxia for 14 days. No calcium response to PHA was observed in splenocytes obtained from rats after intermittent hypoxia for 7 days. After intermittent hypoxia for 14 days, the calcium response to PHA was restored to the level of the controls. Intermittent hypoxia for 7 days was able to induce higher iNOS expression in splenic tissues than hypoxia for 14 days. These results suggested that intermittent hypoxia for 14 days appeared to involve acclimatization that protects the rats from oxidative injury through less hemolysis and iNOS expression in splenic tissues and by the presence of more bilirubin in the plasma. The increase in plasma total bilirubin levels might be the cause of induced adaptation to chronic intermittent hypoxia.     

The effect of ischemia and recirculation, hypoxia and recovery on anti-oxidant factors and beta-adrenoceptor density. Is the damage in the erythrocytes a reflection of brain damage caused by complete cerebral ischemia and by hypoxia?

This investigation was focussed on the gravity of tissue injury caused by complete ischemia (for five min) and hypoxia (for three weeks) in the cerebral cortex (homogenate) and the erythrocyte lysate or the erythrocyte membrane of the rat in order to investigate if the changes that occur in brain tissue are reflected in the erythrocyte. To this end, glutathione (GSH), superoxide dismutase (SOD) and catalase were measured, also alterations in beta-adrenoceptor density under these two conditions were examined. It was found that in ischemia partial parallelism in changes that occur in the central nervous system (cerebral cortex) and the erythrocyte exists. The SOD activity became higher and the beta-adrenoceptor density (measured as specific (-)-[125I] iodocyanopindolol binding) was decreased in both tissues. However after the hypoxic condition we established a decrease in the number of beta-adrenoceptors in the cerebral cortex but an increase in beta-adrenoceptor density in the erythrocyte.     

Effects of in ovo treatment with PACAP antagonist on general activity, motor and social behavior of chickens

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to influence nervous system development. The aim of the present study was to investigate the effects of in ovo treatment with the PACAP antagonist PACAP6-38 during embryonic life (E8 and E16) on motor activity and social behavior in chicken. Our results showed that a single injection of PACAP6-38 during the first half of embryonic life caused subtle transient changes in general behavior and motor control when compared to saline-treated controls. Increased activity and reduced anxiety were observed also in a novel environment at 2 days after hatching. However, most of these behavioral differences disappeared by 2 weeks. PACAP6-38-treatment during the first half of embryonic life resulted in markedly reduced social behavior, which was still present at 2 weeks of age. Treatment during the second half of embryonic life resulted in no behavioral differences between control and PACAP6-38-treated chicken. PACAP content in different brain areas was not different between control and PACAP6-38-treated chicken at 5 days or 3 weeks of age, but it decreased significantly with age in both groups. In summary, our results show that PACAP6-38 treatment at E8 caused transient changes in motor behavior, and long-lasting reduction in social behavior.     

In vitro differentiation of transplantable neural precursors from human embryonic stem cells.

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.     

Effect of acute hypoxia on respiration and brain stem blood flow in the piglet

Changes in local brain stem perfusion that alter extracellular fluid Pco2 and/or [H+] near central chemoreceptors may contribute to the decrease in respiration observed during hypoxia after peripheral chemoreceptor denervation and to the delayed decrease observed during hypoxia in the newborn. In this study, we measured the changes in respiration and brain stem blood flow (BBF) during 2-4 min of hypoxic hypoxia in both intact and denervated piglets and calculated the changes in brain stem Pco2 and [H+] that would be expected to occur as a result of the changes in BBF. All animals were anesthetized, spontaneously breathing, and between 2 and 7 days of age. Respiratory and other variables were measured before and during hypoxia in all animals, and BBF (microspheres) was measured in a subgroup of intact and denervated animals at 0, 30, and 260 s and at 0 and 80 s, respectively. During hypoxia, minute ventilation increased and then decreased (biphasic response) in the intact animals but decreased only in the denervated animals. BBF increased in a near linear fashion, and calculated brain stem extracellular fluid Pco2 and [H+] decreased over the first 80 s both before and after denervation. We speculate that a rapid increase in BBF during acute hypoxia decreases brain stem extracellular fluid Pco2 and [H+], which, in turn, negatively modulate the increase in respiratory drive produced by peripheral chemoreceptor input to the central respiratory generator.     

Activity of Lactate Dehydrogenase in Serum and Cerebral Cortex of Immature and Mature Rats after Hypobaric Hypoxia

In our previous studies we have found both an increase of lipid peroxidation damage (expressed as levels of thiobarbituric acid-reactive substances) in brain and plasma lactate concentration in 21-day-old rats after a 30-min exposure to hypobaric hypoxia. Pretreatment of rats with l-carnitine decreased both parameters. The aim of our present study was to determine if the l-carnitine-dependent decrease of plasma lactate could be due to a modification of lactate dehydrogenase (LDH) activity. We followed brain and blood serum LDH activity of 14-, 21- and 90-day-old Wistar rats. We found an increase of brain LDH activity with age. However, we did not observe any significant differences in LDH activity after exposure to hypobaric hypoxia or l-carnitine pretreatment. In contrast to brain, serum LDH activity did not show any clear age-dependence. The hypoxia exposure increased LDH activity of 21-day-old rats only. Pretreatment of rats with l-carnitine decreased serum LDH activity of 21- and 90-day-old rats probably due to membrane stabilizing role of l-carnitine. In conclusions, acute hypobaric hypoxia and/or l-carnitine pretreatment modified serum but not brain LDH activity.     

低氧脑水肿大鼠脑组织VEGF和AQPs基因及蛋白表达的变化

目的：探讨低氧脑水肿时血管内皮细胞生长因子（VEGF）、水通道蛋白（AQP1和AQP4）基因和蛋白表达变化,为阐明急性低氧对脑组织的损伤及低氧脑水肿的发病机制提供实验依据。方法：Wistar大鼠随机分为4个组：常氧对照组（Control）、低氧暴露4 000 m组（4 000 m）、低氧暴露6 000 m组（6 000 m）和低氧暴露8 000 m组（8 000 m）,低氧组于低压舱中模拟相应海拔高度持续暴露8 h建立低氧脑水肿模型。用干-湿重法测定脑组织水含量,常规光镜观察脑组织形态学的改变;用RT-PCR法和免疫组化法检测低氧脑水肿时大鼠脑组织VEGF、AQP1和AQP4mRNA和蛋白表达的变化。结果：①干-湿重法测定表明,低氧（≥6 000 m）暴露后,大鼠脑组织水含量明显增加（P〈0.01）。②常规光镜检测结果表明,低氧暴露4 000 m时大鼠脑神经细胞、血管内皮细胞和星形胶质细胞足突轻度肿胀,组织中出现漏出液;低氧暴露6 000 m时脑血管内皮细胞和星形胶质细胞足突肿胀加重,血管与组织间隙扩大,组织中漏出液增多;低氧暴露8 000m时脑血管内皮细胞和星形胶质细胞足突重度肿胀,血管与组织间隙进一步扩大,组织中漏出液明显增多。③低氧脑水肿时,VEGF、AQP1、AQP4mRNA表达水平增高,AQP1在内皮细胞异常表达,内皮细胞VEGF和AQP1、星形胶质细胞足突AQP4蛋白质表达水平增高。结论：低氧脑水肿时,VEGF、AQP1和AQP4表达和分布的变化可能是引起血脑屏障损伤、导致低氧脑水肿的发病机制之一。