A. V. Churilova

Preconditioning induces prolonged expression of transcription factors pCREB and NF‐κB in the neocortex of rats before and following severe hypobaric hypoxia

Preconditioning using mild repetitive hypobaric hypoxia is known to increase a tolerance of brain neurons to severe hypoxia and other injurious exposures. In the present study, the effects of mild hypoxic preconditioning on the expression of transcription factors NF‐κB and phosphorylated CREB (pCREB) has been studied in the neocortex of rats exposed to severe hypobaric hypoxia. As revealed by quantitative immunocytochemistry, the injurious severe hypobaric hypoxia (180 Torr, 3 h) remarkably reduced the neocortical levels of pCREB and NF‐κB. The three‐trial hypoxic preconditioning (360 Torr, 2 h, 3 days) induced persistent up‐regulation of pCREB and NF‐κB expression in the neocortex of rats 3–24 h following the severe hypoxia. In addition, the preconditioning alone which was not followed by the severe hypoxia, considerably increased neocortical pCREB and NF‐κB levels. The findings suggest a role for transcription factors cAMP response element‐binding protein and NF‐κB in the neuroprotective mechanisms activated by the hypoxic preconditioning.

Expression of glucocorticoid and mineralocorticoid receptors in hippocampus of rats exposed to various modes of hypobaric hypoxia: Putative role in hypoxic preconditioning.

Effects of mild (preconditioning) and severe injurious hypobaric hypoxia (SH), as well as of their combination on hippocampal expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors and HPA axis activity have been examined in rats. As revealed by quantitative immunocytochemistry, three-trial exposure to mild hypoxia produced robust GR and MR overexpression located mainly in the neuronal nuclei in the dentate gyrus (DG) but only MR overexpression was observed in the CA1. SH induced sharp reduction of MR levels and enhanced GR expression in the CA1, suggesting that the unbalance of GR and MR observed might be at the bottom of the extensive neuronal loss seen in this area in response to SH. Contrastingly, SH in tolerant (preconditioned) rats failed to imbalance GR and MR expression in CA1 and up-regulated GR levels in DG. Radioimmunoassay of serum corticosterone showed that both preconditioning hypoxia itself and SH in tolerant rats produced moderate activation of HPA axis followed by its proper inactivation. In the non-preconditioned rats, HPA axis response to SH was impaired. Taken together, these novel results suggest that modifications of the hippocampal expression of GR and MR produced by preconditioning may contribute to the molecular and neuroendocrine mechanisms of tolerance to severe hypoxic stress.

Neocortical pCREB and BDNF expression under different modes of hypobaric hypoxia: Role in brain hypoxic tolerance in rats

Preconditioning with repetitive mild hypobaric hypoxia is known to increase tolerance of susceptible brain neurons to severe hypoxia, whereas a single trial of mild hypoxia has been ineffective. In the present study, the effects of three-trial and one-trial hypobaric preconditioning on the expression of the protective transcription factor phosphorylated CREB (pCREB) and neurotrophin BDNF, before and after severe hypobaric hypoxia, have been comparatively studied in the neocortex of rats. As revealed by quantitative immunocytochemistry, the severe hypobaric hypoxia (180 Torr, 3h) substantially down-regulated the levels of pCREB and BDNF in cortical neurons assessed 24h after the treatment. One trial of mild hypoxia (360 Torr, 2h) also reduced by half the number of BDNF-expressing cells, but had no effect on pCREB expression in the neocortex. In contrast, the exposure to three trials of mild hypoxia at 24h intervals considerably up-regulated pCREB and BDNF levels in the neocortex of rats. Only preconditioning by three trials of mild hypoxia (360 Torr, 2h, 24h intervals), but not a single trial preconditioning, was neuroprotective significantly enhancing the pCREB and BDNF neuronal expression in response to severe hypoxic challenge. The results of the present study indicate that development of the neuronal hypoxic tolerance induced by the three-trial mild hypoxic preconditioning is apparently associated with activation of CREB and BDNF expression.

Effects of Moderate Hypobaric Hypoxic Preconditioning on the Expression of the Transcription Factors pCREB and NF-κB in the Rat Hippocampus Before and After Severe Hypoxia

Preconditioning using three sessions of moderate hypobaric hypoxia, i.e., hypoxic preconditioning (HP), increased the tolerance of susceptible brain neurons to severe hypoxia and other harmful factors. The study addressed changes in the expression of transcription factors NF-κB (nuclear factor kappa B) and CREB (cAMP response element binding protein) in the hippocampus of rats preconditioned with moderate hypoxia. Immunocytochemical methods demonstrated that HP increased immunoreactivity for NF-κB and phosphorylated CREB (pCREB) in hippocampal fields CA1–CA4 and the dentate gyrus and promoted increases in the expression of these transcription factors in the hippocampus of preconditioned rats 3–24 h after severe hypobaric hypoxia. These data provide evidence that NF-κB and CREB are involved in the mechanisms forming HP-induced tolerance of the brain.

Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance

Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180mm Hg, 3h) and neuroprotective preconditioning mode (three episodes of 360mm Hg for 2h spaced at 24h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II-III, V) and hippocampus (CA1, CA3) by 3-24h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24h, whereas acetylation of H3K24 was intense but transient, peaked at 3h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance.

The effect of different modes of hypobaric hypoxia on the expression of transcription factor pCREB and pro-survival proteins BDNF and BCL-2 in rat neocortex and hippocampus.

Severe hypobaric hypoxia (180 mm Hg) is a harmful stimulus that induces structural and functional injures of susceptible brain neurons in the neocortex and hippocampus. In contrast, moderate hypobaric hypoxia (360 mm Hg) activates endogenous cellular defensive mechanisms. The rate of neuroprotection afforded by the mild hypoxia depends on the quantity of hypoxic sessions. In particular, preconditioning (pre-exposure) by three trials of mild hypoxia protects from deleterious effects of subsequent severe hypoxia whereas preconditioning with one mild hypoxic trial does not. The molecular mechanisms induced by mild hypoxic preconditioning are unclear. Pro-survival proteins, such as neurotrophic factor BDNF and anti-apoptotic factor Bcl-2 are supposed to be involved in this process. In the present study, the effects of three-trial and one-trial hypoxic preconditioning on the expression of pro-survival proteins BDNF and Bcl-2 as well as their up-stream activator pCREB, have been studied in the neocortex and hippocampus of rats. As revealed by quantitative immunocytochemistry, the severe hypobaric hypoxia didn’t affect or down-regulated the neuronal levels of pCREB, BDNF and Bcl-2 at 3-24 h after the exposure. The one-trial preconditioning did not change this effect of severe hypoxia. In contrast, preconditioning by three trials of mild hypoxia (360 Torr, 2h, 24 h intervals, 3 times) significantly enhanced the pCREB, BDNF and Bcl-2 neuronal expression in response to severe hypoxic challenge. Three-trial mild hypoxia alone also up-regulated the expression of molecular factors examined in the neocortex and hippocampus at 24 h whereas one trial of the mild hypoxia did not. The results of the present study indicate that development of the neuronal hypoxic tolerance induced by the three-trial, in contrast to one-trial, mild hypoxic preconditioning is apparently largely associated with the activation of CREB, as well as BDNF and Bcl-2 overexpression.

Changes in the Expression of Antiapoptotic Protein Bcl-2 in the Rat Neocortex and Hippocampus in Different Hypobaric Hypoxia Regimes

Experiments on 72 male Wistar rats using an immunocytochemical method addressed the level of expression of the antiapoptotic factor Bcl-2 in neocortical and hippocampal neurons on exposure to harmful severe (SH) and moderate hypobaric (MHH) hypoxia separately and in combination. SH (180 mmHg) suppressed or had no effect on Bcl-2 expression in neurons in these brain areas. Single episodes of preconditioning (360 mmHg) produced similar effects on Bcl-2 expression as SH. Conversely, repeated preconditioning significantly increased the level of Bcl-2 expression 3–24 h after SH, helping to prevent neuron injury due to SH. MHH itself increased the level of Bcl-2 expression only after multiple (3–6) sessions, while single sessions had no effect on Bcl-2 content. The increases in Bcl-2 expression seen in response to multiple exposures to MHH appear to be important for the formation of the mechanisms increasing the tolerance of cerebral neurons to harmful actions.

The adaptive role of the CREB and NF-κB neuronal transcription factors in post-stress psychopathology models in rats

We studied the dynamics of the activation of the CREB and NF-κB transcription factors in areas of the rat brain after exposure to pathogenic psycho-emotional stress in the model of endogenous depression and the model of posttraumatic stress disorder (PTSD), as well as after application of hypoxic preconditioning, which prevents the formation of anxiety-depressive pathologies in these models. The development of anxiety-depressive pathology in both models was associated with reduced (10 times lower than the control maximum) or basal levels of the activating transcription factors CREB and NF-κB in the hippocampus and the neocortex. However, in the hypothalamus, NF-κB overactivation (up to an 18-fold increase above the control level) was detected in the model of depression. Therefore the lack of activation of these factors in the extra-hypothalamic areas of the brain may be a common component of the pathogenesis of both depression and PTSD, while NF-κB overactivation in the neurosecretory centers of the hypothalamus is obviously involved in the development of depressive conditions. Antidepressant and anxiolytic actions of hypoxic preconditioning were accompanied by a mild 2- to 6-fold increase in CREB and NF-κB levels in the neurons of the brain in both stress paradigms. Obviously, the maintenance of optimum activity of transcription factors in the neurons of the brain plays an important role in nonspecific compensatory processes that enhance the adaptive potential of the brain under stress conditions.

Morphological Differences between the Effects of Different Preconditioning Regimes Correcting Damage to Hippocampal Neurons Due to Exposure to Severe Hypobaric Hypoxia

Studies on five groups of rats (six animals per group) addressed changes in neurons in hippocampal fields CA1 and CA4 seven days after severe hypobaric hypoxia (180 mmHg, 3 h) using different numbers (1, 3, or 6) of sessions of preconditioning (PC) with moderate hypobaric hypoxia (360 mmHg, 2 h, 24 h before severe hypoxia). Single-session PC was found not to prevent damage to neuron structure with neuron death by day 7 after severe hypoxia. At the same time, six and especially three sessions of PC induced protective mechanisms preventing neuron damage. Use of six sessions of PC, in contrast to three, resulted in moderate chromatolysis in hippocampal neurons, which may be a consequence of prolonged hypermetabolic activity of neurons and may be evidence of their functional overloading.

Effects of Different Hypobaric Hypoxia Regimes on the Expression of Corticosteroid Receptors in the Rat Hippocampus

Hippocampal gluco- and mineralocorticoid receptors (GR, MR) have important roles in the mechanisms regulating the activity of the hypothalamo-hypophyseal-adrenocortical system (HHAS), neuron survival/death, learning, and memory. Imbalance in MR and GR contents lead to impairments of HHAS activity and can promote neuron damage/death in extreme conditions. The present study used a quantitative immunocytochemistry method to provide the first comparative analysis of the effects of different regimes of hypobaric hypoxia on the nature of GR and MR expression in the dorsal (CA1) and ventral (dentate gyrus) parts of the hippocampus in rats. The data obtained here showed that severely harmful hypoxia induces marked impairments to the expression of both GR and MR in CA1 and dental gyrus cells, which correlated with damage/death of a significant proportion of neurons in CA1 and dysregulation of the activity of the HHAS. Series of three and six sessions (but not one session) of preconditioning with moderate hypoxia preceding severe hypoxia prevented these impairments.