T. S. Glushchenko

Mild hypobaric hypoxia preconditioning up-regulates expression of transcription factors c-Fos and NGFI-A in rat neocortex and hippocampus

Transcription factors c-Fos and NGFI-A encoded by immediate early genes largely participate in the biochemical cascade leading to genomically driven lasting adaptation by neurons to injurious exposures including hypoxia/ischemia. Present study was designed to examine the involvement of c-Fos and NGFI-A in the development of brain hypoxic tolerance induced by mild hypoxic preconditioning. Earlier we have reported that preconditioning by repetitive mild hypobaric hypoxia (MHH) considerably increases neuronal resistance to subsequent severe injurious exposures. Herein, changes of c-Fos and NGFI-A expression in vulnerable rat brain areas (hippocampus, neocortex) in response to preconditioning MHH itself were studied using quantitative immunocytochemistry. Exposure to MHH differentially enhanced c-Fos and NGFI-A expression in neocortex and hippocampal fields 3-24h following the last MHH trial. The c-Fos up-regulation was the most pronounced in neocortex, CA1, and dentate gyrus, but it was twice lower in CA3/CA4. The up-regulation of NGFI-A in CA1, dentate gyrus and neocortex was 1.5-2-fold lower than that of c-Fos; but in CA3 and CA4 the rates of the c-Fos and NGFI-A induction were comparable. The present findings indicate that cooperative but differential activation of c-Fos and NGFI-A expression in vulnerable brain areas contribute to the development of tolerance achieved by MHH preconditioning.

Expression of early gene proteins, structural changes in brain neurons in hypobaric hypoxia, and the correcting effects of preconditioning.

The Nissl method and immunocytochemistry were used to study the effects of severe hypobaric hypoxia and its actions in combination with the preconditioning actions of moderate hypoxia on the expression of the early gene proteins c-Fos and NGFI-A as well as structural changes in hippocampal and neocortical neurons in the rat brain. Severe hypoxia was found to suppress c-Fos and NGFI-A synthesis (3–24 h after exposure) and to induce delayed (days 3–7) structural damage to neurons, of the “light” and predominantly the “dark” types, which appear to reflect the development of necrotic and apoptotic processes respectively. Preconditioning with the regime used here corrected these derangements, resulting in increases in the expression of early gene proteins and significant reductions in structural damage to neurons after severe hypoxia.

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.

Preconditioning modifies the activities of mitogen-activated protein kinases and c-Jun transcription factor in rat hippocampus after severe hypobaric hypoxia

The effect of preconditioning by a moderate hypobaric hypoxia on changes in the activity of the mitogen-activated (MAP) kinases EPK, JNK 1/2, and p38 and the c-Jun transcriptional factor in rat hippocampus in response to the severe hypoxia was studied using the methods of quantitative immunocytochemistry and Western-blot analysis. Severe damaging hypoxia caused a persistent activation of the JNK cascade, including JNK 1/2 and c-Jun, and also p38 kinase in the hippocampus cells. The preconditioning efficiently inhibited the expression of phosphorylated forms of JNK, c-Jun, and p38 and activated the protein kinase ERK following the severe hypoxia, which obviously promoted the survival of hippocampal neurons. The results indicate the important role of the family of MAP kinases and the c-Jun transcription factor in the processes of neuronal death/survival of the hippocampal neurons after a severe hypobaric hypoxia, and a correcting effect of preconditioning action.

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.

Effects of Hypoxic Postconditioning on the Expression of Antiapoptotic Protein Bcl-2 and Neurotrophin BDNF in Hippocampal Field CA1 in Rats Subjected to Severe Hypoxia

A quantitative immunohistochemical method was used to study the expression of antiapoptotic protein Bcl-2 and neurotrophin BDNF in hippocampal field CA1 in rats subjected to severe hypoxia (SH), the harmful consequences of which were compensated by three subsequent sessions of postconditioning (PostC) with moderate hypobaric hypoxia (360 mmHg, 2 h, three times with 24-h intervals). Decreases in the expression of these proteins were seen in the hippocampus of rats after SH. Hypoxic PostC, which improves structural-functional rehabilitation after severe hypoxia, increased Bcl-2 and BDNF expression in neurons in hippocampal field CA1 in rats subjected to SH. The results provided evidence of the involvement of Bcl-2 and BDNF in the processes of adaptation to SH and compensation of its damaging effects.

Mild hypobaric hypoxic postconditioning increases the expression of HIF-1α and erythropoietin in the CA1 field of the hippocampus of rats that survive after severe hypoxia

Using immunohistochemistry, we studied the expression of the alpha regulatory subunit of the hypoxia-inducible factor (HIF-1α) and the product of its target gene, which encodes the protective cytokine erythropoietin in the hippocampal CA1 field of rats in response to damaging severe hypoxia and severe hypoxia followed by three sessions of postconditioning with mild hypobaric hypoxia. We found that the immunoreactivity to the proteins studied in the hippocampus of rats was reduced in response to severe hypoxia. Hypoxic postconditioning sessions of mild hypobaric hypoxia (360 mmHg, 2 h, three times at intervals of 24 h) up regulated the expression of HIF-1α and erythropoietin in hippocampal CA1 neurons of rats that survived after severe hypoxia. Our results indicate that postconditioning led to compensation of hypoxia-induced neuron damage in the brain, which actively involved HIF-1α and erythropoietin.

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.

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 Hypobaric Hypoxia in Various Modes on Expression of Neurogenesis Marker NeuroD2 in the Dentate Gyrus of Rats Hippocampus

The expression of neurogenesis marker – NeuroD2 transcription factor – in the hippocampal dentate gyrus was studied in rats exposed to severe destructive hypoxia, a single or three episodes of moderate hypobaric hypoxia, preconditioned severe hypoxia, and severe hypoxia followed by 3 sessions of postconditioning by moderate hypobaric hypoxia. All the studied hypoxic exposure modes led to an increase of NeuroD2 level. Three-fold moderate hypoxia per se and in the preconditioning mode (followed by exposure to severe hypoxia) produced most pronounced up-regulatory effect on NeuroD2 expression. The results indicated that stimulation of neurogenesis processes seemed to be one of the aspects of the neuroprotective effect of three-fold preconditioning moderate hypoxia, but not of hypoxic postconditioning.