Tatjana Gluschenko

The preconditioning modified neuronal expression of apoptosis-related proteins of Bcl-2 superfamily following severe hypobaric hypoxia in rats.

The patterns of expression of the Bcl-2, Bax, and Bcl-xL proteins were examined immunocytochemically in rat hippocampus and neocortex after severe hypobaric hypoxia (180 Torr for 3 h) and severe hypoxia preconditioned by intermittent mild hypoxia (360 Torr for 2 h daily, for 3 consecutive days, 24 h prior to severe hypoxia). As revealed by TUNEL assay, severe hypobaric hypoxia produced extensive apoptotic damage to the neurons of hippocampal CA1-CA4 and the neocortex but not the dentate gyrus granule cells. Remarkable posthypoxic up-regulation of Bax expression maximal at 24 h was detected in the CA1-CA4 areas of hippocampus and neocortex 3-72 h after severe hypoxia. The preconditioning to severe hypoxia protected neurons from the posthypoxic apoptotic transformations, the up-regulation of Bax expression, and resulted in persistent overexpression of Bcl-2 and Bcl-xL. We conclude that the protective action of hypoxic preconditioning is at least in part mediated by shifting of neuronal Bax/Bcl-2-Bcl-xL ratio to a favor of antiapoptotic proteins Bcl-2 and Bcl-xL.

Preconditioning enhances the expression of mitochondrial antioxidant thioredoxin-2 in the forebrain of rats exposed to severe hypobaric hypoxia

The impact of severe hypoxia and preconditioning on the expression of the mitochondrial antioxidant thioredoxin‐2 (Trx‐2) in rat hippocampus (CA1, CA2, CA3 fields, and dentate gyrus) and neocortex was studied by immunocytochemistry. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 hr) spaced at 24 hr. The last trial was followed by severe hypobaric hypoxia (180 Torr, 3 hr) 24 hr later. Both in hippocampus and in neocortex, severe hypobaric hypoxia resulted in enhanced Trx‐2 expression at 3 hr, followed by a slight decline in Trx‐2 levels, which nevertheless remained increased at 24 hr elsewhere except for the CA1 region. The preconditioning considerably augmented severe hypoxia‐induced Trx‐2 immunoreactivity, affecting both the number of immunoreactive cells and the intensity of immunostaining. The findings suggest a role for Trx‐2 in the formation of brain hypoxic/ischemic tolerance accomplished by the preconditioning.

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.

The augmentation of brain thioredoxin-1 expression after severe hypobaric hypoxia by the preconditioning in rats

Induction of endogenous antioxidants is one of the key molecular mechanisms of cell resistance to hypoxia/ischemia. The effect of severe hypoxia on the expression of cytosolic antioxidant thioredoxin-1 (Trx) in hippocampus and neocortex was studied in preconditioned and non-preconditioned rats. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. Twenty-four hours after the last trial rats were subjected to severe hypobaric hypoxia (180 Torr, 3 h). Trx expression was studied by immunocytochemistry. In hippocampus severe hypobaric hypoxia rapidly induced Trx expression, which remained elevated still at 24 h. In neocortex the enhanced expression appeared only at 24 h. The preconditioning significantly augmented severe hypoxia-induced Trx-immunoreactivity at 3 h but not at 24 h. These findings point out that Trx contributes to mechanisms of brain tolerance to hypobaric hypoxia, especially in early periods after the exposure.

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.

Differential expression of ADAM15 and ADAM17 metalloproteases in the rat brain after severe hypobaric hypoxia and hypoxic preconditioning

The ADAMs (a disintegrin and metalloprotease) are a family of membrane-anchored glycoproteins capable of shedding a multitude of proteins from the cell surface. Although ADAMs are being considered as crucial modulators of physiological and pathophysiological processes, their roles in neuronal death/survival are largely unexplored. In the present study, changes in brain expression of ADAM15 and ADAM17 (TACE) have been quantitatively examined in rats in response to injurious severe hypoxia (SH) and in animals which acquired hypoxic tolerance through preconditioning to mild hypoxia prior SH. SH persistently up-regulated ADAM15 mRNA and protein levels in hippocampus and neocortex but not in thalamus or hypothalamus. This effect was not observed in the preconditioned rats tolerant to SH. In contrast, hippocampal levels of ADAM17 mRNA and neocortical levels of ADAM17 mRNA and protein were largely reduced following SH in non-preconditioned rats. Hypoxic preconditioning prevented down-regulation of the adam17 gene and considerably enhanced ADAM17 protein expression in hippocampus and neocortex in response to SH. The present findings implicate ADAM15 in the processes of neuronal hypoxic injury. On the other hand, these results also provide evidence for a pro-survival neuroprotective role of ADAM17 and its engagement in the process of preconditioning-induced hypoxic tolerance. The analysis of the protein levels of soluble and membrane-bound forms of APP in the neocortex and hippocampus of rats subjected to SH and SH with preconditioning has demonstrated that an increased ADAM17 expression in preconditioned animals 24h after hypoxia corresponded to a higher level of soluble form of APP and a reduction of the membrane bound fraction which reflects the role of ADAM17 in APP shedding.

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 expression pattern of pro- and antiapoptotic proteins bax and Bcl-2 in rat brain neurons in response to severe hypobaric hypoxia: the correcting effect of hypoxic preconditioning.

In the early 1980s, the phenomenon of delayed neuronal death several days after ischemia was described in hippocampal neurons [1]. It was then demonstrated that the neuronal injury observed developed by the apoptotic mechanism (programmed cell death) with an involvement of genes and killer proteins. This neuronal death caused by severe forms of ischemia/hypoxia has been reported in susceptible brain areas other than hippocampus: neocortex, striatum, and cerebellum [2–4].

Effects of preconditioning by mild hypobaric hypoxia on the expression of manganese superoxide dismutase in the rat hippocampus

The molecular mechanisms of neuroprotection during hypoxic action involve induction of antioxidants, including manganese superoxide dismutase (Mn-SOD). Previously, we demonstrated that severe hypobaric hypoxia (SHH) resulted in some enhancement of the content of Mn-SOD in rat hippocampal neurons 3 h later. Preconditioning by a 3-time repetitive mild hypobaric hypoxia (MHH) considerably increased the expression of Mn-SOD in the CA2 and CA3 areas of the hippocampus (but not in the CA1 and DG areas) in comparison with unconditioned animals. The mechanism for the enhancement of the increase in Mn-SOD in the preconditioned rats remains unclear. This enhancement could be caused by induction of Mn-SOD expression by MHH prior to SHH, or by modification of the reaction to severe hypoxia itself. It was demonstrated that immunoreactivity to Mn-SOD was increased in CA1 and DG, but not in CA2 and CA3 up to the 24th hour after the three courses of MHH (i.e., until the beginning of SHH). Thus, the effect of preconditioning on Mn-SOD expression after SHH was revealed in those hyppocampal areas in which MSS itself did not result in an increase in the expression of this protein. Thus, the neuroprotective effect of preconditioning on the early stages following severe hypoxia was not associated with Mn-SOD accumulation during preconditioning, but was due to modification of the reaction to SHH itself.

Hypoxic preconditioning modifies activity of pro- and antioxidant systems in the rat hippocampus

The effects of repetitive mild hypobaric hypoxic preconditioning on pro- and antioxidant systems in rat hippocampus have been studied. It was found that three-trial preconditioning by mild hypobaric hypoxia (360 mm Hg, 2 h) induced moderate oxidative stress immediately after the last preconditioning trial. In addition, it down regulated the levels of protein antioxidants (Trx-1, Trx-2, Cu,Zn-SOD) and also decreased several lipid peroxidation products 24 h after the preconditioning.