Experimental Modeling of Damaging and Protective Hypoxia of the Mammalian Brain.

Currently, there is a new surge of interest in the problem of hypoxia, almost lost in recent decades. Due to the fact that the circle of competent specialists in this field has significantly narrowed, it is necessary to carry out an intensive exchange of knowledge. In order to inform a wide range of interested researchers and doctors, this review summarizes the current understanding of hypoxia, its pathogenic and adaptogenic consequences, as well as key physiological and molecular mechanisms that implement the response to hypoxia at various levels-from cellular to organismic. The review presents a modern classification of forms of hypoxia, the understanding of which is necessary for the formation of a scientifically based approach to experimental modeling of hypoxic states. An analysis of the literature covering the history and current level of hypoxia modeling in mammals and human experiments, including methods for creating moderate hypoxia used to increase the resistance of the nervous system to severe forms of hypoxia and other extreme factors, is carried out. Special attention is paid to the discussion of the features and limitations of various approaches to the creation of hypoxia, as well as the disclosure of the potential for the practical application of moderate hypoxic effects in medicine.

Acclimatization to Middle Attitude Hypoxia Masks the Symptoms of Experimental Posttraumatic Stress Disorder, but Does Not Affect Its Pathogenetic Mechanisms.

The effects of acclimatization to middle attitude hypoxia on the resistance to acute emotional stress were studied on the model of posttraumatic stress disorder in rats. Anxyolitic, but not anxiogenic effect was observed in acclimatized rats. However, acclimatized rats with posttraumatic stress disorder were characterized by hypofunction of the pituitary-adrenocortical axis, which is typical of this pathology, and reduction in corticosterone/dehydroepiandrosterone ratio. At the neuroendocrine level, up-regulation of glucocorticoid receptors and a decrease in the level of corticotropin-releasing hormone in the hippocampus were revealed. The observed modifications of regulatory mechanisms can underlie hypofunction of the pituitary-adrenocortical axis. It was concluded that acclimatization to middle attitude hypoxia masks behavioral symptoms of posttraumatic stress disorder, but does not alter its pathogenetic neuroendocrine mechanisms.

Pathogenetic Role of the Stress-induced Release of Glucocorticoid Hormones in the Development of Post-traumatic Stress Disorder: An Experimental Study.

In the rat experimental model of posttraumatic stress disorder (PTSD), the level of blood corticosterone was at least eight-fold increased (an overrelease). The use of hypobaric hypoxic preconditioning or short-term inhibition of glucocorticoid synthesis by metyrapone injection prevented development of the experimental PTSD.

Effects of Different Modes of Hypobaric Hypoxia on the Content of Epigenetic Factors in the Rat in Neurons of Rat Neocortex.

We studied the effects of different modes of hypobaric hypoxia on the content of epigenetic factors acH3K24, meH3K9, and meDNA modulating conformational characteristics of chromatin and gene expression in neurons of associative complex of rat parietal neocortex. Severe destructive hypoxia dramatically reduced the level of acH3K24 in 3 h after the end of exposure and increased meH3K9 and meDNA content. By contrast, 3-fold (but not single) adaptive exposure to moderate hypobaric hypoxia that produced a neuroprotective effect enhanced neuronal acH3K24 expression and decreased both meH3K9 and meDNA levels. Elevated acH3K24 content facilitates, while increased content of meH3K9 hampers binding of transcription factors to the target genes. At the same time, increased expression of meDNA suppresses transcription. The role of modification of epigenetic mechanisms in the regulation of proadaptive genes under the effects of hypoxic exposure according to various protocols is discussed.

Cerebral Mechanisms of Hypoxic/Ischemic Postconditioning.

This review analyzes recent data on mechanisms of cerebral hypoxia and the protective methods of hypoxic and ischemic postconditioning, as well as their interrelationship with the key mechanisms responsible for neuroprotection and neuroplasticity. Upregulation of expression of antiapoptotic factors and neurotrophins and modulation of activity of several protein kinases and transcription factors such as hypoxia-inducible factor-1 (HIF-1) are considered as the most important aspects in the neuroprotective potential of postconditioning. The presented information indicates substantial transformative promise of the noninvasive techniques of hypoxic postconditioning as well as significant similarity between the adaptive pathways activated by various postconditioning methods, which are far from being fully understood.

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.

Current Theory on the Cerebral Mechanisms of Hypoxic PRE- and Postconditioning.

An exposure of the organism to several episodes of mild hypoxia results in the development of brain hypoxic/ischemic tolerance, as well as cross-tolerance to the stressful factors of psychoemotional nature. Such kind of preconditioning by mild hypoxia functions as "alarm signalization" by I.P. Pavlov, preparing the organism and, in particularly, brain to the forthcoming harmful event. Dose-dependent action of hypoxia on the brain can be considered as one particular case of the general phenomenon termed hormesis, or neurohormesis. Endogenous defense processes launched by the hypoxic preconditioning and leading to the development of cerebral tolerance are associated with activation of intracellular signal cascades, transcriptional factors, regulatory proteins and expression of pro-adaptive genes and their products in the susceptible brain regions. Important mechanism of systemic adaptation induced by hypoxic preconditioning includes modifications of pituitary-adrenal axis aimed at enhancement of its adaptive resources. All these components are involved in the neuroprotective processes in three sequential phases - initiation, induction, and expression. Important role belongs also to epigenetic mechanisms controlling the activity of pro-adaptive genes. In contrast to the preconditioning, hypoxic postconditioning is comparatively novel phenomenon and therefore its mechanisms are less studied. The involvement of hypoxia-inducible factor HIF-1, and non-specific protective processes as up-regulation of anti-apoptotic factors and neurotrophines.

[A comparison of a neuroprotective effects of hypoxic postconditioning and cerebrolysin in the experimental model].

Hypoxic postconditioning using episodes of mild hypobaric hypoxia is a new neuroprotective technique. We compared the neuroprotective efficacy of hypoxic postconditioning and cerebrolysin in a model of posthypoxic pathology in rats. Animals that survived the severe hypoxia (180 Torr, 3 h) were exposed to hypoxic postconditioning or received cerebrolysin. Postconditioning prevented the injury and loss of hippocampal (fields CA1, CA4) and neocortical neurons whereas cerebrolysin was protective only for CA4 and the neocortex. Besides that, postconditioning, unlike cerebrolysin, led to the complete functional rehabilitation from the severe hypoxia by normalizing the level of anxiety and the pituitary-adrenal axis activity. The findings demonstrate that the elaborated postconditioning technique might provide useful tool for therapy of posthypoxic pathology and stroke.

[Involvement of transcriptional factor induced by hypoxia in the neuronal mechanisms of adaptation to psychoemotional and hypoxic stress].

Using quantitative immunohistochemistry, neuronal expression of alpha-subunit of the transcriptional factor HIF-1 in hippocampus and neocortex of rats in response to pathogenic psychoemotional (model of posttraumatic stress disorder, PTSD) and hypoxic (severe hypobaric hypoxia, 180 Torr, 3 h), as well as to neuroprotective exposures to hypoxic pre- and postconditioning has been studied. Elongated overexpression of HIF-1alpha in hippocampus and neocortex of rats in response to the psychoemotional stress in PTSD paradigm, but not hypoxic stress, has been observed. Hypoxic pre- and postconditioning with mild hypobaric hypoxia (360 Torr, 2 h, 3 trials spaced at 24 h), those induced adaptation to the psychoemotional stress, abolished the elongated HIF-1alpha overexpression. Hypoxic postconditioning which improved structure and functional rehabilitation following severe hypoxic stress up-regulated HIF-1alpha expression in the brain neurons of rats survived severe hypoxia. The findings indicate that transcription factor HIF-1 is particularly involved in the processes of adaptation/ maladaptation to the action of injurious stresses, but its role depends upon the nature of stressor.

[The effect of mild hypobaric hypoxia regime on expression of factor induced by hypoxia in the rat neocortex].

Using quantitative immunohistochemistry, modifications of HIF-1alpha expression in neocortex of rats exposed to various modes of mild hypobaric hypoxia (MHH) (1,3 and 6 episodes) differed in their neuroprotective efficacy have been studied. It has been shown that three-trial MHH being the most effective neuroprotective mode when used as a preconditioning produces most considerable changes in HIF-1 by substantial up-regulation of its regulatory alpha-subunit expression in the rat neocortex. Present findings support the hypothesis on important roles of HIF-1 in the mechanisms of brain hypoxic tolerance induced by the hypoxic preconditioning with three-trial MHH.

[Signal molecular and hormonal mechanisms of formation of the hypoxic preconditioning protective effects].

In the review, results of the long-standing authors'studies and literature data concerning one of the underresearched aspects of actual problem of induced brain tolerance to injurious factors - "preventive" signal function of the hypoxic preconditioning, as well as molecular and hormonal mechanisms underlying its protective effects are presented. Hypoxic preconditioning by using of mild hypobaric hypoxia in special mode mobilizes evolutionary acquired genome determined defense mechanisms of brain neurons and whole organism. This process involves an activation of multiple intracellular components, as well as hypothalamic-pituitary-adrenal axis. Cascade mechanisms of intracellular signaling including receptors, mitochondrial respiratory chain, key intracellular regulatory systems, early genes, superfamilies of the inducible and activation transcription factors are sequentially engaged in the processes of initiation, induction and expression of hypoxic tolerance. The determination of optimal modes of hypoxic preconditioning appears to be of significant importance to assure the effective activation of protective signal mechanisms.

[Hypoxic postconditioning corrects behavioral abnormalities in a model of post-traumatic stress disorder in rats].

Protective effects of the novel technique of hypoxic postconditioning with a hypobaric hypoxia paradigm were studied in "stress-restress" model ofposttraumatic stress disorder in rats. It was shown that repeated (3 times) exposure of rats that survived after severe traumatic stress to mild hypobaric hypoxia (postconditioning mode) efficiently abolished the development of stress-induced anxiety state. Postconditioning had a clear anxiolytic effect both when it was delivered after traumatic stress and after restress, but the intensity of this effect depended on the period ofpathogenesis of the posttraumatic stress disorder, when postconditioning was given. The results indicate that suggested postconditioning model with repetitive mild hypobaric hypoxia exerts potent anxiolytic and stress-protective action.

Antidepressant effect of hypoxic preconditioning is associated with modification of expression of transcription factor c-Fos in rat brain in response to unavoidable stress.

Development of post-stress depression in rats was accompanied by long-term moderate activation of the expression of transcription factor c-Fos in the neocortex, hippocampus, and paraventricular nucleus of the hypothalamus. Hypoxic preconditioning preventing depressive state in rats under conditions of unavoidable stress considerably enhanced c-Fos expression in the studied brain regions during the early stages of stress response (days 1-5) and promoted its normalization at later terms (10 days). Disturbances in the wavy dynamics of c-Fos expression can contribute to the pathogenic mechanisms of depression, in particular and induce hyperproduction of hypothalamic neurohormone corticoliberin, whereas potentiation of early expression of this factor in response to stress is obviously necessary for prevention of post-stress disorders.

[Molecular-cellular and hormonal mechanisms of induced brain tolerance of extreme factors].

This review includes results of own studies and literature data on the topical problem of neurobiology and medicine: discovery of the mechanisms of increased brain resistance to extreme exposures. The emphasis is made on the molecular-cellular and hormonal mechanisms of hypoxic preconditioning-induced brain tolerance to injurious hypoxia, psychoemotional and traumatic stress. A role of basic hormonal and intracellular cascade pro-adaptive processes mediating the neuroprotective action of hypoxic preconditioning is reviewed. A dynamics of the mechanisms of development of induced susceptible brain areas (hippocampus, neocortex) tolerance which includes phases of induction, transformation and expression, is presented. New data on preconditioning-induced cross-tolerance providing increased brain resistance not only to hypoxia but also to other stresses are reported. For the first time neuroprotective effects of hypoxic postconditioning are described.

[Effects of several modes of hypobaric hypoxia on expression profiles of corticosteroid receptors in rat hippocampus].

Gluco- and mineralocorticoid receptors are believed to play important roles in mechanisms of the hypothalamic-pituitary-adrenal axis (HPA) regulation, neuronal death/survival, as well as learning and memory processes. Imbalanced levels of MR and GR result in impairment of HPA activity and can promote neuronal injury and loss following exposures to extreme factors. In the present study, using quantitative immunohistochemistry, the comparative analysis of the effects of hypobaric hypoxia in several modes on expression profiles of GR and MR in dorsal (CA1) and ventral (dentate gyrus) hippocampus was performed. According to the data obtained, severe injurious hypoxia induced prominent disturbances of GR and MR expression in the cells of CA1 and dentate gyrus that correlated to the remarkable neuronal injury/loss in CA1 and dysregulated HPA activity. Sets of three- or six-trial (but not one-trial) preconditioning using mild hypoxia prior to severe hypoxia prevented these abnormalities.

[A test for evaluation of pituitary-adrenal axis disregulation].

In rat models, a modification of the fast feedback paradigm for the pituitary adrenocortical system applied to detect posttraumatic stress disorder (PTSD) was developed. Both standard and modified methods were used. In contrast to the standard method (injection of exogenous cortisol), the new modification suggested measuring blood corticosterone, rather than adrenocorticotropic hormone, at the early stages of development of the stress reaction (3, 10, 30, and 60 min of the exposure to stress factors). With the suggested modification, the fast feedback phenomenon was studied in reliable rat models of PTSD (stress-restress) and depression (learned helplessness). Fast pathological inhibition of the pituitary adrenocortical system by the fast feedback mechanism was revealed only during the simulated PTSD but not in the depressive-like state, which supported the specificity and validity of the developed modification of the test. Thus, the proposed methodological modification is a valid tool for diagnostics of the PTSD-specific fast feedback inhibition of the pituitary adrenocortical system in the animal models of this psychopathology.

Effects of moderate hypobaric hypoxic preconditioning on the expression of the transcription factors pCREB and NF-kappaB 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-kappaB (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-kappaB 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-kappaB and CREB are involved in the mechanisms forming HP-induced tolerance of the brain.

Effects of hypoxic preconditioning on expression of transcription factor NGFI-A in the rat brain after unavoidable stress in the "learned helplessness" model.

We report here our immunocytochemical studies establishing that the development of a depression-like state in rats following unavoidable stress in a "learned helplessness" model is accompanied by stable activation of the expression of transcription factor NGFI-A in the dorsal hippocampus (field CA1) and the magnocellular paraventricular nucleus of the hypothalamus, along with an early wave of post-stress expression, which died down rapidly, in the ventral hippocampus (the dentate gyrus) and a long period of up to five days of high-level expression in the neocortex. In rats subjected to three sessions of preconditioning consisting of moderate hypobaric hypoxia (360 mmHg, 2 h, with intervals of 24 h), which did not form depression in these circumstances, there were significant changes in the dynamics of immunoreactive protein content in the hippocampus, with a stable increase in expression in the ventral hippocampus and only transient and delayed (by five days) expression in field CA1. In the neocortex (layer II), preconditioning eliminated the effects of stress, preventing prolongation of the first wave of NGFI-A expression to five days, while in the magnocellular hypothalamus, conversely, preconditioning stimulated a second (delayed) wave of the expression of this transcription factor. The pattern of NGFI-A expression in the hippocampus, neocortex, and hypothalamus seen in non-preconditioned rats appears to reflect the pathological reaction to aversive stress, which, rather than adaptation, produced depressive disorders. Post-stress modification of the expression of the product of the early gene NGFI-A in the brain induced by hypoxic preconditioning probably plays an important role in increased tolerance to severe psychoemotional stresses and is an important component of antidepressant mechanisms.

Stable modifications to the expression of neurohormones in the rat hypothalamus in a model of post-traumatic stress disorder.

We report studies of the neuroendocrine mechanisms of development of an anxiety state in rats using the "stress-restress" experimental model of post-traumatic stress disorder. Immunocytochemical methods demonstrated significant increases in corticoliberin expression in both the parvo- and magnocellular parts of the paraventricular nucleus persisting to 10 days after presentation of the animals with repeated stress. Decreases in vasopressin expression were seen in the paraventricular nucleus of the animals on the first day after repeated stress. Vasopressin contents in the parvocellular part of the nucleus in animals of the experimental group were no different at 10 days from those in animals of the control group, while levels in the magnocellular part were increased. These data provide evidence for the involvement of the hypothalamic component of the vasopressinergic system (along with the corticoliberinergic system) in the pathogenetic mechanisms of the analog of post-traumatic stress disorder generated in this model.

[The hypoxic preconditioning effect upon expression of the NGFI-A transcription factor in the rat brain following an unavoidable stess in the "learned helpnessness" model].

In this study, by an immunocytochemistry it is established that development of the depression after an inescapable stress in the learned helplessness model in rats is associated with stable induction of the transcription factor NGFI-A in the dorsal hippocampus (CA1 field) and in the paraventricular hypothalamic magnocellular nucleus (PVNm), as well as with rapid and transient stress-induced expression of NGFI-A in the dentate gyrus and, supported on high level till 5 days, in the neocortex. Hypoxic preconditioning using mild repetitive hypobaric hypoxia (360 Torr for 2 hrs each of 3 days) prevented development of the depressive state in rats, and considerably changed the dynamics of the NGFI-A immunoreactivity in the hippocampus: the stable increase of an expression in the dentate gyrus and only transitory and delayed (for 5 day) in the CA1 field was detected. In the neocortex (Layer II) the stress influence was levelled with preconditioning by preventing the prolongation of the first wave of expression NGFI-A untill 5 days, and in PVNm, on the contrary, was stimulated the second (delayed) wave of an expression of this transcription factor. The pattern of NGFI-A expression in the hippocampus, neocortex and hypothalamus of preconditioned rats revealed an obvious pathological response to aversive stress, which results in development of depressive frustration rather than the adaptation. Stress-induced modifications of early gene product NGFI-A expression in the brain caused by hypoxic preconditioning, possibly, play important role in tolerance to hard psychoemotional stresses and may be an important part of antidepressive mechanisms.