[The effect of Mexidol on cerebral mitochondriogenesis at a young age and during aging]. / Vliianie Meksidola na tserebral'nyi mitokhondriogenez v molodom vozraste i pri starenii.

AIM: To study the ability of mexidol to induce cerebral mitochondriogenesis in the brain of young and aging rats. MATERIAL AND METHODS: Expression level of marker proteins of cerebral mitochondriogenesis was evaluated during treatment with mexidol (20, 40, 100 mg/kg; 20 days; intraperitoneally) in the cerebral cortex of young (3 month) and aging (6, 9, 12, and 15 month) outbred male rats, using the Western blot analysis. RESULTS: It has been shown for the first time that the course injections of mexidol in doses of 40 and 100 mg/kg is accompanied by dose-dependent induction of the succinate receptor SUCNR1 and protein markers of mitochondrial biogenesis: transcription coactivator PGC-1α, transcription factors (NRF1, TFAM), catalytic subunits of respiratory enzymes (NDUV2, NDUV2,cytb, COX2) and ATP synthase (ATP5A) in the cerebral cortex of young and aging outbred male rats. Mexidol-dependent overexpression of subunits of mitochondrial enzymes and PGC-1α is observed only with the course of the drug. CONCLUSION: The results indicate the ability of mexidol to induce cerebral mitochondriogenesis and eliminate mitochondrial dysfunction in young and aging animals and, thus, exert an effect on one of the key pathogenetic links of the development of disorders in aging and neurodegenerative diseases.

Peculiarities of Immediate Response of Respiratory Chain Enzymes in Rat Cerebral Cortex to Hypoxia.

We performed a complex study of the dependence of immediate reaction of catalytic subunits in mitochondrial enzymes (NDUFV2, SDHA, Cyt b, COX1, and ATP5A) in rat cerebral cortex (the most hypoxia-sensitive tissue) on the severity and duration of hypoxia in vivo and the role of individual resistance of rats to oxygen deficiency in this process. Three types of responses to hypoxia were revealed. The immediate response of mitochondria to oxygen deficiency appeared after its drop by 30-33% relatively to normal atmosphere level. It manifested in up-regulation of NAD-dependent oxidation, i.e., activation of respiratory chain complex I. Further decrease in oxygen concentration by 50% reprogrammed the work of respiratory chain via activation of respiratory chain complex II in parallel with down-regulation of the electron transport function of the respiratory chain complex I. This response was optimal for the expression of adaptation genes and for the formation of immediate tolerance of rats to hypoxia. The greatest drop of oxygen concentration by 60-62% reversed the Krebs cycle promoting recovery of the electron transport function of respiratory chain complex I. Despite this, the energy efficiency of the respiratory chain and the potency to mobilize the rapid adaptation mechanisms degraded due to abnormalities in cytochrome segment of the respiratory chain.

The Role of Succinate in Regulation of Immediate HIF-1α Expression in Hypoxia.

Hypoxia-induced immediate expression of transcription factor HIF-1α in the brain cortex is regulated by succinate produced in both the tricarbonic acid cycle and GABA shunt reactions and is induced by succinate-containing drugs. These facts prove the existence of succinate-dependent signalling regulation involved in immediate and delayed molecular adaptation and increased body resistance to oxygen deficiency, where succinate acts as a signal molecule. The intensity of this process differs in animals with low and high resistance to hypoxia.

Specific Features of Immediate Ultrastructural Changes in Brain Cortex Mitochondria of Rats with Different Tolerance to Hypoxia under Various Modes of Hypoxic Exposures.

We performed ultrastructural study of cerebral cortex mitochondria in rats with different tolerance to oxygen deficiency (low resistant and highly resistant specimens). Low resistant rats were characterized by the prevalence of mitochondria with lightened matrix due to the nondense packing of cristae. By contrast, mitochondria of highly resistant animals had the dense packing of cristae. The structure of mitochondria underwent adaptive changes at 14-10% O2 in the inspired air. Under these conditions, structural characteristics of the cerebral cortex in hypoxia-sensitive rats resembled those in resistant animals. The decrease in O2 concentration to 8% was accompanied by ultrastructural signs of mitochondrial damage, which correlated with de-energization of the cell and dysfunction of adaptive signaling systems. Ultrastructural features of cerebral cortex mitochondria in animals with low and high tolerance to acute oxygen deficiency confirm the hypothesis that they are associated with two different "functionaland-metabolic portraits".

Specific Features of Immediate Expression of Succinate-Dependent Receptor GPR91 in Tissues during Hypoxia.

Under normoxic conditions, succinate-dependent receptor GPR91 was found in varying amounts in all analyzed aerobic tissues except erythrocytes. The maximum density of the receptor was observed in the myocardium: by 2.3 and 1.7 times higher than in the kidneys and brain cortex, respectively. Hypoxic expression of GPR91 was tissue-specific, depended on the duration and severity of hypoxia, and did not correlate with the basal level of this receptor. It was maximum in the brain cortex, which confirms the high importance of this signal pathway for brain functioning. Single hypoxic exposure induced immediate expression of GPR91 in the brain cortex within 15-60 min, which correlated with the development of urgent tolerance to hypoxia of the body. Induction of immediate expression of GPR91 in brain cortex occurred during first 15-60 min and correlated to the forming of immediate tolerance of organism to hypoxia. Brain cortex-specific immediate expression of GPR91 during hypoxia was related to activity of the GABA-bypass that acts as the source of succinate for the receptor under these conditions.

[Urgent changes in the expression of hypoxia-inducible factor-1α (HIF-1α) in the neocortex of rats with different tolerance to acute hypoxia underwent focal ischemic stroke prefrontal cortex].

Using immunohistochemical method, it was demonstrated that neurons of the cerebral cortex have the capacity to express hypoxia-inducible factor-1 alpha (HIF-1α) in normoxia. Intensity of this process is different for rats having unequal tolerance to hypoxia. Basal HIF-1α expression in neurons of rats with low-resistance (LR) to hypoxia is higher compared to rats with high-resistance (HR). Bilateral photochemically induced focal ischemic insult in the rat prefrontal cortex completely suppressed HIF-1α neuronal expression the in the ischemic zone and only partially--in the area of the penumbra. Neuronal injury was more pronounced in cortex of LR rats compared to HR rats. These findings suggest that functional significance of HIF-1α is greater in neurons of the cerebral cortex of LR rats compared to HR rats.

[Role of the glutathione system in regulation of redox status in the rat cerebral cortex under hypoxia].

The study compared effects of different hypobaric hypoxia regimens (14% O2, 10.5% O2, and 8% O2; 1 hour; 15 days) on parameters of the glutathione system, intensity of free radical oxidation, and intensity of lipid peroxidation in cerebral cortex (CC) of rats with different, genetically predetermined resistance to acute hypoxia. In normoxia, baseline concentrations of oxidized glutathione (0.155 ± 0.011 nmol/mg protein) and hydroperoxide metabolites (50.4 ± 2.62 cumene hydroperoxide equivalents/g tissue) were 20% lower in CC of low-resistance (LR) rats than in high-resistance (HR) rats (0.191 ± 0.013 nmol/mg protein; 63.0 ± 3.46 cumene hydroperoxide equivalents/g tissue, respectively). Baseline activities of the glutathione cycle enzymes, glutathione peroxidase (31.2 ± 1.59 nmol/min/mg protein) and glutathione reductase (28.5 ± 1.49 nmol/min/mg protein), were 30% lower in LR rat CC than in HR rat CC (47.0 ± 2.41; 433 ± 2.26 nmol/min/mg protein, respectively). These data suggested more efficient antioxidant defense of CC tissue in LR rats than in HR rats. The phenotypic difference in CC tissue redox properties between two rat phenotypes remained in hypoxia. The efficiency of glutathione system in regulation of CC redox homeostasis was shown to depend on both severity and duration of hypoxic exposures and on individual tolerability of hypoxia, i.e., this efficiency was genetically predetermined. The glutathione system maintains its regulatory properties in a broader range of lowered pO2 values and during longer hypoxic exposures. in LR rat CC than in HR rat CC. For this reason, activation of free radical processes and development of oxidative stress induced by single or repeated hypoxic exposures are prevented or alleviated in LR rat CC but not in HR rat CC. The obtained data strongly justify the need for selection of hypoxic exposure regimens for therapeutic hypoxic preconditioning. Apparently the hypoxic exposures not associated with signs of oxidative stress should be considered optimum.

Role of proinflammatory factors, nitric oxide, and some parameters of lipid metabolism in the development of immediate adaptation to hypoxia and HIF-1α accumulation.

The development of immediate and delayed long-term resistance to hypoxia during a course of intermittent normobaric hypoxia (15 daily sessions of alternating exposure to 10% O2 and atmospheric air for 1 h) correlated with biphasic expression of HIF-1α in neocortex of hypoxia-intolerant rats, which suggests involvement of this protein factor not only in the formation of long-term adaptation, but also in triggering immediate adaptation to hypoxia. Both processes develop under conditions promoting down-regulation of oxidative modification of LDL and increasing tolerance of biological membranes to hypoxia in the absence of activation of the free radical processes, which therefore do not trigger HIF-1α expression under these conditions. Neither cytokines nor NO are the inducers of immediate adaptation, and they are not related to HIF-1α expression during the early post-hypoxic period. In contrast, long-term adaptation in response to the course of intermittent normobaric hypoxia develops against the background of enhanced NO production, activation of pro- and anti-inflammatory factors, and expression of VEGF, the marker of angiogenesis. Therefore, all these factors can promote activation of transcription processes required to form the long-term adaptation.

Phenotypic features of the dynamics of HIF-1α levels in rat neocortex in different hypoxia regimens.

There are tissue-specific and phenotypic differences in the basal levels of HIF-1α under normoxic conditions. Induction of short-term adaptation to hypoxia and formation of long-term adaptation are genetically determined. These phenomena are observed only in animals with low resistance to hypoxia and are associated with biphasic expression of HIF-1α in the neocortex only during hypoxic preconditioning. Severe hypoxia disorders HIF-1α expression and impairs the formation of short-term and long-term resistance. In animals with high resistance to hypoxia, neither short-term nor long-term resistance develops in response to hypoxic exposure, and this correlates with the absence of changes in post-hypoxic HIF-1α levels in the neocortex.

Effect of hypoxic preconditioning on free radical processes in tissues of rats with different resistance to hypoxia.

We studied the effect of single hypoxic preconditioning exposure (hypobaric hypoxia, 5000 m, 60 min) on free radical processes, glutathione system, and antioxidant defense enzymes in tissues of rats with different resistance to acute hypoxia. The intensity of free radical processes was shown to increase or decrease on day 1 after hypoxic preconditioning. These changes were tissue-specific and opposite in animals with genetically determined differences in the resistance to hypoxia. Hypoxic preconditioning contributes to the immediate resistance. The effect was more pronounced in low resistant animals, who did not exhibit signs of oxidative stress in tissues during the early posthypoxic period. By contrast, hypoxic preconditioning was followed by activation of free radical processes in tissues of highly resistant animals. These rats were characterized by low ability for the development of immediate resistance. Activation of free radical processes in the early period of adaptation (first hours after hypoxic preconditioning) does not play a role in the induction of immediate adaptive mechanisms for hypoxia.

Urgent reaction of the complement system to hypoxic exposure in rats sensitive to hypoxia.

Different modes of hypoxic exposure led to phasic changes in activities of the complement system components in rats sensitive to hypoxia starting from the first minutes of the posthypoxic period and persisting for 24 h and longer. The direction of shifts in the complement system depended on the duration and intensity of oxygen deficiency. Single one-hour interval hypoxia led to a moderate elevation of activities of virtually all the studied components. A more intense hypoxic exposure (1-h hypobaric hypoxia at a height of 5000 m) induced a biphasic response: reduction of activities of the majority of complement system components during the first hour of posthypoxic period and subsequent elevation of these activities above the normal. Exposure to severe hypobaric hypoxia (7000 m) led to a longer and more pronounced primary reduction of complement components activities, while the phase of their activity increase was blurred. Animal capacity to the formation of urgent tolerance of hypoxia was retained and increased with increasing the severity of hypoxic exposure. The complement consumption during the posthypoxic period was presumably a programmed reaction preventing hyperactivation of complement system components and essential for tolerance formation.