[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.

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.

[Phenotypic characteristics of factor expression induced by hypoxia and redox status of the rat neocortical cells at different stages of adaptation to hypoxia].

Hypoxic preconditioning induces two-phase increase of HIF-1alpha expression in the neocortex of low-resistance rats. The first, brief phase appears after each hypoxic episode and rapidly disappears in normoxic conditions. The second increase in of HIF-1alpha expression occurs in 24 hours after the hypoxic episode. The phase-nature of HIF-1alpha expression corresponds to the dynamics of urgent and long-term resistance in low-resistance rats, which suggests the HIF-1alpha involvement in mechanisms of urgent and long-term adaptation. It was found that in the mode of preconditioning, hypoxic treatments mobilized the anti-oxidant system (activated Cu, Zn-SOD) and had no effect on the intensity of lipid peroxidation processes in neocortex (INH, 10% O2) or even decreased the content of lipid peroxidation products and oxidized glutathione in neocortical cells in the early post-hypoxic period (HBH-5000, 10.5% O2). Thus, ROS do not play a key role in the induction of HIF-1alpha expression and fast-response/long-term adaptation to O2 deficiency in hypoxia-sensitive animals. In high-resistance rats, hypoxia preconditioning does not influence the HIF-1alpha protein expression and the adaptation. Severe hypoxic modes (HBH-7000, 8% O2) caused activation of lipid peroxidation processes in neocortex of hypoxia-sensitive rats. With the pro-oxidant systems dominating over the anti-oxidant ones, the neocortical expression of HIF-1alpha was found to decrease, which was accompanied by the impairment of the mechanisms of fast-response/long-term adaptation to hypoxia.

Activity of mitochondrial ATP-dependent potassium channel in animals with different resistance to hypoxia before and after the course of hypoxic training.

Activity of mitochondrial ATP-dependent potassium channel in rats with high genetically determined resistance to hypoxia was higher than in sensitive animals. Adaptation of low resistant rats to hypoxia was accompanied by activation of the channel, facilitation of potassium recycling in mitochondria, and a decrease in the rate of H2O2 formation. Our results indicate that mitochondrial ATP-dependent potassium channel plays an important role in the delayed mechanisms of animal's adaptation to hypoxia.

Effects of preconditioning on the resistance to acute hypobaric hypoxia and their correction with selective antagonists of nicotinic receptors.

Hypobaric hypoxic preconditioning increased the resistance of low resistant and highly resistant rats to acute hypobaric hypoxia at a critical height. Intergroup differences in the resistance of rats to acute hypobaric hypoxia were not observed after hypobaric hypoxia and one variational series with a wide range of resistance (4.5-24.5 min) appeared. Methyllycaconitine, an antagonist of subtype α(7) nicotinic cholinergic receptors, abolished the influence of hypobaric hypoxia on low resistant rats, but had no effect on highly resistant animals. Mecamylamine, a preferential antagonist of subtype α(4)ß(2) and α(3)-containing cholinergic receptors, did not modulate the effect of hypobaric hypoxia. By contrast, hypobaric hypoxia abolished the effect of mecamylamine on the resistance of rats that were not trained under conditions of hypobaric hypoxia (low resistant and highly resistant animals with low sensitivity to hypobaric hypoxia). We conclude that the same effect of hypobaric hypoxia is mediated by various mechanisms, which involve different nicotinic cholinergic receptors. They differ from the resistance mechanisms in non-trained rats.

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.

Development of resistance of an organism under various conditions of hypoxic preconditioning: role of the hypoxic period and reoxygenation.

Single exposure to moderate (10% O(2)) hypobaric, normobaric, and intermittent hypoxia is followed by a preconditioning response of the organism. The mechanisms for immediate adaptation are activated during the hypoxic period. Intermittent reoxygenation not only delays, but even suppresses this process. However, periods of oxygenation during the course of hypoxic training reduce the effect of hypoxia and prevent the possibility for "overdosage" of the adverse stimulus. Hence, they have a regulatory or normalizing role under these conditions. Our results indicate that hypoxitherapy in intermittent hypoxia mode provides optimum conditions for long-term adaptation.

Energotropic effect of succinate-containing derivatives of 3-hydroxypyridine.

Succinate-containing derivatives of 3-hydroxypyridine, mexidol and proxypin, serve as succinate donors for the respiratory chain and contribute to activation of the succinate oxidase pathway of oxidation. Under conditions of hypoxia, these changes promote recovery of aerobic energy production, normalization of intracellular ATP concentration, and development of the antihypoxic effect. Succinate-free analogues of the test compounds exhibit no such properties. Both agents are considered as energotropic substances. The specific effect of these compounds is manifested in direct interaction with the respiratory chain and normalization of ATP synthesis under conditions of hypoxia/ischemia. The test compounds can be used for the correction of energy metabolism disorders during acute oxygen deficiency. Moreover, they can be used for the treatment of associated functional disturbances.

Effect of several flavonoid-containing plant preparations on activity of mitochondrial ATP-dependent potassium channel.

Flavonoid-containing plant preparations (water soluble extracts of Pentaphylloides fruticosa [Extralife], Emblica officinalis Gaerth [Amla], and Bergenia crassifolia [Bergenia]) produced a dose-dependent and tissue-specific effect on activity of mitochondrial ATP-dependent potassium channel. The effect of these preparations was biphasic (activation and inhibition). The activating effect of Extralife was one order of magnitude higher than that of Amla and Bergenia and was observed in a wider concentration range. The activating effect of preparations was abolished by inhibitors of the mitochondrial ATP-dependent potassium channel, which attested to specificity of their influence on mitochondrial channel. Under in vivo conditions, the antihypoxic effect of Extralife was partially abolished by mitochondrial ATP-dependent potassium channel inhibitor 5-hydroxydecanoate.

[The regulatory role of mitochondrial dysfunction in hypoxia and its interaction with transcription activity].

The mitochondrial respiratory chain participates in the performance of the signal system, which activates the realization of metabolic compensatory processes and coupled functional response to both single and repeated, long-term exposure to acute hypoxia. Under the conditions of reduced oxygen delivery to cells the mitochondrial respiratory chain is involved in the process of oxygen homeostasis regulation and modulates oxygen consumption, the rate of oxygen delivery from the extracellular milieu to mitochondria, and energy synthesis, activating hypoxia-specific transcription factors as well.

[Energotropic, antihypoxic, and antioxidative effects of flavonoids].

Phytogenous flavonoid-containing agents (PFCA) are able to initiate electron flow bypassing the NAD-dependent region of respiratory chain, which is related with the activity of DT-diaphorase catalyzing two-electron reduction of quinones to hydroquinones and hydrogen peroxide in the presence of NADH and oxygen. This property is dramatically potentiated under the conditions of suppressed electron transport function of mitochondrial enzyme complex I (MEC I). In this process, part of the flow goes to the cytochrome region of respiratory chain and provides recovery of the MEC II and MEC III coupling function. The other part forms a flow of free oxidation which can perform as an additional mechanism normalizing the cell redox potential and aimed at decreasing intracellular acidosis under the conditions of MEC I bypassing. The energotropic effect of PFCA under the conditions of blocked MEC I is best evident at low PFCA concentrations. The ratio of coupled to free oxidation in the presence of PFCA depends on PFCA concentration. At low PFCA concentrations and oxidation of NAD-dependent substrates, both pathways become potentiated to an approximately similar extent, although the coupled oxidation pathway is generally activated earlier. At high PFCA doses, the increase in free oxidation pathway predominates and may result in toxic side effects.

Effect of intermittent normobaric hypoxia on kinetic properties of mitochondrial enzymes.

We studied the effect of intermittent normobaric hypoxia on the formation of adaptive signs and state of mitochondrial enzymes in the cerebral cortex of rats with different resistance to hypoxia. Kinetic parameters for mitochondrial enzymes in the substrate region of the respiratory chain of the cerebral cortex underwent various changes in low resistant and highly resistant rats over the first 2 h after 1-h intermittent normobaric hypoxia. Low resistant animals were characterized by more effective functioning of rotenone-sensitive NADH-cytochrome C reductase and succinate-cytochrome C reductase under conditions of increased reduction status of the cell. These features correlated with the increase in the general resistance of animals. Significant changes in kinetic properties of mitochondrial enzymes and signs of the development of resistance were not found in highly resistant rats. Reciprocal relations between mitochondrial enzyme complexes in the substrate region of the respiratory chain probably play a role of the signal regulatory mechanism, which mediates tissue-specific and general resistance of rats under conditions of intermittent normobaric hypoxia. These effects did not depend on oxygenation of the inhaled gas mixture during the inter-hypoxic period.

Behavioral peculiarities and reactions of brain dopaminergic systems in rats with different resistance to acute hypobaric hypoxia.

Locomotor activity in the open field test did not correlate with rat resistance to acute hypobaric hypoxia; there was a correlation between this resistance and rat behavior during acute stress. Immobility was characteristic of rats with low and particularly medium resistance to hypoxia; this reaction can be abolished by antidepressants. by contrast, highly resistant rats were mainly hyperactive. The resistance to hypoxia was associated with extreme parameters of dopaminergic neuron functioning. In low-resistant rats locomotor stereotypia was maximal, while perioral stereotypia was the minimal; highly resistant rats were characterized by an opposite pattern, and medium-resistant rats occupied an intermediate position.