С момента эпохального открытия микроглии Пио дель Рио Ортегой минуло столетие многоплановых исследований, которые подтвердили гениально предсказанные испанским гистологом свойства и функции микроглиальных клеток. Однако причины возрастзависимого снижения гомеостатического/репаративного потенциала микроглии, также как подходы к модуляции цитотоксического состояния микроглии стареющего мозга остаются нерешенными вопросами на текущем этапе развития нейробиологии, в то время как возраст-ассоциированные нейровоспаление, нейродегенерация и нейродисфункция представляют собой угрожающий вызов современному «стареющему» социуму. Цель обзора – анализ представлений о причинах развития дистрофического/старческого фенотипа микроглии и подходов к увеличению ее нейрорепаративного потенциала в стареющем мозге. В работе обсуждаются прогрессирующая с возрастом глюкокортикоидная гиперпродукция и недостаточность проразрешающих/про-анаболических факторов как ведущие механизмы в развитии возрастных дистрофических изменений микроглии. Since the epoch-making discovery of microglia by Pio del Rio Ortega, a century of multifaceted studies has passed, which confirmed the properties and functions of microglial cells ingeniously predicted by the Spanish histologist. However, the reasons for the steady age-dependent decrease in the homeostatic/reparative potential of microglia, as well as approaches to modulating the cytotoxic state of microglia in the aging brain, remain unresolved issues at the current state of neurobiology, while age-associated neuroinflammation, neurodegeneration and neurodysfunction represent a threatening challenge to the modern “aging” society. The aim of this review was to analyze the ideas about the causes for the development of the microglia dystrophic/senescent phenotype of microglia and approaches to enhancing its neuroreparative potential in the aging brain. The authors discussed the age-related progression of glucocorticoid hyperproduction and the shortage of pro-resolving/pro-anabolic factors as the leading mechanisms in the development of age-related dystrophic changes in microglia.

В развитии возраст-ассоциированных нейродегенеративных заболеваний ключевая роль отводится устойчивой провоспалительной активации микроглии и опосредованным реактивной микроглией окислительному стрессу, нейровоспалению, астроглиальной дисфункции. Конститутивная экспрессия микроглиоцитами сукцинатного рецептора SUCNR1/GPR91, формирование новых представлений о сукцинате как иммунометаболите (метабокин) и отсутствие исследований влияния сукцинатной сигнализации на морфофункциональное состояние резидентных иммунных клеток ЦНС предопределили проведение исследования. Цель работы - выявление морфофункциональных особенностей и количества астро- и микроглиоцитов в стареющем мозге крыс при курсовом применении сукцинатсодержащего препарата мексидол (этилметилгидроксипиридина сукцинат). Методика. Исследование выполнено на самцах беспородных белых крыс 3, 6 и 18 мес. Мексидол вводили в/б в дозе 100 мг/кг ежедневно, в течение 3, 7 и 14 сут. Методом вестерн-блот-анализа в лизате коры головного мозга (КГМ) определяли содержание высокоспецифичных маркеров микроглиальной (Iba1, ionized calcium binding adapter molecule 1) и астроглиальной (GFAP, glial fibrillary acidic protein) активации, а также уровень синаптофизина (SYP, маркер синаптогенеза). Методом иммуногистохимического окрашивания Iba1 и GFAP на парафиновых срезах префронтальной коры (ПФК) и гиппокампа оценивали морфологические особенности и осуществляли подсчет астро- и микроглиоцитов у молодых и старых крыс. Результаты. У старых животных уровень GFAP и Iba1 увеличивался на 30% и 20% соответственно, а содержание SYP снижалось на 25% в сравнении с молодыми животными, что свидетельствует об активации воспалительного процесса и снижении синаптогенного потенциала у старых животных. Морфологические признаки провоспалительной поляризации (короткие, слаборазветвленные, малочисленные отростки) были характерны для микро- и астроглии ПФК старых крыс. Курс мексидола вызывал в КГМ старых крыс снижение содержания GFAP и Iba1, увеличение экспрессии SYP до уровня молодых животных, увеличение количества, длины, разветвленности отростков у GFAP- и Iba1-положительных клеток, что свидетельствует о сукцинат/SUCNR1-зависимой противовоспалительной трансформации микро- и астроглии в стареющем мозге. Заключение. Исследование впервые раскрывает новые аспекты активности мексидола и сукцинатной сигнализации в мозге, что проявляется в ограничении воспалительной реакции и усилении синаптической пластичности. Сукцинатсодержащий препарат мексидол может быть использован в комплексной терапии различных неврологических патологий, ассоциированных с нейровоспалением и когнитивным дефицитом. In the development of age-associated neurodegenerative diseases, a key role is played by sustained pro-inflammatory activation of microglia and reactive microglia-mediated oxidative stress, neuroinflammation, and astroglial dysfunction. The constitutive expression of the succinate receptor SUCNR1/GPR91 by microgliocytes, formation of new ideas about succinate as an immunometabolite (metabokine) and the lack of studies of the effect of succinate signaling on the morpho-functional state of CNS resident immune cells stimulated this study. The aim of the study was to assess the morpho-functional features and the number of astro- and microgliocytes in the aging brain of intact rats and during the application of the succinate-containing drug, mexidol (ethylmethylhydroxypyridine succinate). Methods. Experiments were performed on outbred white rats aged 3, 6, and 18 months. Mexidol was administered intraperitoneally at 100 mg/kg/day for 3, 7, and 14 days. The content of highly specific markers of microglial activation (Iba1, ionized calcium-binding adapter molecule 1) and astroglial activation (GFAP, glial fibrillary acidic protein), and the content of synaptophysin (SYP, a marker of synaptogenesis) were determined in cerebral cortex (CC) lysate by Western blot analysis. Immunohistochemical staining of Iba1 and GFAP in paraffin-embedded sections of the prefrontal cortex (PFC) and hippocampus was used to evaluate morphological features and to count astro- and microgliocytes in young and aged rats. Results. In aged rats, the contents of GFAP and Iba1 were increased by 30% and 20%, respectively, and the content of SYP was decreased by 25%, compared with that of young animals. These findings indicate activation of inflammation and a reduction of synaptogenic potential in aged animals. Morphological features of proinflammatory polarization, i.e., short, poorly branched, few processes, were observed for micro- and astroglia in PFC of aged rats. Mexidol caused a decrease of the GFAP and Iba1 content in CC of aged rats, an increase of SYP expression to its level in young animals, an increase in the number, length, branching of processes in GFAP- and Iba1-positive cells. These findings demonstrate succinate/SUCNR1-dependent anti-inflammatory transformation of micro - and astroglia in the aging brain. Conclusion. The findings demonstrate, for the first time, new aspects of mexidol activity and succinate signaling in the brain that limit inflammatory responses and enhance synaptic plasticity. The succinate-containing drug mexidol can be used in the complex therapy of various neurological pathologies associated with neuroinflammation and cognitive deficit.

Alloxan diabetes was modeled in August rats with high activity of the NO system and in Wistar rats, and the effects of NO system blockade (by a course treatment with L-NNA) on Langerhans islet β cells were studied in 15 days. The toxic effects of diabetes on the rat β cells and islets were similar: the content of active β cells in the islets decreased to 15-20%, the number of islets to 24-29% of control. A course of L-NNA reduced the β cell and islet death, in August cells greater than in Wistar: the number of islets in August rats was restored to 81%, in Wistar rats to 60% of initial level; the activity of β cells remained at the control level in the former and 2-fold lower than in the control in the latter. It seems that a less pronounced protective effect of L-NNA in Wistar rats was explained by excessive reduction of NO level essential for β cell regeneration.

Previously we have shown that adaptation to hypoxia (AH) is cardio- and vasoprotective in myocardial ischemic and reperfusion injury and this protection is associated with restriction of nitrosative stress. The present study was focused on further elucidation of NO-dependent mechanisms of AH by identifying specific NO synthases (NOS) that could play the major role in AH protection. AH was performed in a normobaric hypoxic chamber by breathing hypoxic gas mixture (9.5-10% O2) for 5-10 min with intervening 4 min normoxia (5-8 cycles daily for 21 days). Expression of neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) protein was measured in the left ventricular myocardium using Western blot analysis with respective antibodies. AH educed iNOS protein expression by 71% (p < 0.05) whereas eNOS protein expression tended to be reduced by 41% compared to control (p < 0.05). nNOS protein expression remained unchanged after AH. Selective iNOS inhibition can mimic the AH-induced protection. Therefore protective effects of AH could be at least partially due to restriction of iNOS and, probably, eNOS expression.

UNLABELLED:The mechanisms of the protective effect of oligonucleotides (OGN) during pathological processes are poorlyunderstood. The goal of this work was to study the effect of OGN on arrhythmias induced by myocardial ischemia and reperfusion, and the HSP70 level in the heart. As a source of OGN was used the drug "Derinat" ("Technomedservis", Russia). In male Wistar rats were pre-treated the drug for 7 days (i/m, 7.5 mg/kg).The intensity of the arrhythmias was assessed by ECG during 10 min occlusion of the left coronary artery and subsequent 5 min of reperfusion. Protein HSP70 determined in the left ventricle of the heart by Western-blot analysis. During ischemia, this drug reduced duration of extrasystolia by 13 times and the incidence of ventricular tachycardia by 1.5 times. During reperfusion the drug reduced the incidence of ventricular fibrillation, a more than 2-fold, as compared with the control (respectively 23% vs 56%) and by 5 times its duration (8,4 ± 2,3 48,1 ± sec vs 18 7 sec). "Derinat" increased the HSP70 level in the heart by 65% compared with control.CONCLUSION:These data support the fact that the activation of HSP70 synthesis, induced by OGN is one of the mechanisms that increases the heart resistance to the ischemic and reperfusion damages.

We have previously shown that the innate increased activity of the NO- system, typical for the August rats, increases vulnerability to alloxane diabetes (ALD). The purpose of this study was to investigate the effect of ALD on the cardiovascular system and lipid peroxidation in rats with different activity of NO-system. The August rats and Wistar rats treated with alloxan (125 mg/kg, s/c, once) were studied 3.5 months after. In August-ALD the double production significantly decreased to a greater extent (by 35%) than in Wistar-ALD (by 17%) compared with the control. As in August-ALD and in Wistar-ALD was observed the similar fall of the relaxation (-dp/dt) of the left ventricle (by 45-49%), but not the contraction rate (+dp/dt). LPO activation in the heart and liver, as well as NO-system (level of nitrates and nitrites in the blood plasma) in August rats were more pronounced than in Wistar rats. The hsp32 level in August rats fell significantly more (by 93% ) than in Wistar rats (by 61%). Pathological changes in the microvasculature of the mesostenium were identical in compared rats. Thus, more pronounced cardiac dysfunction in August-ALD, compared with Wistar-ALD, associated with greater activation of lipid peroxidation and NO-system.

Recently we demonstrated that intermittent, normobaric hypoxia conditioning (IHC) prevented injuries of myocardium and coronary blood vessels induced by myocardial ischemia and reperfusion (IR). This cardio‐ and vasoprotection of was associated with alleviation of nitric oxide overproduction. The aim of this study was to identify specific NO synthase(s) responsible for the IR‐induced NO overproduction and to determine the effect of IHC on these NO synthases. IHC of rats was performed in a normobaric chamber (5‐8 cycles/d for 20 d, FIO2 9.5 ‐ 10% for 5 ‐ 10 min/cycle, with intervening 4‐min normoxia). IR was produced by ligation of the left anterior descending coronary artery for 30 min followed by 60‐min reperfusion. The protein nitration marker, nitrotyrosine (3‐NT) and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthases were measured by immunoblot. IR induced appreciable 3‐NT accumulation in the left ventriclular free wall, increasing the 3‐NT content by 42% (p<0.01), but not in septum. In IHC rats, 3‐NT after IR was similar to that of control rats without IR. IHC decreased iNOS by 71% (p<0.05) and eNOS by 41% (p<0.05) in the left ventricular myocardium; the myocardial content of nNOS remained unchanged. Therefore, IHC prevents IR‐induced NO overproduction in myocardium by restricting myocardial expression of iNOS and eNOS.Grant Funding Source: Supported by the Russian Foundation for Basic Research grant 10‐04‐00980

Adaptation to hypoxia is known to be cardioprotective in ischemic and reperfusion (IR) injury of the myocardium. This study was focused on investigating a possibility for prevention of endothelial dysfunction in IR injury of the rat heart using adaptation to intermittent hypoxia, which was performed in a cyclic mode (5-10 min of hypoxia interspersed with 4 min of normoxia, 5-8 cycles daily) for 21 days. Endothelial function of coronary blood vessels was evaluated after the in vitro IR of isolated heart (15 min of ischemia and 10 min of reperfusion) by the increment of coronary flow rate in response to acetylcholine. Endothelium-dependent relaxation of isolated rat aorta was evaluated after the IR myocardial injury in situ (30 min of ischemia and 60 min of reperfusion) by a relaxation response of noradrenaline-precontracted vessel rings to acetylcholine. The following major results were obtained in this study: 1) IR myocardial injury induced endothelial dysfunction of coronary blood vessels and the aorta, a non-coronary blood vessel, remote from the IR injury area; and 2) adaptation to hypoxia prevented the endothelial dysfunction of both coronary and non-coronary blood vessels associated with the IR injury. Therefore, adaptation to hypoxia is not only cardioprotective but also vasoprotective in myocardial IR injury.

We studied the effects of Nω-nitro-L-arginine (L-NNA), a nonselective inhibitor of NO synthases, on the severity of type 1 diabetes mellitus induced by subcutaneous injection of 130 mg/kg alloxan in August rats with high activity of NO system and in Wistar rats. Five days after alloxan injection, hyperglycemia levels after overnight fasting in August and Wistar rats were 27.1±3.7 and 22.0±1.1 mmol/liter, respectively (p<0.03). The mortality over 15 days after alloxan injection in August rats was higher than in Wistar rats (36 and 26%, respectively). L-NNA normalized glucose levels in diabetics of both groups. It completely prevented mortality in August and reduced it to 13% in Wistar rats. Body weight loss and polydipsia after LNNA injection were also less pronounced in August rats. Plasma nitrite/nitrate concentrations in August rats were 32% higher than in Wistar rats, both in intact and diabetic rats. These data attest to an important role of NO in the pathogenesis of alloxan diabetes.

Prior adaptation to intermittent hypoxia (AIH) is cardioprotective during myocardial ischemia‐reperfusion injury (IR). This study investigated the possibility that AIH would lessen endothelial dysfunction (EDF) in rat hearts during IR. AIH was performed in a normobaric chamber (5–8 cycles/d for 20 d, FIO2 9.5–10% for 5–10 min/cycle, with intervening 4‐min normoxia). Endothelium‐dependent (ED) function of coronary blood vessels was evaluated by the flow response to acetylcholine (ACH) in Langendorf isolated hearts after in vitro IR. Also, ED relaxation of noradrenaline‐precontracted isolated aortic rings was evaluated by response to ACH after in situ myocardial IR. With no IR, coronary flow and response to ACH were similar in hearts of non‐AIH (n=9) and AIH (n=10) rats. IR injury decreased ED dilation of coronary arteries to 10.4±1.7% of the pre‐IR response to ACH in non‐AIH (n=10) rats and to 76±20% in AIH (n=9) rats (p<0.05). In the aorta, IR reduced ED relaxation from 45±4% (n=8) of the precontraction magnitude to 9.7±1.2% (n=10) in non‐AIH rats and to 30±3% (n=10) in AIH (p<0.05). Therefore, 1) IR heart injury induced EDF of both coronary and remote, non‐coronary blood vessels; and 2) AIH blunted EDF of both coronary and noncoronary vessels. These data suggest that adaptation to hypoxia is not only cardioprotective but also vasoprotective during myocardial IR injury. Supported by RFBR grant 10–04‐00980.

Favorable versus detrimental cardiovascular responses to intermittent hypoxia conditioning (IHC) are heavily dependent on experimental or pathological conditions, including the duration, frequency and intensity of the hypoxia exposures. Recently, we demonstrated that a program of moderate, normobaric IHC (FIO2 9.5-10% for 5-10 min/cycle, with intervening 4 min normoxia, 5-8 cycles/day for 20 days) in dogs afforded robust cardioprotection against infarction and arrhythmias induced by coronary artery occlusion-reperfusion, but this protection has not been verified in other species. Accordingly, in this investigation cardio-as well as vasoprotection were examined in male Wistar rats completing the normobaric IHC program or a sham program in which the rats continuously breathed atmospheric air. Myocardial ischemia and reperfusion (IR) was imposed by occlusion and reperfusion of the left anterior descending coronary artery in in situ experiments and by subjecting isolated, perfused hearts to global ischemia-reperfusion. Cardiac arrhythmias and myocardial infarct size were quantified in in situ experiments. Endothelial function was evaluated from the relaxation to acetylcholine of norepinephrine-precontracted aortic rings taken from in situ IR experiments, and from the increase in coronary flow produced by acetylcholine in isolated hearts. IHC sharply reduced cardiac arrhythmias during ischemia and decreased infarct size by 43% following IR. Endothelial dysfunction in aorta was marked after IR in sham rats, but not significant in IHC rats. Similar findings were found for the coronary circulations of isolated hearts. These findings support the hypothesis that moderate, normobaric IHC is cardio-and vasoprotective in a rat model of IR.

Previously, we have shown that nitric oxide (NO) plays an important role in the pathogenesis of alloxan diabetes (ALD). In this study in August rats, with the congenital increased activity of NO, and in Wistar rats was induced ALD (130 mg/kg, p/c) and 15 days after were examined the effects of the NO-blockade synthesis, induced by administration of Nω-nitro-L-arginine (L-NNA) cour- se on the activity of lipid peroxidation (LP), HIF-1α level, the degree of NO-system activation. The activation of iNOS, HIF-1a expression and 3-nitrotyrosine accumulation in liver were more pronounced in August-ALD rats than in Wistar-ALD rats. The level of TBA-active products in the heart and liver was increased in both diabetic groups only in the first 3 days ofALD and then this indicator of LP sharply was decreased as compared with the control. This effect was pronounced more in August rats. The inhibition of NO overproduction reduced significantly the severity of ALD and prevented the activation of LP, iNOS and HIF-1a. Thus, these data suggest, that NO plays an important role in the pathogenesis of ALD and in the regulation of oxygen homeostasis.

Adaptation to intermittent normobaric hypoxia is cardioprotective and can stimulate nitric oxide (NO) synthesis. However the role of nitric oxide (NO) in prevention of ischemia-reperfusion (IR) injury of myocardium is controversial. This study was focused on evaluating the effect of adaptation to hypoxia and IR on NO production and development of nitrative stress in the myocardium. Adaptation to hypoxia tended to increase NO production, which was determined by the total level of plasma nitrite and nitrate, and prevented IR-induced NO overproduction. The IR-induced NO overproduction was associated with significant 3-nitrotyrosine (3-NT) accumulation in the left ventricle but not in septum or aorta. In hypoxia-adapted rats, 3-NT after IR was similar to that of control rats without IR. IHC induced marked accumulation of HIF-1alpha in the left ventricle. We suggest that HIF-1alpha contributes to NO-synthase expression during adaptation to hypoxia and thereby facilitates the increase in NO production. NO, in turn, may subsequently prevent NO overproduction during IR by a negative feedback mechanism.

Similar degree of glycemia (28-31 mmol/liter) and similar mortality (37-42%) were revealed in August rats exhibiting enhanced activity of NO system and in Wistar rats 3 weeks after alloxan treatment. Under conditions of myocardial ischemia caused by 10-min coronary artery ligation, the intensity of arrhythmias did not differ from the control in Wistar rats with diabetes mellitus and increased in August rats. Under conditions of reperfusion, diabetes produced an antiarrhythmic effect in Wistar rats and did not affect arrhythmia in August rats. Plasma concentrations of nitrates and nitrites in Wistar and August rats increased by 82 and 143%, respectively, compared to the control. The level of hemoxygenase-1 (hsp32) in the myocardium remained unchanged in Wistar rats and decreased by 26% in August rats. Thus, the absence of antiarrhythmic effect of acute diabetes in August rats is probably related to elevated NO content and reduced antioxidant activity.

The role of nitric oxide (NO) in prevention of ischemia and reperfusion (IR) injury is controversial. Earlier we showed that IHC protects the heart and endothelium from IR injury. Now, the role of NO‐dependent mechanisms in this protection was studied. Rats were subjected to normobaric IHC (5–8 cycles/d for 20 d, FIO2 9.5–10% for 5–10 min/cycle, with intervening 4 min normoxia), and control rats were sham‐conditioned. IR was produced by ligation of the left coronary artery for 30 min with 60‐min reperfusion. NO production was evaluated from plasma nitrite+nitrate (NOx). 3‐Nitrotyrosine (3‐NT) and HIF‐1α were measured by immunoblot. IHC tended to increase basal NOx (6.8±0.5 vs 8.1±0.4 μmol/L NOx, p=0.23) and prevented IR‐induced NO overproduction (14±1 vs 9.1±1.1 μmol/L NOx, p<0.005). Without IHC, IR‐induced NO overproduction was associated with significant 3‐NT accumulation in the left ventricle (142±8% vs 100±12% in control, p<0.01) but not in septum or aorta. In IHC rats, 3‐NT after IR was similar to that of control rats without IR. IHC induced marked accumulation of HIF‐1α in the left ventricle (220±38% vs 100±7% in control, p<0.005). HIF‐1α‐induced increase in NO production may prevent subsequent NO overproduction by negative feedback. Thus, IHC cardioprotection is associated with prevention of toxic effects of NO overproduction in the myocardium during IR. Support: RFBR grant 10‐04‐00980.

UNLABELLEDHeart function was studied in the August rats with innate raised sympathetic-adrenal system and in the Wistar rats through the period of 3 month after myocardial infarction. The sizes of the postinfarction scars were similar in the rats under comparison (56-62%) but end-diastolic pressure in Wistar rats and in August rats was 18.7 +/- 2.2 mm Hg and 11.8 +/- 0.7 mm Hg. Under the maximum isometric load induced by the aorta coarctation, the work efficiency of the heart in the August rats was greater than in the Wistar rats. During the postinfarction period, plasma catecholamine (CA) in August rats was higher than in Wistar rats. In the adrenal glands, the CA contents in August rats increased and in Wistar rats decreased. The activity of CA resynthes in the adrenal glands and in the hypothalamus in August rats did not change and in Wistar rats increased. The blood contents of nitrate and nitrite and hemine oxygenase-1 level in the myocardium of August rats were increased in contrast to Wistar rats.THE CONCLUSIONthe higher viability of the myocardium in August rats with long existing postinfarction cardiasclerosis is to a considerable extent associated with lowered activation of the sympathetic-adrenal system under more expressing activation of NO-system and antioxidant protection.