Experimental model of resection craniotomy with subsequent reconstruction of the defect with a polymer implant enables comprehensive assessment of functional and ultrastructural changes during replacement of the damaged tissue. Reconstruction of a skull defect was accompanied by transient motor disturbance in the acute period and did not cause functional disorders and neurological deficits in a delayed period. Histological examination of osteal and brain tissue revealed no pathological reactions that could be associated with the response to the chemical components of the implant.

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.

The SUCNR1 succinate receptor (GPR91) is constitutively expressed by the main populations of immunocytes. Succinate, as an immunometabolite (metabokine), performs SUCNR1-dependent modulation of the morphofunctional state of immune cells and is an important regulator of innate and adaptive immunity, as well as tissue homeostasis. Contradictory interpretations of the role of succinate signaling in the functioning of macrophages, the lack of studies of the effect of SUCNR1 on the morphofunctional transformation of microglia, the problem of low permeability of the blood-brain barrier (BBB) for any promising analogues of succinate have predetermined the conduct of this study. The aim of this study was to evaluate the effect of ethylmethylhydroxypyridine succinate on the polarization of microglia in chronic inflammation in the aging brain. The work was performed on white mongrel male rats aged 3 (young), 6 (middle-aged), and 18 months (old). The drug Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate) was used as a succinate derivative that overcomes BBB. Mexidol was administered intraperitoneally at a dose of 100 mg/kg daily for 3, 7, and 14 days. Surface markers of the pro- (M1) and anti-inflammatory (M2) phenotype of microglia (CD86 and CD206, respectively), Iba1 cytoplasmic marker of microglia, as well as IL-1β and TNF-α pro-inflammatory cytokines (expressed by M1 microglia), TGF-β1 immunosuppressive cytokine and BDNF neurotrophin (expressed by M2 microglia) were detected in the samples of the cerebral cortex by immunoblotting. It was shown that the expression of cellular markers of pro- (CD86) and especially anti-inflammatory (CD206) microglia decreased in old rats; however, the level of the marker Iba1 that was expressed independently of the phenotype remained unchanged in all the studied groups, which indicated the maintenance of quantitatively equivalent populations of microglia in different age groups. At the same time, the levels of TNF-α and IL-1β increased, and the content of TGF-β1 and BDNF was significantly lower in old rats compared with young and middle-aged ones. In general, the data obtained indicated the dominance of the pro-inflammatory status of microglia in the aging brain. A 14-day course of Mexidol caused a slight decrease in the level of CD86 in old rats and a significant increase of 45% in the content of CD206 up to the level of CD206 in 6-month-old rats. The increase in the CD206 expression was associated with an increase in the levels of TGF-β1 and BDNF by 60% and 35%, respectively, which indicates the involvement of succinate/SUCNR1 signaling in the anti-inflammatory polarization of microglia in the aging brain. The obtained data develop ideas about the cerebral effects of succinate/SUCNR1 signaling and reveal a new component of the mechanism of the neuroprotective action of Mexidol.

The pharmacological induction and activation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), a key regulator of ischemic brain tolerance, is a promising direction in neuroprotective therapy. Pharmacological agents with known abilities to modulate cerebral PGC-1α are scarce. This study focused on the potential PGC-1α-modulating activity of Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate) and Semax (ACTH(4–7) analog) in a rat model of photochemical-induced thrombosis (PT) in the prefrontal cortex. Mexidol (100 mg/kg) was administered intraperitoneally, and Semax (25 μg/kg) was administered intranasally, for 7 days each. The expression of PGC-1α and PGC-1α-dependent protein markers of mitochondriogenesis, angiogenesis, and synaptogenesis was measured in the penumbra via immunoblotting at Days 1, 3, 7, and 21 after PT. The nuclear content of PGC-1α was measured immunohistochemically. The suppression of PGC-1α expression was observed in the penumbra from 24 h to 21 days following PT and reflected decreases in both the number of neurons and PGC-1α expression in individual neurons. Administration of Mexidol or Semax was associated with preservation of the neuron number and neuronal expression of PGC-1α, stimulation of the nuclear translocation of PGC-1α, and increased contents of protein markers for PGC-1α activation. This study opens new prospects for the pharmacological modulation of PGC-1α in the ischemic brain.

Обзор посвящен рассмотрению современных подходов к фармакологической модуляции структурно-функционального состояния митохондриального аппарата нейронов как перспективной стратегии терапии ишемического инсульта головного мозга, составляющего по современным оценкам до 85% всех случаев острого нарушения мозгового кровообращения. В обзоре проведен анализ классических представлений о нейропротекции как терапии, направленной на блокирование ключевых патогенетических звеньев «ишемического каскада», в контексте современного понимания приоритетной роли коррекции митохондриальной дисфункции в ограничении механизмов повреждения при церебральной ишемии. Обсуждаются возможности применения препаратов плейотропного нейропротекторного действия, реализующих митохондриально-направленные защитные эффекты через модуляцию активности транскрипционного коактиватора PGC-1α (peroxisome proliferator-activated receptor-1γ coactivator-1α), контролирующего процессы биогенеза митохондрий, ангиогенеза, ферментативное звено антиоксидантной системы. The review addresses modern approaches to pharmacological modulation of structure and function of the neuronal mitochondrial apparatus. This is a promising strategy for therapy of ischemic stroke, which accounts for up to 85% of all cases of acute cerebrovascular disease. The review analyzes classical concepts of neuroprotection as a therapy aimed at blocking key pathogenetic components of the “ischemic cascade”. These concepts are based on current ideas about the importance of correcting mitochondrial dysfunction for alleviation of damage in cerebral ischemia. The authors discussed possibilities of using pleiotropic neuroprotectors that implement mitochondrial-targeted protective effects by modulating the activity of transcriptional coactivator, peroxisome proliferator-activated receptor-1γ coactivator-1α (PGC-1α), which controls mitochondrial biogenesis, angiogenesis, and the enzymatic antioxidant system.

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.

Цель исследования - изучение влияния комбинированной терапии (мутантные молекулы эритропоэтина (EPO) и дипептидный миметик фактора роста нервов ГК-2H) на воспроизведение условного рефлекса пассивного избегания (УРПИ) и объем поражения коры мозга у крыс с двусторонним ишемическим повреждением префронтальной коры. Методика. Мутантные молекулы EPO (MЕРО-TR и MЕPО-Fc) с значительно редуцированной эритропоэтической и выраженной цитопротекторной активностью созданы методом генной инженерии. Используемый миметик фактора роста нервов человека, эндогенного регуляторного белка, в экспериментах in vitro проявлял отчетливые нейропротективные свойства. Двустороннюю фокальную ишемию префронтальной коры головного мозга крыс создавали методом фотохимического тромбоза. Выработку и оценку УРПИ проводили по стандартной методике. Объем повреждения мозга оценивался при помощи МРТ. MEPO-TR и MEPO-Fc (50 мкг/кг) вводили интраназально однократно через 1 ч после фототромбоза, ГК-2Н (1 мг/кг) - внутрибрюшинно через 4 ч после фототромбоза и далее в течение 4 послеоперационных суток. Результаты. Выявлено статистически значимое сохранение выработанного до ишемии УРПИ, а также значимое снижение объема повреждения коры при комплексной терапии. Полученные данные свидетельствуют об антиамнестическом и нейропротекторном эффектах примененной комбинированной терапии, которые наиболее отчетливо выражены в дозах: МEPO-Fc (50 мкг/кг) и ГК-2Н (1 мг/кг). Заключение. Подтвержден нейропротекторный эффект и усиление антиамнестического эффекта при сочетанном применении мутантных производных эритропоэтина - MEPO-TR и MEPO-Fc и дипептидного миметика фактора роста нервов человека ГК-2H. The aim of this study was to investigate the effect of combination therapy, including mutant erythropoietin molecules (EPO) and a dipeptide mimetic of the nerve growth factor, GK-2H, on the conditioned passive avoidance (PA) reflex and the volume of injury induced by bilateral ischemia of the prefrontal cortex in rats. Using the method of genetic engineering the mutant molecules of EPO, MERO-TR and MEPO-Fc, with strongly reduced erythropoietic and pronounced cytoprotective activity were created. The used human nerve growth factor mimetic, an endogenous regulatory protein based on the b-bend of loop 4, which is a dimeric substituted dipeptide of bis- (N-monosuccinyl-glycyl-lysine) hexamethylenediamine, GK-2 human (GK-2H), has proven neuroprotective in in vitro experiments. Methods. Bilateral focal ischemic infarction was modeled in the rat prefrontal cortex by photochemically induced thrombosis. The PA test was performed according to a standard method. Volume of brain injury was estimated using MRI. MEPO-TR, and MEPO-Fc (50 mg/kg, intranasally) were administered once, one hour after the injury. GK-2Н (1 mg/kg, i.p.) was injected four hours after the injury and then for next four days. Results. The study showed that the complex therapy provided statistically significant retention of the PA reflex developed prior to ischemia and a significant decrease in the volume of injury. The anti-amnestic and neuroprotective effects of combination therapy were most pronounced at doses of MEPO-Fc 50 mg/kg and GK-2H 1 mg/kg. Conclusion. This study has confirmed the neuroprotective effect and enhancement of the anti-amnestic effect exerted by the combination of mutant erythropoietin derivatives, MEPO-TR and MEPO-Fc, and the dipeptide mimetic of human growth factor GK-2H.

Ишемическое повреждение префронтальной коры головного мозга сопровождается ранним и прогрессирующим во времени снижением активности сукцинатдегидрогеназы (СДГ) митохондрий перифокальной зоны. Суточное дробное введение синтетического аналога фрагмента АКТГ - Семакса в дозе 25 мкг/кг сопровождается нормализацией активности СДГ через 24 ч после фототромбоза коры. Исследование показало, что определение активности СДГ является информативным методом, позволяющим оценить тяжесть ишемического повреждения головного мозга и эффективность нейропротекторной терапии.Ischemic damage to the prefrontal cortex is accompanied by early and progressive decrease in the succinate dehydrogenase (SDH) activity of the mitochondria of the perifocal area. The daily fractional administration of a synthetic analogue of the ACTH fragment - Semax at a dose of 25 µg / kg is accompanied by normalization of the activity of SDH 24 hours after photothrombosis of the cortex. The study showed that the determination of the activity of SDH is an informative method to assess the severity of ischemic brain damage and the effectiveness of neuroprotective therapy.

New hybrid proteins based on erythropoietin (EPO), EPO-TR and EPO-Fc, and their mutant forms, MEPO-Fc and MEPOTR, which lack hematopoietic activity but retain the cytoprotective properties of erythropoietin, were created via genetic engineering. The assessment of the antiamnesic efficiency of the obtained proteins was conducted on the 4th day after bilateral photothrombosis of the medial prefrontal cortex of rats and it was dependent on the strength of the conditioned passive avoidance reflex before ischemia. The concentration of S100b protein, a glial marker of brain tissue damage in the serum of rats, was assessed using enzyme-linked immunosorbent assay (ELISA) within the same period. A one-time intranasal administration of erythropoietin derivatives EPO-Fc and EPO-TR, as well as their mutated forms, MEPO-Fc and MEPO-TR, at a dose of 50 mg/kg, one hour after ischemic brain cortex injury, was associated with the preservation of the skill that was developed before ischemia. A significant decrease in the level of S100b protein in serum was found when EPO-TR was administered. Administration of the other tested derivatives showed a tendency to decrease the S100b level, which was most pronounced in animals treated with MEPO-TR. Our results confirm the neuroprotective efficacy of these novel proteins as potential drugs for the treatment of experimental focal ischemic brain damage.

A correlation between the severity of morphofunctional disturbances and the volume of brain tissue injury determined by MRT was demonstrated on the model of open traumatic brain injury in rats. A relationship between the studied parameters (limb placing and beam walking tests and histological changes) and impact force (the height of load fell onto exposed brain surface) was revealed.

It was stated with model of bilateral photochemically induced thrombosis of the prefrontal cortex by injected intranasally or intraperitoneally in 1h after operation new derivatives of eritropoetine: Epo, Epo-Fc, Epo-Tr provoked neuroprotective and antiamnestic action. Epo-Fc demonstrated more effective action by intranasal injection.

Mutant EPO molecules, deprived of erythropoietic activity, but possessing cytoprotective action, were created by the method of genetic engineering. The assessment of the therapeutic effectiveness of the received mutant proteins was carried out by the retention of the conditioned reflex of passive avoidance (PA), developed before the ischemic injury of rat brain prefrontal cortex, and by the MRI-analysis of ischemic damage volume. Antiamnestic and neuroprotective action of mutant molecules - MERO-Fc and MEPO-TR is investigated on model of photothrombosis of rat brain prefrontal cortex at single intranasal introduction in 1 h after cortex ischemic damage. The neuroprotective (MRI) and antiamnestic (PA) effects of mutant molecules of erythropoietin derivatives are shown.

Contemporary approaches to experimental traumatic brain injury modeling, the principles of functioning and techinical characteristics of appropriate equipment are reviewed. The methods describing traumatic brain injury modeling and assessment of brain structural and functional changes caused by the weight drop method are given.

Using the model of bilateral photothrombosis of the blood vessels in the prefrontal cortex we have shown that new hybrid proteins derived from recombinant human erythropoietin, carbamylated EPO-Fc and EPO-TR fusion proteins, injected intraperitoneally 1 h after ischemic injury contribute to restoration of passive avoidance response formed before photothrombotic injury and reduction in the volume of the ischemic focus. These data attest to nootropic and neuroprotective activities of these hybrid proteins. Carbamylated glycopeptide derivative ЕPO-TR exhibited prolonged neuroprotective properties.

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.

Experiments on the model of bilateral photothrombosis in prefrontal cortex showed that antibodies to glutamate one-time administered intranasally 1h after ischemic damage to the brain cortex lead to decrease of neurodegenerative influence of excitatory neurotransmitter after photothrombosis. It was showed the change of the level of dopamine, serotonin and their metabolites in hippocampus and prefrontal cortex.

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.

On the model of acute ischemic damage of prefrontal areas of the rats brain cortex was shown, that intranasal injection of glutamate antibodies over one hour after ischemic damage of brain prefrontal areas leads to diminishing of glutamate content in hippocampus and prefrontal cortex.