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.

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.

The aim of the study was to identify the potential succinate/SUCNR1-mediated mechanism of induction and activation of transcription coactivator PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1 alpha) – key activator of the mitochondriogenesis, angiogenesis, fusion of mitochondria, antioxidant defense system that are basis of neuroprotective mechanisms – in the peri-infarction zone of the rat prefrontal cortex (PFC). Focal bilateral damage of the PFC was modeled by photo-induced thrombosis. Ethylmethylhydroxypyridine succinate (a form of succinate passing through the blood-brain barrier; trade name of the drug Mexidol) was injected intraperitoneally 2 hours after infarction and then every day at a dose of 100 mg/kg (7 day course). The tissue of the peri-infarction zone was taken 1, 3, 7 days after the start of therapy, as well as 14 days after discontinuation of the drug. Brain samples were stored in liquid nitrogen, a cytosolic extract was obtained, expression levels of SUCNR1, PGC-1α, transcription factors (NRF1, TFAM), VEGF, catalytic subunits of mitochondrial respiratory enzymes (NDUFV2, SDHA, cyt c1, COX2) and ATP synthases (ATP5A) were determined by Western blot analysis. The study showed that the content of PGC-1α in the peri-infarct zone decreased one day after the induction of ischemic damage by 40% and remained reduced for 21 days after a ischemia. The course of injection of mexidol stabilized PGC-1α in the peri-ischemic zone at a level comparable to the control, both at the stage of treatment and drug withdrawal, and in intact rats induced PGC-1α by 30%. Induction of NRF1, TFAM, NDUFV2, SDHA, cyt c1, ATP5A, indicating the activation of PGC-1α, was observed in rats of the three compared groups, however, the highest expression levels were found in animals subjected to the course of mexidol. Also it was revealed overexpression of SUCNR1 and VEGF in ischemic and intact rats, were injected with mexidol. The study demonstrates for the first time that succinate/SUCNR1 signaling is involved not only in the mechanisms of cerebral angiogenesis, but also in the mechanisms of PGC-1α-dependent neuroprotection. The study opens up new perspectives for pharmacological modulation of PGC-1α levels 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.

Event Abstract Back to Event MORPHOFUNCTIONAL PECULIARITIES OF ISCHEMIC AND HEMORRHAGIC INJURIES OF THE BRAIN IN RATS AT THE MODELING OF THE EFFECTS OF MICROGRAVITATION Mikhail Baranov1*, Alexander Paltsyn2, Galina Romanova2 and Fatima Shakova2 1 Federal Research and Clinical Center, Federal Medical-Biological Agency, Russia 2 Research Institute of General Pathology and Pathophysiology, the Russian Academy of Medical Sciences, Russia Information on the effects of microgravity at the central nervous system (CNS) is still fragmentary. At the same time, cognitive functions: memory, ability to learn and analyze situations, make decisions - can also change under microgravity. Besides, cerebro-vascular changes can impact the onset and course of both cerebral ischemic and hemorrhagic stroke if it occurs during the space flight. Purpose: To assess microgravity effects at the formation of specific features in the development of brain injuries of various etiology. The rat suspension model was used for modeling microgravity. Several experiment series were made in which suit suspension (SS) of various duration preceded to or was made after experimental brain injury. For the experimental model of ischemic stroke, photochemical thrombosis of blood vessels in the prefrontal cortex was used. Cognitive functions of rats' brain were studied by developing a conditioned passive avoidance reflex (CPAR) [1]. CPAR stability was defined with latent period (LP). Model of hemorrhagic stroke. A transplant consisting of crushed hemostatic collagen sponge and blood plasma of rabbit was injected into the cortex motor zone in the left hemisphere into the area which is responsible for movements of animal's right front paw. There were several groups of rats in this experiment: Group1. Typical hemorrhagic stroke. Transplant contains acellular blood plasma. Group 2. Typical hemorrhagic stroke. Transplant contains blood plasma rich with platelets containing angiogenesis stimulation factors (PRP). Group 3. Typical stroke. Transplant (PRP). 7 days of SS after stroke. Group 4. 7 days of SS. Typical stroke. Transplant (PRP). Group 5. 2 days of SS. Typical stroke. Transplant (PRP). Group 6. Vivarium control. Animals' functional state was assessed with their motor activity. Motor activity was examined before surgery and on day 7 after it using test "walking on bar". Morphological findings were used for defining: vasculature development, blood vessels caliber, neurons content, gliocytes, infiltrate cells; location density of binuclear neurons. Results: Microgravity negative impact at rats' cognitive functions was revealed even without additional pathological damage. After exposure to microgravity in EG, average values of motor activity and CPAR indices were not significantly different from the baseline values. It was noted decrease in LP CPAR. In CG, motor activity was reliably reduced in comparison to initial levels after 14 days of staying in normal vivarium conditions, while CPAR latent period remained unchanged. Decreased motor activity in the control group and a conditioned passive avoidance reflex demonstrate that cognitive functions were preserved, since animals show less "interest" to repeated presentations to familiar "open field" device environment and keep in their memory unpleasant sensations of electric shock (CPAR). On the other hand, findings in experimental animals can be regarded as an adverse SS effect at cognitive functions. Less transition latent period indicates weakened conditioned passive avoidance reflex; animals have motor activity on the "open field" similar to the pre-experimental level what shows weak fixation of the information obtained during the first testing. Than we studied an impact of ischemic stroke at cognitive functions and at the structure of brain prefrontal cortex. Animals were divided into control and experimental groups. For 14 days, controls were in vivarium, while experimental animals were in SS. On day 7, ischemic stroke was induced with the described procedure in both groups. We can state that stroke in the prefrontal cortex causes significant memory impairments in rats of both groups, but there are no differences between the groups. At the next stage, hemorrhagic stroke was chosen as a model of pathological process in brain. Number of binuclear neurons per unit area in rat's cerebral cortex was taken as an indicator of regenerative processes. The highest mortality rate was in the group in which SS started after stroke modeling; and the lowest mortality rate was in Group 5 in which stroke was preceded by a short-term simulated microgravity. The best restoration of motor function was in animals from Groups 4 and 5 in which hemorrhagic stroke was preceded by exposure to SS. The worst results were in Group 3. Morphological examination of brain preparations from animals of Group 1 showed extravasates in the infiltrate, neoangiogenesis. However, vessels grow slowly; they are mostly of small diameter. Group 2 - much more blood vessels. Vessels were of larger caliber and they were arterioles and venules. Significantly more macrophages; they were most often located in the vessel wall. Morphological changes in Group 3 demonstrated signs of acute circulatory disorders. Serious circulation impairments - extravasates- which were not seen in Group 2, are often met in Group 3. Many longitudinally cut capillaries which are invisible under normal circulation, now become clearly seen and are indicative of disorders in blood outflow and blood stasis. Group 4 has a larger number of newly formed vessels than Groups 1 and 3. These vessels are capillaries and more matured vessels - venules and arterioles. All animals of this group had extravasates at the border of infiltrate and penumbra which mainly consisted of loosely lying erythrocytes and rarely met dense clusters. There were no other circulatory disorders, such as blood stasis. Group 5 has the largest number of newly formed vessels, both in the infiltrate and penumbra. There are no circulatory disorders, such as blood stasis or extravasates. Groups 3 and 5 significantly differ from the controls with density of binuclear neurons locations. In Group 5, density of fusion locations in the stroke area significantly exceeds similar density at the same part of intact hemisphere. Injury triggers regeneration, but simultaneously it inhibits this regeneration with pathogenic changes in the stroke focus. That is why, regeneration intensity is always a ratio of stimulating and inhibiting factors. In Group 5, this ratio promoted regeneration what was manifested both morphologically and functionally. The overall good result in Group 5 can be probably explained with effects of short-term microgravity which preceded damage; it increased venous pressure and thereby, contributed to collapse or dilatation of collaterals and anastomoses. References 1. Bures J., Burešová O., Huston J.P. Techniques and Basic Experiments for the Study of Brain and Behavior., Elsevier, 1976. Keywords: ischemic stroke, Hemorragic stroke, microgravity, Rats, morphology Conference: 39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018. Presentation Type: Extended abstract Topic: Animal Models Citation: Baranov M, Paltsyn A, Romanova G and Shakova F (2019). MORPHOFUNCTIONAL PECULIARITIES OF ISCHEMIC AND HEMORRHAGIC INJURIES OF THE BRAIN IN RATS AT THE MODELING OF THE EFFECTS OF MICROGRAVITATION. Front. Physiol. Conference Abstract: 39th ISGP Meeting & ESA Life Sciences Meeting. doi: 10.3389/conf.fphys.2018.26.00035 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Dec 2018; Published Online: 16 Jan 2019. * Correspondence: Dr. Mikhail Baranov, Federal Research and Clinical Center, Federal Medical-Biological Agency, Moscow, Russia, baranovm2000@mail.ru Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mikhail Baranov Alexander Paltsyn Galina Romanova Fatima Shakova Google Mikhail Baranov Alexander Paltsyn Galina Romanova Fatima Shakova Google Scholar Mikhail Baranov Alexander Paltsyn Galina Romanova Fatima Shakova PubMed Mikhail Baranov Alexander Paltsyn Galina Romanova Fatima Shakova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

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.

The neuroprotective effects of dipeptide GK-2h, a mimetic of nerve growth factor, in bifocal photoinduced ischemia in rat brain prefrontal cortex was studied. It was shown that GK-2h, injected intraperitonealy in dose 0.1 mg/kg in 1 h or 4 h after operation and then on 2nd, 4th and 8th days, prevented significantly on 9th day from increasing volume of cortical infarction.

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.