O. G. Deryabina

Aim. To evaluate the effect of mesenchymal stem cells of various origins, rat embryonic fibroblasts and Wharton's jelly MSCs lysate on the dynamics of destructive changes in the somatosensory cortex of rats with artificially induced acute cerebrovascular injury. Materials and methods. Modelling of transient bilateral ischemia-reperfusion (IR) in rats by ligation of the internal carotid arteries. Transplantation of mesenchymal stem cells (MSCs) of different origins into the femoral vein of experimental animals. Cytological study on 7th and 14th days after IR with counting the total number of neuronal nuclei in 1 mm 2 in the somatosensory cortex of animals and the ratio of the intact neuronal nuclei to the pathologically altered nuclei. Results. Transplantation of MSCs, rat embryonic fibroblasts, MSC lysate from human umbilical cord Wharton's jelly (hUC-WJ) and administration of citicoline led to an increase in the number of intact neurons and nuclei without pathological changes in the somatosensory cortex of rats. The most effective in terms of Cit at ion:

Background. The treatment of cerebral blood circulation disorders remains a pressing issue due to their pre­valence in the elderly. Brain tissue ischemia caused by such disorders leads to necrotic and neuroapoptotic changes. To mitigate neuroapoptosis in the ischemic zone during the subacute period of the process, neuroprotectors are used. In recent years, the neuroprotective properties of mesenchymal stromal cells (MSCs) have been actively studied. Objective. To compare effect of MSCs of different origin and the cell lysate of human MSCs from Wharton's jelly (hWJ-MSC) on neuroapoptotic changes in the hippocampus of the rat brain after ischemia-reperfusion (IR). Methods. A 20-minute bilateral transient IR of the internal carotid arteries was performed on 165 four-month-old male Wistar rats. Following IR modeling, MSCs derived from hWJ-MSCs, as well as human and rat adipose tissue, were injected intravenously into the femoral vein of the rats. Other groups of rats received intravenous injections of fetal rat fibroblasts and cell lysate from hWJ-MSCs. Only an intravenous injection of physiological solution was administered to the control group of rats. The level of DNA fragmentation in the nuclei of hippocampal neurons on the 7th day after IR was assessed via flow cytometry. Results. Experimental IR caused a 4.9-fold increase in the level of fragmented DNA in the operated rats compared to the sham-operated animals. The use of MSCs of various origins and hWJ-MSC lysate reduces the intensity of DNA fragmentation in the nuclei of rat hippocampal neurons, with the most pronounced effects observed in groups treated with rat fetal fibroblasts (by 4.8 times), human adipose tissue MSCs (by 2.5 times), and hWJ-MSC cell lysate (by 2 times). Conclusions. A persistent focus of necrotic and apoptotic death of neurons in the hippocampus of rats is formed after experimental 20-minute IR of rats' brain, as evidenced by increased levels of fragmented DNA. Intravenous transplantation of MSCs of various origin and cell lysate from hWJ-MSC demonstrated a significant effect in the IR model: neurodestruction and neuroapoptosis at the area of the ischemic brain damage get less intensive. MSCs derived from human adipose tissue demonstrated superior neuroprotective potential compared to rat adipose tissue MSCs in the IR model of the rat brain.

Correlation analysis allows evaluating the statistical relationship between two random variables or two-dimensional data.Aim: The aim of the study is to analyse the correlations between biochemical indicators determined in the somatosensory cortex and hippocampus, morphological manifestations of neuroapoptosis, and parameters of CNS functioning in acute cerebral ischemia in rats. Materials and methods: To model an acute cerebral ischemia, ligatures were placed on the internal carotid artery bilaterally rats, after 20 minutes the blood flow through the specified vessels was restored. Experimental rat group received MSCs obtained from the human umbilical cord Wharton jelly at a dose 106 cells/rat. McGrow stroke-index scale, the functional state of the central nervous system, flow metric, immune histochemical and biochemical indicators in the tissue of the somatosensory cortex and hippocampus were analysed. Statistical processing of the results was carried out using the Statistica 7.0 computer program (StatSoft Inc., USA). Spearman's rank correlation analysis was performed to determine correlational dependencies. Results: Biochemical indicators (in particular, lactate and glucose content, MDA, SDH, SOD and NOS activity determined in the brain tissue of the hippocampal and somatosensory cortical areas), reliably correlated with functional indicators of the central nervous system in rats both with and without brain ischemia.. After acute brain ischemia at day 7 (which corresponds to the sub-acute period of ischemia) specific direct correlation between protein content in brain tissue and morphological indicators appear as well as between protein content and neurological deficit both in the somatosensory cortex and in the hippocampus. At day 14 (the recovery period) after brain ischemia high specific inverse correlation takes place between MDA activity in the somatosensory cortex and morphological indicators in the hippocampus. Conclusion: 1. Multiplex correlations between biochemical and morphological indicators determined in the somatosensory cortex and hippocampus indicate their close functional interaction. 2. No new specific correlations emerged when human umbilical cord Wharton jelly MSCs were transfused for neuro-protection immediately after brain reperfusion beginning, but some correlational dependencies disappeared.

In neurodegenerative processes of the brain, the hippocampus is primarily damaged with subsequent cognitive impairments as a result of increased generation of reactive oxygen species and reactive nitrogen species, which lead to apoptosis and necrosis of neurons. Cell therapy with the use of mesenchymal stromal cells (MSCs) has shown encouraging results regarding endogenous mechanisms of neuroregeneration in response to ischemic injury of brain structures. The work aimed to study the therapeutic potential of MSCs of various origins and sources, MSCs lysate and reference drug citicoline on the energy component of neuronal metabolism, oxidative and nitrosative stress in the rat hippocampus under conditions of cerebral ischemia-reperfusion (IR). Material and methods. The experiment was carried out on 126 Wistar rats with modeled pathology (20-minute IR of the internal carotid arteries), which were injected with MSCs derived from Wharton’s jelly of the human umbilical cord, MSCs derived from human and rat adipose tissues, rat embryonic fibroblasts, MSCs lysate and citicoline immediately after removal of the ligatures. Biochemical parameters of carbohydrate metabolism (glucose, lactate), oxidative (NADPH oxidase activity) and nitrosative stress (NO synthase activity) in the rat hippocampus were determined on days 7 and 14 after IR under the conditions of cerebral IR and on the background of its correction. Results. It was established that during IR in the rat hippocampus of the brain, the content of glucose and lactate increases, the process of aerobic oxidation of glucose is inhibited, anaerobic glycolysis increases, lactic acidosis develops in hippocampal cells, the level of NADPH oxidase activity decreases, and an imbalance occurs in the functioning of the nitrogen monoxide system. A positive effect of human umbilical cord Wharton’s jelly-derived MSC and rat embryonic fibroblasts transplantation, and the use of citicoline on the stabilization of glucose, lactate, NADPH-oxidase and nitric oxide levels was found. Transplantation of human and rat adipose-derived MSCs was significantly less effective than citicoline and demonstrated no statistically significant modulatory effect on biochemical parameters in the hippocampus of experimental animals with IR. Conclusions. Transplantation of the human umbilical cord Wharton’s jelly-derived MSCs was not inferior to the citicoline and, better than other tested MSCs and their lysate, contributed to the recovery of disturbed energy processes (glucose level) and eliminated metabolic acidosis (lactate level) in the hippocampus of rats, i.e. had a positive modulating effect on the oxidant-antioxidant balance (NADPH oxidase activity level).

In neurodegenerative processes of the brain, the hippocampus is primarily damaged with subsequent cognitive impairments as a result of increased generation of reactive oxygen species and reactive nitrogen species, which lead to apoptosis and necrosis of neurons. Cell therapy with the use of mesenchymal stromal cells (MSCs) has shown encouraging results regarding endogenous mechanisms of neuroregeneration in response to ischemic injury of brain structures. The work aimed to study the therapeutic potential of MSCs of various origins and sources, MSCs lysate and reference drug citicoline on the energy component of neuronal metabolism, oxidative and nitrosative stress in the rat hippocampus under conditions of cerebral ischemia-reperfusion (IR). Material and methods. The experiment was carried out on 126 Wistar rats with modeled pathology (20-minute IR of the internal carotid arteries), which were injected with MSCs derived from Wharton’s jelly of the human umbilical cord, MSCs derived from human and rat adipose tissues, rat embryonic fibroblasts, MSCs lysate and citicoline immediately after removal of the ligatures. Biochemical parameters of carbohydrate metabolism (glucose, lactate), oxidative (NADPH oxidase activity) and nitrosative stress (NO synthase activity) in the rat hippocampus were determined on days 7 and 14 after IR under the conditions of cerebral IR and on the background of its correction. Results. It was established that during IR in the rat hippocampus of the brain, the content of glucose and lactate increases, the process of aerobic oxidation of glucose is inhibited, anaerobic glycolysis increases, lactic acidosis develops in hippocampal cells, the level of NADPH oxidase activity decreases, and an imbalance occurs in the functioning of the nitrogen monoxide system. A positive effect of human umbilical cord Wharton’s jelly-derived MSC and rat embryonic fibroblasts transplantation, and the use of citicoline on the stabilization of glucose, lactate, NADPH-oxidase and nitric oxide levels was found. Transplantation of human and rat adipose-derived MSCs was significantly less effective than citicoline and demonstrated no statistically significant modulatory effect on biochemical parameters in the hippocampus of experimental animals with IR. Conclusions. Transplantation of the human umbilical cord Wharton’s jelly-derived MSCs was not inferior to the citicoline and, better than other tested MSCs and their lysate, contributed to the recovery of disturbed energy processes (glucose level) and eliminated metabolic acidosis (lactate level) in the hippocampus of rats, i.e. had a positive modulating effect on the oxidant-antioxidant balance (NADPH oxidase activity level).

Ischemic stroke is an extremely important pathology with high mortality, in which more than 50 % of patients with occlusion of the main vessels remain disabled, despite early reperfusion therapy by thrombolysis or thrombectomy. As part of the regenerative strategy, stem cell transplantation in ischemic stroke became a new impetus. Cell therapy with the use of mesenchymal stromal cells demonstrated encouraging results regarding endogenous mechanisms of neuroregeneration in response to ischemic damage to brain structures. The aim of the research is to study the influence of mesenchymal stromal cells of various genesis, lysate of mesenchymal stromal cells obtained from Wharton’s jelly umbilical cord and citicoline on the dynamics of morphological changes in the hippocampal CA1 region of rats with acute cerebral ischemia-reperfusion according to light microscopy and micromorphometry data. The experiment was carried out using 200 male Wistar rats, which were subjected to ischemia-reperfusion by reversible 20-minute bilateral occlusion of the internal carotid arteries. Animals with modeled pathology were intravenously transplanted with mesenchymal stromal cells of various genesis (from Wharton’s jelly of the human umbilical cord, human and rat adipose tissue), and rat embryonic fibroblasts, lysate of mesenchymal stromal cells and citicoline were injected. Histological analysis of rat brain sections was performed on the 7th and 14th day of the experiment. Statistical analysis was performed using “Statistica 6.0” (StatSoft® Snc, USA). The significance of differences was assessed using the Student’s t-test and the nonparametric Mann-Whitney U test. During the study, it was found that modeled ischemia-reperfusion in rats caused almost complete degeneration of the structure of the pyramidal layer of hippocampal CA1 region, gave uniformity to the structure of the radiant layer, infiltration of microglia, contributed to the disruption of the arrangement of apical dendrite bundles and narrowing of blood vessels as a result of perivascular edema. Also, the modeled pathology reduced the total number of neuronal nuclei in the hippocampal CA1 area, the overwhelming majority of which had signs of pathological changes. Transplantation of mesenchymal stromal cells of various origins, lysate of mesenchymal stromal cells and citicoline contributed to a significant increase in the number of neuronal nuclei in the hippocampal CA1 zone and nuclei that did not undergo pathological changes. The most positive effect was found in the transplantation of mesenchymal stromal cells from human Wharton’s jelly-derived cells. Thus, both in the subacute and recovery periods of ischemic stroke in rats, the transplantation of human Wharton’s jelly-derived mesenchymal stromal cells was significantly surpassed the reference drug citicoline in its ability to reduce the number of pathologically changed nuclei by 1.5 times (p<0.05). At the same time, the number of pathologically unchanged nuclei significantly exceeded the number of nuclei with signs of karyorrhexis and karyopyknosis, so it would be advisable to use mesenchymal stromal cells of various genesis, lysate or citicoline in conditions of acute cerebral ischemia-reperfusion, taking into account their ability to reduce the volume of the infarct. In the future, an injectable drug will be created from the most effective culture of mesenchymal stromal cells in terms of cerebroprotective properties for cell therapy of patients with acute ischemic stroke.

Aim: The aim of this work was to study the effects of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) on inflammation-impaired cognitive functions and the brain of mice. Methods: Young mice (~3-month-old) and aged mice (~18-month-old) were injected with bacterial lipopolysaccharide (LPS) and obtained intravenously donor 106 human umbilical cord MSCs, EVs isolated from a similar amount of MSCs or conditioned medium (CM) of MSCs. Subsequently, the mice were examined in behavioral tests and the mouse brains were analyzed for the levels of pro-inflammatory cytokines, α7 nicotinic acetylcholine receptors (α7 nAChRs) and amyloid beta 1-42 (Aβ1-42). Results: EVs prevented LPS-induced memory impairment in mice, whereas CM provided a weaker and temporal effect. Both EVs and MSCs injected once after regular injections of LPS stably improved memory of young mice. In contrast, both cells and EVs provided only transient effect in aged mice injected with LPS. The brains of aged LPS-treated mice contained elevated amounts of IL-1β and IL-6; both MSCs and EVs decreased them significantly. The brains of non-treated aged mice contained decreased levels of α7 nAChRs and increased levels of Aβ1-42 and α7-bound Aβ1-42 compared to the brains of young mice. LPS treatment decreased α7 nAChRs in both young and aged mice, while both MSCs and EVs restored them up to the control level. In young mice, LPS treatment increased the level of Aβ1-42 and α7-bound Aβ1-42, whereas MSCs and EVs decreased it. In contrast, neither LPS nor MSCs/EVs influenced the elevated level of Aβ1-42 but increased α7-bound Aβ1-42 in the brains of aged mice. Conclusions: Regenerative potential of MSCs and MSC-derived EVs is sufficient to support cognitive functions of LPS-treated young mice but is quite poor for aged animals, possibly, due to decreased levels of α7 nAChRs and accumulated Aβ1-42 in their brains.

Neuroprotective therapy in acute cerebrovascular disorders is directly aimed at preserving neurons in the penumbra zone, but it also emphasizes the importance of gliogenesis in the affected area. Glial cells exhibit rapid reactivity and are highly sensitive to cerebral ischemic damage. Recent experimental studies have demonstrated the successful application of multipotent mesenchymal stromal cells (MMSCs) in stroke to modulate microglial activation. The aim of the study was to investigate the impact of MMSCs of different origins, MMSC lysate, and citicoline on glial components in a model of cerebral ischemia-reperfusion in rats. Materials and methods. The experiments were conducted on 190 male Wistar rats aged 4 months, weighing 160-190 g. After modeling cerebral ischemia-reperfusion through bilateral 20-minute occlusion of the internal carotid arteries, rats were intravenously administered MMSCs derived from human Wharton’s jelly, human adipose tissue, and rat Wharton’s jelly at a dose of 106 cells per animal. Other groups received fetal rat fibroblasts (106 cells/animal in 0.2 ml of physiological solution) and lysate from human Wharton’s jelly at a dose of 0.2 ml per animal. Control animals received 0.2 ml of physiological solution intravenously. The last group of rats received a single dose of the reference drug citicoline at a dose of 250 mg/kg. On the 7th and 14th days, the area and fluorescence intensity of cells expressing markers of astrocytes (GFAP), microglia (Iba1), and oligodendrocytes (Rip) were quantitatively assessed in CA1 hippocampal region slices using immunohistochemical examination and confocal microscopy. Results. On the 7th and 14th days after cerebral ischemia-reperfusion in rats, the intensity of fluorescence of GFAP-positive astrocytes and Iba1-positive microglial cells increased, indicating pronounced reactive astrogliosis and microglial activation in the CA1 region of the hippocampus. Meanwhile, ischemia-reperfusion had a significant impact on the content of Rip-positive oligodendrocytes in brain slices. The application of all treatments (transplantation of MMSCs of different origins, their lysate, or the reference drug citicoline) had a cytoprotective effect, reducing reactive astrogliosis and microgliosis both on the 7th and 14th days after injury. The best result was demonstrated with the treatment using MSCs from human Wharton’s jelly.s. Conclusion. Cerebral ischemia-reperfusion induces reactive gliosis through the activation of GFAP- and Iba1-positive glial cells in all layers of the hippocampus. The application of MSCs from human Wharton’s jelly and fetal rat fibroblasts significantly reduces its intensity on both the 7th and 14th days after injury modeling.

Transplantation of mesenchymal stromal cells (MSCs) is a promising direction in the therapy of ischemic stroke, which promotes the regeneration of neurons in experimental and clinical studies. We evaluated the neuroprotective and angioprotective properties of intravenously transplanted allogeneic and xenogeneic MSCs obtained from human and rat adipose tissue, human umbilical cord Wharton jelly and its cells lysate compared with citicoline in a rat brain ischemia-reperfusion model. We studied the somatosensory cortex and CA1 zone of the hippocampus in rats using immunohistochemical analysis. It was found that the CA1 zone of the hippocampus was the most sensitive to ischemic damage. Analysis of brain sections 7 and 14 days after ischemia-reperfusion showed that all treatment options increased the staining intensity of NeuN-positive neurons, but did not reach the control value in sham-operated rats. To quantify the obtained results, we used the integrated fluorescence density indicator, which demonstrated a decrease in the fluorescence intensity of NeuN-positive neurons in 4.2 (7 days) and 3.2 times (14 days). All treatment variants significantly increased the intensity of fluorescence, but human umbilical cord Wharton's jelly MSCs gave the best result. There was no difference in the fluorescence intensity of RECA-1-positive blood vessels. Thus, immunohistochemical analysis using a broad panel of antibodies against neurons and endothelial cells demonstrated that ischemia-reperfusion resulted in the death of NeuN-positive neurons in the CA1 zone of the hippocampus. Transplantation of stem cells of different origin, lysate of umbilical cord Wharton's jelly MSCs and administration of citicoline had a neuroprotective effect on the CA1 zone of the hippocampus. However, the best result has been shown by the treatment with Wharton's jelly MSCs.

Relevance: One of the main causes of stroke in acute cerebrovascular accident (ACVA) is ischemia, which begins with the formation of an acute neuronal energy deficit with subsequent activation of the "ischemic cascade" reactions that lead to irreversible damage to nervous tissue. Aim: To compare the effect of mesenchymal stromal cells (MSCs) of different origin and human MSCs from Wharton's jelly lysate on neuroapoptotic changes in the somatosensory cortex of the rat brain in conditions of model ischemia-reperfusion (IR) performed by ductal cytoflowmetry. Materials and methods: The experiment was carried out using 165 four-month-old male Wistar rats weighing 160-190 g, which were subjected to bilateral 20-minute transient ischemia-reperfusion (IR) of the internal carotid arteries. After modeling the pathology, the animals were injected into the femoral vein (iv) with MSCs obtained from umbilical cord Wharton jelly, human and rat adipose tissue in the amount of 10 6 cells/animal. Other groups of experimental animals were intravenously injected with fetal rat fibroblasts in the amount of 10 6 cells/animal (in 0.2 ml of physiological solution) and MSCs from umbilical cord Wharton's jelly lysate in a dose of 0.2 ml/animal. Control animals were injected intravenously with 0.2 ml of physiological solution. The level of DNA fragmentation in the nuclei of neurons of the somatosensory cortex of rats on the 7th day after ischemiareperfusion was studied by flow cytometry. The research was carried out on a flow cytometer "Partech РАС" of the company Partech, Germany. The statistical significance of the differences was assessed by Student's t-test. Results: The study noted an increase in the level of fragmented DNA in a group of animals with IR by 3.25 times 7 days after model IR. The performed treatment showed that in groups with transplanted MSCs of various origins and MSC lysate from human Wharton's jelly cells, the intensity of DNA fragmentation in the nuclei of neurons in rat brain somatosensory cortex reliably decreased in1.8-2. 6 times compared with the group of control pathology (IR without treatment). Conclusions: Experimental 20-minute IR of the brain of rats forms a persistent focus of necrotic and apoptotic death of neurons, which is manifested by an increase in fragmented DNA (3.25 times). Intravenous transplantation of MSCs of various origin and lysate of MSCs from human Wharton jelly has a therapeutic effect in model IR, which is manifested by a decrease in the processes of neuro-destruction and neuroapoptosis in the area of ischemic brain damage Such effect is a link to the polytrophic mechanism of MSCs neuroprotective action.