The possibility of transferring exosomes of neural (NSC) and mesenchymal (MSC) mouse stem cells labeled with the fluorescent dye PKH26 into the cerebral cortex and hippocampus after their intranasal administration to mice, and the accumulation and localization of exosomes in cultured brain cells of various types, and also the effect of exosomes of NSCs and MSCs on the parameters of the cell cycle and the level of apoptosis of cultured NSCs after irradiation at a dose of 4 Gy. The accumulation of exosomes obtained from the culture medium of NSCs and MSCs from the adipose tissue of C57BL/6 mice was shown both in the hippocampus and in the cerebral cortex after their intranasal administration to syngeneic mice. Exosomes were found predominantly in the perinuclear region of brain cells. When culturing NSCs and differentiated from NSCs neurons and astrocytes, exosomes accumulate more intensively in astrocytes and are also localized in the perinuclear region of cells, and in astrocytes also in the cytoplasm. Exosomes accumulated most intensively in astrocytes. When studying the effect of stem cell exosomes on the cell cycle of irradiated NSCs, it was shown that the cultivation of NSCs irradiated at a dose of 4 Gy in the presence of exosomes of both NSCs and mouse MSCs does not lead to the restoration of cell cycle parameters, but provides a decrease the number of apoptotic cells in 24 h after exposure.

Purpose: To investigate the effect of γ, n-irradiation of the mice head on the brain cells damage, behavior and cognition, and to examine the possibility of using lactoferrin (LF) to alleviate radiation-induced impairments. Material and methods: Mice heads were irradiated in a beam of neutrons and gamma rays from the IR-8 nuclear reactor. The brain cells of control and irradiated mice were isolated using Percoll. Neurons and resting and activated microglia cells were analyzed using the fluorescently labeled antibodies and flow cytometry. The level of DNA double-strand breaks in neurons was determined by γH2AX histone content. Cytokine gene expression in the hippocampus was studied by RT-PCR. Behavior and cognitive functions were studied using the open field, Morris water maze and novel object recognition tests. LF was isolated from female colostrum by preparative ion-exchange chromatography and purified by affinity chromatography on heparin-sepharose. Results: γ, n-Irradiation of the mice head at a dose of 1.5 Gy led to an increase in the level of DNA double-strand breaks in neurons. Twenty-four hours after irradiation the total number of cells and the number of neurons in the isolated fraction of brain cells decreased, but the number of microglial cells remained unchanged. The number of resting and activated microglia did not change within 3–72 h after γ, n-irradiation. The expression level of the TNFα, IL-1β, and IL-6 genes increased 2 months after γ, n-irradiation of the mice head at a dose of 1.5 Gy, indicating the development of neuroinflammation. At this time, irradiated mice demonstrated the anxiety-like behavior and impaired spatial and recognition memory. A single i.p. administration of human LF to mice immediately after γ, n-irradiation of the head did not affect the observed radiation-induced disturbances, but decreased the gene expression levels of TNFα, IL-1β and IL-6 pro-inflammatory cytokines and increased the gene expression level of TGFβ anti-inflammatory cytokine in the hippocampus 2 months after radiation exposure. The obtained results indicate a partial decrease in the level of hippocampal neuroinflammation of irradiated animals treated with LF. Conclusion: γ, n-Irradiation of the mice head at a dose of 1.5 Gy leads to DNA damage of neurons and the decrease in the number of neurons. Microglia cells are more resistant to such radiation exposure. Late after head-only γ, n-irradiation, mice develop neuroinflammation, which is detected by an increase in the pro-inflammatory cytokine gene expression in the hippocampus and also by anxiety-like behavior and impaired cognitive functions. A single LF administration leads to a partial decrease in the neuroinflammation level, but does not affect the other studied parameters. The optimal dosing regimen of LF remains to be determined to preserve cognitive functions after γ, n-irradiation of the brain.

The study aimed to investigate the effect of low-dose rate (2.13 mGy/h) prolonged γ,n-irradiation at 0.05 and 0.5 Gy on brain microglia, expression of pro- and anti-inflammatory cytokine genes, behavior and cognition of C57Bl/6 mice two months and one year after exposure. The decrease in the number of resting microglia and the increase in the proportion of activated microglia in brain cell preparations were observed after γ,n-irradiation at a dose of 0.5 Gy. Immunohistochemical analysis revealed the increased number of microglia cells and unaltered number of astrocytes in the hippocampus after γ,n-irradiation at both doses. Expression of TNFα and IL-1β pro-inflammatory cytokine genes in the hippocampus was increased after irradiation at both doses. Expression of TGFβ and IL-4 anti-inflammatory cytokine genes was decreased two months after irradiation only at a dose of 0.5 Gy, suggesting more intensive neuroinflammation. Analysis performed a year later revealed an age-related decrease in the number of resting microglia and increase in proportion of activated microglia compared to young animals, with more profound changes in male mice. The state of microglia in control and irradiated mice was similar one year after exposure. Neither two months nor one year after irradiation the impairments of motor activity and spatial memory of mice were detected; nevertheless, mice irradiated at a dose of 0.5 Gy demonstrated anxious behavior and decreased spatial learning in Morris water maze one year after exposure. In summary, we showed that γ, n-irradiation at doses of 0.05 and 0.5 Gy induced neuroinflammation two months after exposure. No difference from the control mice was observed one year after irradiation. Disturbances of behavior and spatial memory of irradiated mice were not detected.

Рецептор колониестимулирующего фактора-1 (CSF1R) обладает тирозинкиназной активностью, в головном мозге он представлен на клетках микроглии, и его активация обеспечивает их репопуляцию. Введение мышам ингибитора CSF1R пексидартиниба (ПД, 40 мг/кг, внутрижелудочно, 7 сут) снижало количество клеток микроглии на 90 %. Облучение головы в дозе 8 Гр приводило через 2 мес к развитию нейровоспаления, о чем свидетельствовало повышение доли активированной микроглии в препаратах клеток мозга, и к нарушению когнитивных функций. Введение ПД перед облучением предотвращало активацию микроглии. При анализе эпизодической памяти в тесте «Распознавание нового объекта» и пространственной гиппокамп-зависимой памяти в тесте «Водный лабиринт Морриса» показано, что снижение количества клеток микроглии перед облучением с помощью ПД обеспечивает сохранение эпизодической и пространственной памяти у облученных мышей.

Colony-stimulating factor 1 receptor (CSF1R) possesses tyrosine kinase activity. Activation of CSF1R expressed on the surface of brain microglia allows repopulation of microglial cells. Administration of pexidartinib (PD, 40 mg/kg, gavage, 7 d), a CSF1R inhibitor, to mice decreased the number of microglial cells by 90%. Head irradiation at a dose of 8 Gy caused the development of neuroinflammation, as evidenced by the increased fraction of activated microglia in brain-cell preparations, and cognitive impairments two months after the exposure. Administration of PD prior to head irradiation prevented microglia activation. An analysis of episodic memory in the novel-object recognition test and of hippocampus-dependent spatial memory in the Morris water maze test showed that decreasing the number of microglial cells by PD administration prior to irradiation preserved episodic and spatial memory in irradiated mice.

PURPOSE:Currently there are no effective diagnostic methods for the control of neuroinflammation before manifestation of cognitive impairment after head irradiation. The translocator protein (TSPO) is highly expressed in glial cells upon brain damage, therefore we compared the changes in the number of cells with high TSPO expression in the brain and peripheral blood during radiation-induced neuroinflammation.MATERIALS AND METHODS:Hippocampal cytokines mRNA expression and the content of cells with high TSPO expression in the brain and peripheral blood monocytes were analyzed up to eight months after mice head γ-irradiation at a dose of 2 Gy or 8Gy.RESULTS:Mice irradiation at a dose of 8 Gy causes neuroinflammation, accompanied by an increase of M1 microglia and TSPOhigh cells in the brain, elevated gene expression of pro-inflammatory and decreased of anti-inflammatory cytokines along with an increased number of microglia and astrocytes in the hippocampus. The content of TSPOhigh cells in the brain correlates with the level TSPOhigh monocytes in three days, one month and two months after exposure.CONCLUSIONS:An increase in the level of the monocytes with high expression of TSPO may be considered as a marker for an early diagnostics of post-radiation brain damage leading to cognitive impairment.

Reactive oxygen species (ROS), which are formed under the action of ionizing radiation and inflammation factors, react with lipids and lead to the formation of toxic metabolites; one of the most active among them is 4-hydroxy-2-nonenal (4-HNE). It modifies cell proteins by lysine, histidine, and cysteine residues and disrupts their functions. The purpose of this research is to assess the number of neurons containing 4-HNE-modified proteins two months after γ-irradiation of the head at doses of 2, 8, and 20 Gy and determine the development of neuroinflammation at this period. Mouse brain cells were isolated by centrifugation through Percoll solution after brain tissue treatment with accutase. The number of neurons and neurons containing 4-HNE-modified proteins, as well as the number of cells of resting and activated microglia, were analyzed using flow cytometry; the level of expression of proinflammatory cytokine TNFα gene in the hippocampus was analyzed using RT-PCR. It is shown that 14.3 ± 2.3

Purpose: To investigate the effect of fractionated whole-brain γ-irradiation at a cumulative dose of 20 Gy on cognitive functions, the state of brain glial cells and expression of multiple cytokines in mice 2 months after exposure. Material and methods: Male C57Bl/6 mice were exposed to fractionated head γ-irradiation with 5 doses of 4 Gy. Two months after irradiation the behavior and cognitive functions of animals were assessed. After isolation of cells from mice brains the content of resting and activated microglia, microglial cells with M1- and M2-phenotype, astrocytes, proliferating cells were evaluated, and the hippocampal mRNA levels of pro- and anti-inflammatory cytokines (TNFα, IL-1β, IL-6, IL-4, TGFβ) were determined. Results: It was shown that fractionated head γ-irradiation didn’t alter the locomotor activity and associative (context fear) memory, but reduced the episodic memory in novel object recognition test (discrimination index was 0.44 ± 0.08 и 0.02 ± 0.09 in control and irradiated groups, respectively) and spatial memory in Morris water maze (time in target quadrant was 46,8 ± 2,4 % and 37,4 ± 2,8 % in control and irradiated groups, respectively). Exposure of γ-radiation significantly reduced the brain contents of microglial cells (Iba1+) and astrocytes (GFAP+) with concurrent 2.5 times increase in proportion of activated microglia (from 2.0 ± 0.2 % in control to 4.9 ± 0.5 % in irradiated mice), changed the M1- / M2-microglia ratio and significantly decreased the number of proliferating cells (BrdU+) and proliferating microglial cells (BrdU+/Iba1+). An increase in mRNA level of pro-inflammatory cytokine TNFα, a decrease in mRNA level of anti-inflammatory cytokine TGFβ and concurrent increase in mRNA level of IL-4 were detected in hippocampus 2 months after irradiation. Conclusion: We show that fractionated head γ-irradiation at a cumulative dose of 20 Gy reduces the episodic and spatial memory in mice 2 months after exposure. Cognitive dysfunctions detected are associated with neuroinflammation characterized by increasing proportion of activated brain microglia and altered hippocampal pro- and anti-inflammatory cytokine profile.

The 3D reconstruction of 100 μm- and 600 μm-thick fibrous poly-L/L-lactide scaffolds was performed by confocal laser scanning microscopy and supported by scanning electron microscopy and showed that the density of the fibers on the side adjacent to the electrode is higher, which can affect cell diffusion, while the pore size is generally the same. Bone marrow mesenchymal stem cells cultured in a 600 μm-thick scaffold formed colonies and produced conditions for cell differentiation. An in vitro study of stem cells after 7 days revealed that cell proliferation and hepatocyte growth factor release in the 600 μm-thick scaffold were higher than in the 100 μm-thick scaffold. An in vivo study of scaffolds with and without stem cells implanted subcutaneously onto the backs of recipient mice was carried out to test their biodegradation and biocompatibility over a 0–3-week period. The cells seeded onto the 600 μm-thick scaffold promoted significant neovascularization in vivo. After 3 weeks, a significant number of donor cells persisted only on the inside of the 600 μm-thick scaffold. Thus, the use of bulkier matrices allows to prolong the effect of secretion of growth factors by stem cells during implantation. These 600 μm-thick scaffolds could potentially be utilized to repair and regenerate injuries with stem cell co-culture for vascularization of implant.

Mesenchymal stem cells from the adipose tissue (AT MSC) and the bone marrow (BM MSC) stimulated migration of melanoma B16 cells, while mammary adenocarcinoma Ca755 cells stimulated migration of mesenchymal stem cells. Mesenchymal stem cells retained these properties at late terms after γ-irradiation in vitro . Tumors that developed after injection of Ca755 cells alone and in combinations with BM MSC or AT MSC had similar histological structure corresponding to breast adenocarcinoma. Only AT MSC stimulated tumor growth, which was determined by more intensive secretion of factors stimulating proliferation of tumor cells, including chemokine CCL2. The use of AT MSC in regenerative medicine requires careful monitoring of the absence of tumors in patients.

ABSTRACТPurpose: To explore if the total body γ-irradiation at a dose of 0.1 Gy 7 days prior to acute mixed γ, n-irradiation of the head at the dose of 1 Gy can reduce the harmful effects of neutron irradiation on the hippocampal functions, neuroinflammation and neurogenesis.Materials and methods: Mice were exposed to γ-radiation alone, mixed γ,n-radiation or combined γ-rays and γ,n-radiation 7 days after γ-irradiation. Two months post-irradiation, mice were tested in Open Field and in the Morris water maze. The content of microglia, astrocytes, proliferating cells and cytokines TGF-β, TNF-α, IL-1β, GFAP levels, hippocampal BDNF, NT-3, NT-4, NGF mRNA expression were evaluated.Results: Two months after combined irradiation, we observed impaired hippocampus-dependent cognition, which was not detected in mice exposed to γ,n-irradiation. Combined exposure and γ,n-irradiation led to a significant increase in the level of activated microglia and astrocytes in the brains. The level of pro- and anti-inflammatory cytokines in the brain and hippocampal neurotrophine's genes changed differenly after the combined exposure and γ,n-irradiation. The quantity of DCX-positive cells was reduced after γ,n-irradiation exposer alone, but increased after combined irradiation.Conclusions: Our results indicate radio-adaptive responses in brains of mice that were exposed to low-dose gamma irradiation 7 days prior to acute 1 Gy γ,n-irradiation.

Tissue regeneration following radiotherapy occurs due to the survival and regenerative abilities of tissue stem cells and resident or invading mesenchymal stem cells (MSCs), and their functional characteristics depend significantly on their sensitivity to ionizing radiation. The aim of this work was to study the effect of long-term cultivation of adipose tissue–derived MSCs on their radiosensitivity and the ability to repair DNA double-strand breaks (DSBs). To determine whether in vitro expansion influences the sensitivity of MSCs to low and sublethal doses of γ-radiation, we maintained short- and long-term MSC cultures and analyzed their radiosensitivity at early and late passages. MSCs possessed a high telomerase activity at passage 6 (P6) and an extremely low one at passage 30 (P30). The doubling time of cells by passage 30 decreased by 7 h. MSCs irradiated at P30 demonstrated significantly decreased survival seven days after irradiation at 0.1 and 1 Gy compared with the control and P6 cultures. The clonogenic activity of MSCs irradiated at P30 and P6 was the same. MSC cell cycle analysis at P30 and P6 indicated G0/G1 arrest after 24 h, but G2/M arrest at 6 Gy after seven days. The P30 MSCs were able to repair DNA DSB efficiently after γ-irradiation. Even after irradiation at a dose of 6 Gy, P30 MSCs maintained their ability for adipocyte differentiation and cytokine secretion. These results demonstrate that, after long-term cultivation, adipose tissue-derived MSCs maintain the ability to repair DNA DSB and possess stable functional characteristics and differentiation potential after irradiation, suggesting their resistance to sublethal doses of γ-radiation. They can also retain their functions in the tissue surrounding the irradiated organ.

We studied the effect of mesenchymal stem cells from the bone marrow and adipose tissue on the growth rate of melanoma B16 and mammary adenocarcinoma Ca755 tumors after their co-administration with tumor cells to syngeneic mice. Stimulation of tumor growth and formation of melanoma metastases in the lungs was found under the influence of adipose tissue-derived, but not bone marrow-derived stem cells. At delayed terms after irradiation in sublethal doses, the adipose tissue-derived mesenchymal stem cells also stimulated the tumor growth. Stimulation of the tumor growth by adipose tissue-derived mesenchymal stem cells was caused by factors secreted by these cells. Transplantation of mesenchymal stem cells to humans is possible only after accurate exclusion of malignant tumors.