In the treatment of ischemic stroke, an innovative approach is the use of neuroprotective compounds. Natural peptides, including adrenocorticotropic hormone (ACTH), can serve as the basis for such drugs. Previously, a significant effect of non-hormonal ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP peptides on the functions of the nervous system was shown. Also, while using RNA-Seq, we firstly revealed differentially expressed genes (DEGs) that associated with peptides in the penumbra-associated region of the frontal cortex (FC) of rats at 24 h after transient middle cerebral artery occlusion (tMCAO) model. Peptides significantly reduced profile disturbances caused by ischemia for almost two-thousand DEGs in FC related to the neurotransmitter and inflammatory response. Here, we studied how peptides affected the expression of genes in the striatum with an ischemic focus, predominantly. The same animals from which we previously acquired FC were used to collect striatum samples. Peptides generated fewer DEGs in the striatum than in the FC. Both peptides tended to normalize the profile of disturbances caused by ischemia for hundreds of DEGs, whereas 152 genes showed an even more affected profile in the striatum under ACTH(6-9)PGP action. These DEGs were associated with inflammation, predominantly. About hundred genes were overlapped between both peptides in both tissues and were associated with neuroactive ligand-receptor interaction, predominantly. Thus, genes that are associated with the ACTH-like peptide action in rat brain regions with varying levels of ischemia injury were identified. Moreover, differential spatial regulation of the ischemia process in the rat brain at the transcriptome levels was discovered under peptides with different ACTH structures. We suppose that our results may be useful for selecting more effective neuroprotective drug structures in accordance with their specific tissue/damage therapeutic impact.

Ischemic stroke is a multifactorial disease that leads to brain tissue damage and severe neurological deficit. Transient middle cerebral artery occlusion (tMCAO) models are actively used for the molecular, genetic study of stroke. Previously, using high-throughput RNA sequencing (RNA-Seq), we revealed 3774 differentially expressed genes (DEGs) in the penumbra-associated region of the frontal cortex (FC) of rats 24 h after applying the tMCAO model. Here, we studied the gene expression pattern in the striatum that contained an ischemic focus. Striatum samples were obtained from the same rats from which we previously obtained FC samples. Therefore, we compared DEG profiles between two rat brain tissues 24 h after tMCAO. Tissues were selected based on magnetic resonance imaging (MRI) and histological examination (HE) data. As a result, 4409 DEGs were identified 24 h after tMCAO in striatum. Among them, 2609 DEGs were overlapped in the striatum and FC, whereas more than one thousand DEGs were specific for each studied tissue. Furthermore, 54 DEGs exhibited opposite changes at the mRNA level in the two brain tissues after tMCAO. Thus, the spatial regulation of the ischemic process in the ipsilateral hemisphere of rat brain at the transcriptome level was revealed. We believe that the targeted adjustment of the genome responses identified can be the key for the induction of regeneration processes in brain cells after stroke.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

Today, photodynamic therapy (PDT) is one of the most promising methods for the complete eradication of a number of superficial solid malignant neoplasms. In this work, we studied the ef-fectiveness of PDT on Ehrlich carcinoma of mice with the injections of a new photosensitizer (PS) Photoran E6. To determine the optimal drug-light interval, from the time of PS injection to laser ir-radiation, we studied the distribution kinetics of Photoran E6 in tumors and healthy tissues sur-rounding them. In assessing the response of the inhibitory effect of Ehrlich carcinoma to PDT, the following indicators were used: inhibition of tumor growth (ITG, %), complete regression (CR, %), absolute growth rates of tumors (K) in mice with continued growth of neoplasia, and an increase in life expectancy (ILE, %) of individuals in the experimental groups compared with the control, according to the criterion of curing animals: the absence of signs of tumor recurrence within 90 days after therapy. The therapeutic potential of PDT for tumor cell devitalization was determined by histological examination of the areas of exposure to laser radiation at 21d after treatment. The results showed that Photoran E6 has a high antitumor activity for PDT of solid Ehrlich carcinoma. Conducting PDT in the optimal time after the injection of PS with certain parameters of laser ex-posure allows to achieve the maximum inhibitory effect on malignant neoplasms. A pathomor-phological study in the areas of photodynamic exposure of the surviving tumor cells was not found.

Photodynamic therapy (PDT) is a local form of treatment for the complete eradication of solid malignant neoplasms (PDT). In this work, we studied the efficiency of PDT with the photosensi-tizer (PS) chlorin e6 dimethyl ether (DME Che6) in the treatment of positive for the mutant p53 gene sarcoma M-1. To assess the optimal time for tumor irradiation with laser the kinetics of PS accumulation in tumor and surrounding healthy tissues was studied. The tumor response to PDT was assessed with the use of the following criteria: tumor growth inhibition index (%); tumor complete regression (%); absolute growth rate (K) index, it is used in rats with tumor growth after the treatment; increase in the life expectancy in group of treated animals and the control group of tumor-bearing rats; the treatment response criterion – means the absence of signs of tumor re-currence within 90 days after therapy. On the 21st day after the treatment the PDT efficiency for tumor cells devitalization was assessed by examining the areas exposed to laser radiation and data of histological analysis. Results of the study allow us to state that DME Che6 has a high anti-tumor activity in the treatment of M-1 sarcoma. The maximum inhibitory effect of the PS on ma-lignant neoplasms after PDT is achieved if time between the sensitizer introduction and the laser irradiation beginning is optimal. As a result of pathomorphological assessment of material after PDT, no survived tumor cells with expression of the mutant p53 protein were detected.

We studied the effectiveness of photodynamic therapy with the photosensitizer Photoran E6 on the model of rat sarcoma M-1 positive for mutant p53 gene. Experiments showed that Photoran E6 exhibits high antitumor activity in photodynamic therapy of solid tumor of the connective tissue. Photodynamic therapy carried out during the optimal period after injections of Photoran E6 with the determined parameters of laser exposure allows achieving the maximum inhibitory effect on sarcoma M-1: 100% cured animals. Immunohistochemical study revealed no live tumor cells with expression of the mutant p53 protein in areas of photodynamic exposure.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

3D-bioprinting is a promising technology for a tissue scaffold fabrication in the case of damaged tissue/organ replacement. Collagen is one of the most appropriate hydrogel for the purpose, due to its exceptional biocompatibility. However, the use of collagen with conventionally low concentration makes bioprinting process difficult and does not provide its high accuracy. The purpose of the study was evaluation of suitability of collagen with high concentration in case of chondrocyte-laden scaffold fabrication via 3D-bioprinting for cartilage regeneration in vitro and in vivo. The results of the study showed that inherent porosity of 4% collagen was not enough for cell survival in the case of long-term incubation in vitro. With the beginning of the scaffold incubation, cell migration to the surface and out of the scaffold was observed. The residual cells died mostly within 4 weeks. As for in vivo study, in 2 weeks after implantation of the scaffold, a weak granulomatous inflammation was observed. In 6 weeks, a connective tissue was formed in the area of implantation. In the tissue, macrophages and groups of small cells with round nuclei were found. In accordance with morphological criteria, these cells could be considered as young chondrocytes. However, its amount was not enough to initiate the formation of cartilage.

The efficacy of a new photosensitizer of chlorin E6 conjugated with a prostate-specific membrane antigen (PSMA) in photodynamic therapy of murine melanoma B16 was studied in in vivo experiments. The dynamics of photosensitizer accumulation in the tumor and surrounding tissues was evaluated and antitumor efficacy of photodynamic therapy was assessed by parameters of regression and morphological characteristics of experimental transplanted melanoma B16. The inhibitory effect of photodynamic therapy on melanoma was evaluated by complete regression of the tumor, absolute tumor growth coefficient in animals with continuation of tumor growth, and the increase in life span in comparison with the control; the criterion of cure was the absence of signs of tumor recurrence in mice within 90 days after therapy. The therapeutic potential of photodynamic therapy was determined by devitalization of tumor cells (histological examination of the zones of laser exposure on day 21 after treatment). The photosensitizer with PSMA-ligand exhibited high antitumor activity in photodynamic therapy for melanoma B16. Photodynamic therapy carried out at the optimum time after photosensitizer injection with experimentally determined parameters of laser exposure allows achieving the maximum inhibitory effect on melanoma. Pathomorphological study in the zones of exposure detected no survived tumor cells.

Due to its nootropic, neuroprotective, and immunomodulatory effects, the peptide Semax is utilized in the treatment of ischemic stroke. Our earlier RNA-Seq analysis of the transcriptome in an ischemic model of transient occlusion of the middle cerebral artery showed an increase in the mRNA levels of many proinflammatory genes, and the suppression of their induction by Semax. However, for many relevant genes, including Il1a, Il1b, Il6 and Tnfa, the levels of their expression were too low for detailed quantitative evaluation. Here we utilize qRT-PCR to analyze the effects of the Semax peptide on the expression of weakly expressed mRNAs encoding several proinflammatory mediators, and show that exposure to Semax leads to a statistically significant decrease in the Il1a, Il1b, Il6, Ccl3, and Cxcl2 mRNAs, which compensates for the increase in the transcription of these genes induced by ischemia-reperfusion. We conclude that the observed protective effect of Semax in the model of stroke may be due to its anti-inflammatory effects. We also discuss the limitations of the RNA-Seq when applied to quantifying less abundant transcripts as compared to the real-time RT-PCR method.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)—1.6–1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5–1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4–1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors—T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

Cardiovascular diseases, along with cancer, are the leading causes of death worldwide. Although modern pharmacological treatment of various cardiomyopathies can slow the development of myocardial dysfunction, they have limited effectiveness in patients with end-stage disease. Many researchers believe that heart transplantation is the only radical treatment in this case. However, the lack of donors and the high operation cost require careful selection of surgical candidates. With the introduction of molecular and cell biology into medical practice, today, stem cell therapy can become an alternative method of nonsurgical restoration of myocardial functions. The most studied and attractive is the use of mesenchymal stem cells (MSCs). MSCs differ from hematopoietic stem cells used as support for hematopoiesis in high-dose chemotherapy by the following features: pronounced trophic effect, immune tolerance, the ability to suppress alloreactivity and autoimmune disorders. An important stage in the implementation of cell therapy is the creation of a cell biobank of MSCs. In A.F.Tsyb Medical Radiological Research Center, this work has been carried out since1984. Asignificant number of experimental studies have been carried out, confirming the possibility of clinical implementation of this approach. A method for obtaining stable cultures of MSCs and cardiomyoblasts from bone marrow cells was developed and approvals were obtained. Experimental studies of cell therapy are also being conducted to overcome anthracycline-induced cardiotoxicity in cancer patients.This article is devoted to practical application of MSC-based therapy, in particular, in cancer patients with cardiotoxicity, as well as to the issues of creating a cell biobank for treatment with MSCs.

The purpose of the work was to study the ability of the NOS inhibitor T1023 to prevent late radiation injuries. Methods: the effects of T1023 (75 mg / kg, once i.p. 30 minutes before the irradiation) on the development of post-radiation pulmonitis and pneumofibrosis in rats with thoracic exposure to g-radiation at a dose of 12.5 Gy were studied histopathologically and morphometrically. The results of the studies showed that there wasn’t a significant objective effect of T1023 on the development of early radiation-induced lung injuries (9 weeks after irradiation). But it prevented late radiation induced lung injuaries (26 weeks after irradiation) – there were a significant lesser pathomorphological manifestations of post-radiation pulmonitis, proliferation of connective tissue and the development of fibrotic changes in the lung parenchyma. At this stage, the action of T1023 clearly contributed to the preservation of the normal histostructure of the lungs, reducing by 40% the content of compaction zones in the parenchyma. The ability of the NOS inhibitor T1023 to significantly limit the development of lungs late radiation reaction confirms the promise of further development of this compound as a means for prevention radiation therapy complications.

Cerebral ischaemia is the most common cause of impaired brain function. Biologically active peptides represent potential drugs for reducing the damage that occurs after ischaemia. The synthetic melanocortin derivative, ACTH(4-7)PGP (Semax), has been used successfully in the treatment of patients with severe impairment of cerebral blood circulation. However, its molecular mechanisms of action within the brain are not yet fully understood. Previously, we used the transient middle cerebral artery occlusion (tMCAO) model to study the damaging effects of ischaemia–reperfusion on the brain transcriptome in rats. Here, using RNA-Seq analysis, we investigated the protective properties of the Semax peptide at the transcriptome level under tMCAO conditions. We have identified 394 differentially expressed genes (DEGs) (>1.5-fold change) in the brains of rats at 24 h after tMCAO treated with Semax relative to saline. Following tMCAO, we found that Semax suppressed the expression of genes related to inflammatory processes and activated the expression of genes related to neurotransmission. In contrast, ischaemia–reperfusion alone activated the expression of inflammation-related genes and suppressed the expression of neurotransmission-related genes. Therefore, the neuroprotective action of Semax may be associated with a compensation of mRNA expression patterns that are disrupted during ischaemia–reperfusion conditions.

Фотодинамическая активность липосомального борированного хлорина е6Бурмистрова Н.В., Дрожжина В.В., Каплан М

The results of a complex morphofunctional study with markers of proliferation (PCNA and Ki-67), angiogenesis (CD31 and CD34), and structural integrity of mesenchymal cells (vimentin) suggest that the pool of primordial follicles was well preserved in vitrified ovarian tissue and that viability of its cellular components can be rapidly restored during incubation at 37°C within 4 h after thawing.

The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion.