A Comparative Kidney Transcriptome Analysis of Bicarbonate-Loaded insrr-Null Mice.

The maintenance of plasma pH is critical for life in all organisms. The kidney plays a critical role in acid-base regulation in vertebrates by controlling the plasma concentration of bicarbonate. The receptor tyrosine kinase IRR (insulin receptor-related receptor) is expressed in renal ß-intercalated cells and is involved in alkali sensing due to its ability to autophosphorylate under alkalization of extracellular medium (pH > 7.9). In mice with a knockout of the insrr gene, which encodes for IRR, urinary bicarbonate secretion in response to alkali loading is impaired. The specific regulatory mechanisms in the kidney that are under the control of IRR remain unknown. To address this issue, we analyzed and compared the kidney transcriptomes of wild-type and insrr knockout mice under basal or bicarbonate-loaded conditions. Transcriptomic analyses revealed a differential regulation of a number of genes in the kidney. Using TaqMan real-time PCR, we confirmed different expressions of the slc26a4, rps7, slc5a2, aqp6, plcd1, gapdh, rny3, kcnk5, slc6a6 and atp6v1g3 genes in IRR knockout mice. Also, we found that the expression of the kcnk5 gene is increased in wild-type mice after bicarbonate loading but not in knockout mice. Gene set enrichment analysis between the IRR knockout and wild-type samples identified that insrr knockout causes alterations in expression of genes related mostly to the ATP metabolic and electron transport chain processes.

Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas.

The use of relevant, accessible, and easily reproducible preclinical models of diffuse gliomas is a prerequisite for the development of successful therapeutic approaches to their treatment. Here we studied the gene expression profile of 3D spheroids in a comparison with 2D cell cultures and tissue strains of diffuse high-grade gliomas. Using real time PCR, we evaluated the expression of Gfap, Cd44, Pten, S100b, Vegfa, Hif1a, Sox2, Melk, Gdnf, and Mgmt genes playing an important role in the progression of gliomas and regulating tumor cell proliferation, adhesion, invasion, plasticity, apoptosis, DNA repair, and recruitment of tumor-associated cells. Gene expression analysis showed that 3D spheroids are more similar to tumor tissue strains by the expression levels of Gfap, Cd44, and Pten, while the expression levels of Hif1a and Sox2 in 3D spheroids did not differ from those of 2D cell cultures, the expression levels S100b and Vegfa in 3D spheroids was higher than in other models, and the expression levels of Melk, Gdnf, and Mgmt genes changed diversely. Thus, 3D spheroid model more closely mimics the tumor tissue than 2D cell culture, but still is not the most relevant, probably due to too small size of spheroids, which does not allow reproducing hypoxia and apoptotic and necrotic processes in the tumor tissue.

Influence of Sucrose on the Efficiency of Cryopreservation of Human Umbilical Cord-Derived Multipotent Stromal Cells with the Use of Various Penetrating Cryoprotectants.

We studied the influence of sucrose applied in combination with different concentrations of penetrating cryoprotectants (DMSO, ethylene glycol, and glycerol) on the efficiency of cryopreservation of umbilical cord-derived multipotent stromal cells (MSC). The results indicate that these cells can be cryopreserved with the use of 5-10% DMSO or ethylene glycol with equal efficiency; addition of 0.2 M sucrose does not affect cell survival after thawing. The efficiency of glycerol as a cryoprotectant increases with increasing its concentration from 5 to 10%, but remains significantly lower than the efficiency of DMSO or ethylene glycol. Addition of sucrose to a final concentration of 0.2 M increases the efficiency of glycerol. The efficiency of combination of 10% glycerol and sucrose was comparable with that of combinations of DMSO and ethylene glycol with sucrose. The mechanism of the observed enhancement is apparently related to the influence of sucrose on the dynamic properties of the lipid membranes and facilitation of glycerol diffusion into the cells.

Expression of Metalloproteinases and Type I and III Collagens during Healing of Excisional Skin Wound on the Abdomen and Back in Rats.

The study was carried out using a novel rat model developed in our laboratory, namely16 mm diameter circular excisional wounds were generated on the abdomen which resulted in minimal scarring. Restoration of the skin integrity was completed by day 60 after the wounding surgery. By this time, regenerates on the abdomen were stronger than on the back (at, respectively, 58 and 17.4 % of the tensile strength of the intact skin at corresponding location) and the ratio of type I and type III collagens in regenerates on the abdomen reached the level of intact skin at the same location. On days 3 to 14, the ratio of Mmp9/Timp1 expression levels on the abdomen was higher than on the back. On days 20 and 30, the Mmp9/Timp1 ratio on the abdomen was identical to the level of intact skin, whereas the increased MMPs expression levels on the back were maintained until day 30. It has been shown for the first time that according to functional and molecular characteristics, wound healing on the abdomen of an adult rat is more similar to complete regeneration than scarring repair of the back skin.

Analysis of the Expression of Regulator Genes in Kupffer Cells and Monocytes.

Differences in the gene expression profiles in resident macrophages (in particular, Kupffer cells) and monocytes were revealed. However, these differences in gene expression profiles do not allow considering resident liver macrophages as purely M2 macrophages and monocytes as purely M1 macrophages. At the same time, a significant number of the genes upregulated in Kupffer cells are associated with normal regulation of liver functions (Arg 1, Flt, iNOs, and Kng). In monocytes, the expression of genes Alox15, Alox12, Tlr2, Tlr4, Tlr7, and Tlr8 (typical functional genes of macrophages) was also upregulated in comparison with Kupffer cells.

Quantitative and Qualitative Characterization of Phagocytic Activity of Macrophages of Bone Marrow and Fetal Origin.

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.

[The use of blood plasma enriched with platelet growth factors during experimental perforation of the tympanic membrane]. / Primenenie plazmy krovi, obogashchennoi trombotsitarnymi faktorami rosta, pri éksperimental'noi perforatsii barabannoi pereponki.

OBJECTIVE: To study morphofunctional tissue changes during perforation of the tympanic membrane using blood plasma enriched with platelet growth factors in the experiment. MATERIAL AND METHODS: Examined 36 rats (72 tympanic membranes), which were divided into 2 groups, experimental (1st) and control (2nd). Injected blood plasma enriched with platelet-derived growth factors into the external auditory canal once by application after perforation of the tympanic membrane in the rats of the experimental group. Sacrificed rats on the 5th, 10th, 15th day after surgery (6 animals in each group at each stage). Evaluated the condition of the tympanic membrane otoscopically during the experiment. Evaluated the signs of acute otitis media, the presence of 'dry' perforation of the tympanic membrane (without signs of inflammation), and the closure of perforation of the tympanic membrane. RESULTS: In the experimental group, on the 5th day after the surgery, there were no signs of acute otitis media, closure of perforation of the tympanic membrane was identified in 4 cases, 'dry' perforation of the tympanic membrane (without signs of inflammation) was preserved in 8 cases. In the control group, on the 5th day after the surgery, the signs of acute otitis media were identified in 9 cases, cases of closure of the perforation of the tympanic membrane were not noted, 'dry' perforation of the tympanic membrane was preserved in 3 cases. In the 1st group, on the 10th day after surgery, the signs of acute otitis media were identified in 1 case, 'dry' perforation of the tympanic membrane was preserved in 1 case, closure of perforation of the tympanic membrane was identified in 10 cases. At the same time in the 2nd group, the signs of acute otitis media were identified in 6 cases, the 'dry' perforation of the tympanic membrane was preserved in 6 cases, there were no cases of closure of the perforation of the tympanic membrane. In the 1st group, on the 15th day after the surgery, there were no signs of acute otitis media, 'dry' perforation of the tympanic membrane was preserved in 1 case, closure of perforation of the tympanic membrane was identified in 11 cases. In the 2nd group, on the 15th day after surgery the signs of acute otitis media was identified in 1 case, 'dry' perforation of the tympanic membrane was preserved in 10 cases, the closure of perforation of the tympanic membrane was noted in 1 case. CONCLUSION: Preliminary results show a favorable effect of blood plasma enriched with platelet growth factors on the regeneration of the tympanic membrane under conditions of its traumatic perforation in the rat experiment, which is manifested in a decrease in the incidence of acute otitis media on the 5th day after perforation and in an increase in the incidence of closure of tympanic membrane perforation both on the 10th and 15th day, and in general in the experimental group.

Expression of Cytokine Genes and Growth Factors in Rat Lungs and Kidneys after Subtotal Hepatectomy.

Expression of il1b, il6, il10, tnfa, hgf, tgfb, vegf, and fgf2 genes in the lungs and kidneys was examined on rat model of liver regeneration after subtotal hepatectomy. Enhanced expression of il6, il10, tnfa, hgf, and fgf2 genes was detected at the early terms after 80% liver resection.

Reparative regeneration of rat fetal liver after partial hepatectomy.

A reproducible model of fetal liver regeneration was created. Resection of 20% liver was carried out in rat fetuses on day 17 of prenatal development. The organ weight was restored after 2 days at the expense of an increase in hepatocyte mitotic activity; cell hypertrophy was minor. After recovery, the cell composition of the operated liver did not differ from the control, i.e. the regeneration was organotypical.

Dynamics of hepatocyte proliferation in regenerating fetal rat liver.

We studied hepatocyte proliferation in the regenerating liver of 17-day fetuses of outbred albino rats. The animals were sacrificed every 3 h over 2 days after resection of 20% liver. The number of mitoses beyond the zone of injury increased sharply 12 and 24 h after resection. The hepatocyte mitotic index in the perifocal zone did not surpass the mitotic index in regions distant from the focus of injury during any of the studied periods. No circadian rhythm of hepatocyte mitotic activity was detected for resected or intact fetal liver. The injury caused virtually no changes in hepatocyte mitosis phase ratio in the operated compared to intact liver, which attested to stable course of mitosis. The weight of fetal liver recovered at the expense of enhanced mitotic activity of hepatocytes in the entire liver.