Morphological Characteristics of Colon Tumors in Mice with Different Tolerance to Hypoxia.

In adult male C57BL/6 mice with high (HR) and low (LR) resistance to hypoxia, morphological features of colon tumors and blood parameters were evaluated 70 days after intraperitoneal injection of azoxymethane and subsequent consumption of 3 cycles of dextran sulfate sodium. On macroscopic analysis, tumors were found in the distal colon in 35% (7 of 20 animals) of HR and 31% (4 of 13 animals) of LR animals. Microscopic analysis of the distal colon revealed tumors in 75% (15 of 20 animals) of HR and 69% (9 of 13 animals) of LR mice. The tumors were presented by areas of glandular intraepithelial neoplasia and adenocarcinomas; the incidence and the area of the tumors did not differ in groups of HR and LR mice. The number of neuroendocrine and goblet cells in the distal colon mucosa in the areas of tumors was similar in the compared groups. However, in both HR and LR mice of the experimental groups, the content of goblet cells in tumors was lower and the content of endocrine cells was higher than in the corresponding control groups. In the peripheral blood, the erythrocyte count and hemoglobin content decreased in HR and LR mice of the experimental groups; the relative number of monocytes increased only in HR mice and the absolute number of lymphocytes and monocytes decreased in LR mice. Thus, 70 days after azoxymethane administration and dextran sulfate sodium consumption, the tumors in mice were presented by glandular intraepithelial neoplasia and adenocarcinomas, and their incidence and area did not differ between animals with different tolerance to hypoxia.

Morphological and molecular-biological features of glioblastoma progression in tolerant and susceptible to hypoxia Wistar rats.

Hypoxia is a major pathogenetic factor in many cancers. Individual resistance to suboptimal oxygen availability is subject to broad variation and its possible role in tumorigenesis remains underexplored. This study aimed at specific characterization of glioblastoma progression in male tolerant and susceptible to hypoxia Wistar rats. Hypoxia resistance was assessed by gasping time measurement in an 11,500 m altitude-equivalent hypobaric decompression chamber. Based on the outcome, the animals were assigned to three groups termed 'tolerant to hypoxia' (n = 13), 'normal', and 'susceptible to hypoxia' (n = 24). The 'normal' group was excluded from subsequent experiments. One month later, the animals underwent inoculation with rat glioblastoma 101.8 followed by monitoring of survival, body weight dynamics and neurological symptoms. The animals were sacrificed on post-inoculation days 11 (subgroup 1) and 15 (subgroup 2). Relative vessels number, necrosis areas and Ki-67 index were assessed microscopically; tumor volumes were determined by 3D reconstruction from histological images; serum levels of HIF-1α, IL-1ß, and TNFα were determined by ELISA. None of the tolerant to hypoxia animals died of the disease during observation period, cf. 85% survival on day 11 and 55% survival on day 15 in the susceptible group. On day 11, proliferative activity of the tumors in the tolerant animals was higher compared with the susceptible group. On day 15, proliferative activity, necrosis area and volume of the tumors in the tolerant to hypoxia animals were higher compared with the susceptible group. ELISA revealed no dynamics in TNFα levels, elevated levels of IL-1ß in the susceptible animals on day 15 in comparison with day 11 and tolerant ones. Moreover, there were elevated levels of HIF-1α in the tolerant animals on day 15 in comparison with day 11. Thus, the proliferative activity of glioblastoma cells and the content of HIF-1α were higher in tolerant to hypoxia rats, but the mortality associated with the tumor process and IL-1ß level in them were lower than in susceptible animals. Specific features of glioblastoma 101.8 progression in tolerant and susceptible to hypoxia rats, including survival, tumor growth rates and IL-1ß level, can become the basis of new personalized approaches for cancer diseases treatment in accordance to individual hypoxia resistance.

Morphofunctional Changes in the Thymus in Prepubertal Male Wistar Rats in LPS-Induced Systemic Inflammatory Response in Relation to Hypoxia Tolerance.

The dynamics of morphofunctional changes in the thymus during the LPS-induced systemic inflammatory response was assessed in prepubertal male Wistar rats in relationship with the resistance to hypoxia. The systemic inflammatory response was modeled by intraperitoneal administration of E. coli O26:B6 LPS. In histological sections of the thymus, the relative number of thymic bodies and proliferative activity of cells were evaluated. The relative number of CD3+CD4+, CD3+CD8+, and CD4+CD8+ cells in the thymus was determined by flow cytometry. The content of HIF-1α and endotoxin was determined in the blood serum. The expression level of Nfkb mRNA was assessed in the liver. The most pronounced changes in the indicators of the functional state of the thymus were detected 3 and 6 h after LPS administration following the increase in the content of HIF-1α and endotoxin in blood serum and Nfkb mRNA expression in the liver. In the thymus, a decrease in the number of thymic bodies consisting of 3-5 epithelial cells and an increase in the number of bodies consisting of 5 or more cells was observed. In 24 h after LPS administration, the relative number of CD3+CD4+ and CD3+CD8+ cells in the thymus decreased. At the same time, the number of Ki-67+ cells in the subcapsular zone of the thymus increased 6 and 24 h after LPS administration. These data should be taken into account in the development of approaches to the treatment of infectious and inflammatory diseases in prepubertal children.

Age-Specific Features of the Levels of Reference Proteins Actin, Tubulin, and GAPDH in Different Organs of Male Wistar Rats.

Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target protein is normalized to the corresponding signal of the reference protein. The data on the intensity of actin, tubulin, and GAPDH synthesis are fragmentary: their expression differs in different organs and can vary depending on age, which is often not taken into account in experimental studies. We studied the features of the production of reference proteins in the liver, heart, brain, and lungs of newborn, prepubertal, and adult male Wistar rats. Age-related differences in the expression of ß-actin, ß-tubulin, and GAPDH in the myocardium and dorsal prefrontal cortex were revealed. GAPDH expression in the dorsal prefrontal cortex in adult rats was significantly higher than in prepubertal rats; GAPDH expression in the myocardium of adult rats was significantly higher than in newborns. The level of actin in the dorsal prefrontal cortex in newborn rats was significantly higher than in prepubertal and adult rats. In the liver and lungs, the expression of actin, tubulin, and GAPDH did not differ in newborn, prepubertal, and adult rats. When choosing the reference protein for Western blotting, animals age and the studied organ should be taken into account.

Barnase*Barstar-guided two-step targeting approach for drug delivery to tumor cells in vivo.

For precise ligation of a targeting and cytotoxic moiety, the use of Barnase-Barstar pair as a molecular glue is proposed for the first time. Targeting was mediated through the use of a scaffold protein DARPin_9-29 specific for the human epidermal receptor 2 (HER2) antigen that is highly expressed on some types of cancer and Barnase*Barstar native bacterial proteins interacted with each other with Kd 10-14 M. The approach proposed consists of prelabeling a target tumor with hybrid protein DARPin-Barnase prior to administration of cytotoxic component-loaded liposomes that have Barstar covalently attached to their surface. Based on in vivo bioimaging we have proven that DARPin-based Barnase*Barstar-mediated pretargeting possesses precise tumor-targeting capability as well as antitumor activity leading to apparent tumor-growth inhibition of primary tumors and distant metastases in experimental animals. The results obtained indicate that the new system combining DARPin and Barnase*Barstar can be useful both for the drug development and for monitoring the response to treatment in vivo in preclinical studies.

Morphofunctional Changes in Colon after Cold Stress in Male C57BL/6 Mice Susceptible and Tolerant to Hypoxia.

There are individual differences in the tolerance to hypoxia and stress. Stress can contribute to the development of various diseases, including inflammatory bowel disease. It was found that inflammatory bowel diseases in animals susceptible to hypoxia runs more severe course than in tolerant animals. We studied morphofunctional changes in the colon under conditions of modeled cold stress in male C57BL/6 mice susceptible and tolerant to hypoxia. The animals were daily subjected to cold stress (20 min at -20°C) for 2 weeks. Cold stress was followed by an increase in the volume fraction of goblet cells in the colon and production of mucins by these cells in mice tolerant to hypoxia and an increase in cell content in the lamina propria of the colon mucous membrane in animals susceptible to hypoxia. The number of serotoninproducing endocrine cells increased in both groups, but these changes were more pronounced in mice susceptible to hypoxia.

Morphological and Functional Peculiarities of the Immune System of Male and Female Rats with Different Hypoxic Resistance.

The morphological and functional peculiarities of the immune system are studied in adult male and female Wistar rats with high and low hypoxic resistance. Sex-specific differences in the subpopulation composition of the peripheral blood lymphocytes, not depending on the hypoxic resistance of animals, are detected: the males have lower absolute counts of T helpers and higher percentage of regulatory T cells than the females in the diestrus phase. Comparison of the morphofunctional status of the immune system in male and female (diestrus) rats with high resistance to hypoxia has shown a better developed subcapsular zone of the thymus, higher levels of anti-inflammatory cytokine TGF-ß, and lower absolute counts of cytotoxic T lymphocytes in the peripheral blood in the males. Males with low hypoxic resistance have higher counts of phase II thymic bodies in comparison with low-resistant females. Hence, morphofunctional differences in the immune system of male and female rats with different hypoxic resistance are detected, which can determine the course of inflammatory diseases.

Morphological Characteristic of Melanoma B16 Progression in C57BL/6 Mice with High and Low Resistance to Hypoxia.

The features of B16 melanoma progression in male C57BL/6 mice with initially high and low resistance to hypoxia were studied. To assess the resistance to hypoxia, the mice were placed in a low-pressure chamber at a simulated altitude of 10,000 m. One month after testing, B16 melanoma was inoculated to high- and low-resistant animals. In 19 days after melanoma transplantation, the severity of melanoma progression was assessed by morphological and immunofluorescent methods. The expression of vegf-a and hif-1a in the liver of melanomabearing and control mice was evaluated by real-time PCR. Tumor growth progression was more pronounced in low-resistant mice, which was seen from high weight of the primary tumor node, relative necrosis area, proliferation rates (mitotic index and number of Ki-67+ cells), and expression of vegf-a gene in the liver. In high-resistant to hypoxia animals, the number of caspase-3+ cells dying by apoptosis was higher. The data on more rapid melanoma progression in mice with low resistance to hypoxia should be considered during the search of new prognostic markers and methods for therapy of malignant neoplasms.

Expression of Mucins and Claudins in the Colon during Acute and Chronic Experimental Colitis.

We studied changes in the expression of mRNA for mucins and claudins in the medial part of the colon in male C57Bl/6 mice on the model of acute and chronic colitis induced by substitution of drinking water with 1% solution of dextran sodium sulphate for 5 days. In acute colitis, the expression of the main structural component of glycocalyx, mucin Muc3, decreased and expression of pore-forming claudin Cldn2 increased, which reflected enhanced permeability of tight junctions. In the chronic colitis group, in comparison with the normal group, we observed an increase in expression of mRNA of main structural mucus component Muc2, enhanced of expression of Muc1 associated with carcinogenesis, and reduced expression of Muc13, which led to a more severe course of colitis; the expression of pore-forming claudin Cldn2 was elevated. These findings indicate that the imbalance in the expression of mucins and claudins plays an important role in the mechanisms of development of acute and chronic colitis.

Morphological Changes in Mesenteric Lymph Nodes and Lymphocyte Subpopulation Composition in Experimental Ulcerative Colitis.

Morphological changes in the mesenteric lymph nodes of male C57Bl/6 mice and subpopulation composition of lymphocytes in these nodes were studied in experimental acute and chronic ulcerative colitis induced by sodium dextran sulfate. Acute and chronic ulcerative colitis was associated with the development of reactive changes in the mesenteric lymph nodes. These changes were of mixed type and were characterized by follicular hyperplasia and sinus reaction. The content of CD19(+) B cells in the mesenteric lymph nodes decreased in acute ulcerative colitis, while the content of CD3(+)CD8(+) cytotoxic T cells increased, which presumably reflected activation of Th1 reactions. The increase in the count of CD4(+)CD25(+)FOXP3(+) regulatory T cells and CD3(+)CD8(+) cytotoxic T cells was due to intensive migration of lymphocytes from the thymus and the colonic compartment of the local immune system. Chronic ulcerative colitis was associated with higher levels of CD19(+) B cells and CD3(+)CD4(+) T helper cells in the mesenteric lymph nodes, which was characteristic of adoptive immunity reactions and chronization of the inflammatory process.

[Colonic morphological changes in experimental chronic colitis induced by dextran sulfate sodium].

OBJECTIVE: To characterize morphological changes in the colonic wall in experimental chronic colitis induced by dextran sulfate sodium (DSS). MATERIAL AND METHODS: Experiments were carried out on sexually mature male C57B1/6 mice. Drinking water in the animal dishes was replaced by 2% DSS solution for 6 days and the animals were taken away from the experiment on day 21 after initiation of DSS consumption. Hematoxylin- and eosin-stained colonic histological specimens were prepared by Masson's trichrome and Nissl methods. RESULTS: Histologically, the colonic wall displayed epithelialized ulcers; mucosal remodeling; reductions in the count of goblet cells, crypts and their depth; mucosal inflammatory infiltration with lymphocytes, plasmocytes, and histiocytes; lymphoid tissue hyperplasia; diffuse and focal sclerosis of all colonic membranes; neuronal destructive changes in the myenteric and submucosal nerve plexuses. CONCLUSION: The experimental model of DSS-induced chronic colitis corresponds to human colitis in morphological characteristics to a certain degree and may be used to study of the pathogenesis of this disease and to preclinically evaluate of the efficacy of medications.