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.

Comparative Molecular and Biological Characteristic of the Systemic Inflammatory Response in Adult and Old Male Wistar Rats with Different Resistance to Hypoxia.

Morphological, molecular, and biological features of the systemic inflammatory response induced by LPS administration were assessed in adult and old male Wistar rats with high and low resistance to hypoxia. In 6 h after LPS administration, mRNA expression levels of Hif1a, Vegf, Nfkb, and level of IL-1ß protein in old rats were higher than in adult rats regardless of hypoxia tolerance. The morphometric study showed that the number of neutrophils in the interalveolar septa of the lungs was significantly higher in low-resistant adult and old rats 6 h after LPS administration. Thus, in old male Wistar rats, systemic inflammatory response is more pronounced than in adult rats and depends on the initial tolerance to hypoxia, which should be considered when developing new approaches to the therapy of systemic inflammatory response in individuals of different ages.

Morphofunctional Characteristics of Lung Macrophages in Rats with Acute Respiratory Distress Syndrome.

A comprehensive morphofunctional study of the lungs and alveolar macrophages was carried out in Sprague-Dawley rats with acute respiratory distress syndrome (n=10) induced by intratracheal administration of E. coli LPS 0111:B4 in a dose of 15 mg/kg. On the first day after LPS administration, bronchopneumonia was observed in the lungs, the number of macrophages of the bone marrow origin and the number of M1 macrophages with the proinflammatory phenotype in the bronchoalveolar lavage increased, the expression of proinflammatory cytokines increased and the expression of anti-inflammatory cytokines decreased, which was accompanied by an increase in LPS and C-reactive protein in the blood serum. The revealed changes correspond to the development of acute respiratory distress syndrome in humans, and the decrease in the number of macrophages in the lungs and their predominant polarization to the M1-proinflammatory phenotype substantiate the use of cell therapy with reprogrammed M2 macrophages.

Morphological and Molecular Biological Features of the Systemic Inflammatory Response in Old Wistar Rats with High and Low Resistance to Hypoxia.

Morphological and molecular biological features of LPS-induced systemic inflammatory response were assessed in old male Wistar rats with high (HR) and low (LR) resistance to hypoxia. The systemic inflammatory response was modeled by intraperitoneal injection of E. coli O26:B6 LPS; the animals were sacrificed after 6 h. In histological sections, the number of neutrophils in the interalveolar septa and the area of necrosis in the liver were determined. The expression levels of Hif1a, Hif2a, Nfkb, Vegf, Il1b, Il6, Il10, and Tgfb mRNA in the liver and serum concentrations of HIF-1α and IL-1ß were determined. In 4-6 h after LPS injection, 3 (43%) of 7 HR rats died, whereas no deaths were observed among LR rats. At 6 h after LPS injection, the number of neutrophils in the interalveolar septa of the lungs in LR rats was significantly higher than in HR rats, while the area of necrosis in the liver did not differ. At the same time, the mRNA expression levels of the proinflammatory cytokine genes Il1b and Il6 increased in the liver of both HR and LR rats, whereas the expression of Il10 increased only in HR rats. The expression of the Hif1a gene in the liver was higher in LR rats, but the content of HIF-1α protein in blood serum was higher in HR animals. These data should be taken into account when developing new approaches to the therapy of systemic inflammatory response in infectious and inflammatory diseases in the elderly.

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.

Experience of Using Tripeptide Leu-Ile-Lys for Experimental Therapy of Chronic 16-Week Oxalate Nephrolithiasis in Rats.

We studied the effect of tripeptide Leu-Ile-Lys on the course of chronic 16-week oxalate nephrolithiasis in rats modeled by administration of 1% ethylene glycol solution in drinking water for 16 weeks. The tripeptide Leu-Ile-Lys obtained by chemical synthesis (sample purity ≥98%) was administered intragastrically through a probe in a dose of 11.5 mg/kg in 1 ml saline. It was found that during tripeptide Leu-Ile-Lys significantly alleviated the course of experimental pathology, which was confirmed by characteristic biochemical and morphological indicators. We observed a decrease in the concentration of calcium ions by 4.4 times, weakening of oxidative stress in the renal tissue due to a decrease in the total prooxidant activity by 1.2 times, normalization of increased catalase activity, and reduction of superoxide dismutase activity by 2.4 times relative to disease control. Histological signs of nephrolithiasis were recorded in 9% cases (vs. 75% cases in disease control).

Morphological and Biochemical Characteristics of Prostate Hyperplasia during Sulpiride Treatment.

We studied morphological changes in the prostate ventral lobe, proliferative activity of the epithelium in prostate acini, and the levels of prolactin and prostate-specific antigen in the blood serum of Sprague-Dawley rats after repeated injections of sulpiride in a dose of 40 mg/ kg over 30 and 60 days and in 10 and 30 days after withdrawal. Morphological and morphometrical analysis of hyperplastic changes in the prostate ventral lobe was performed. Ki-67+ proliferating epithelial cells in the acini were counted. The dynamics of serum concentrations of prolactin and prostate-specific antigen was evaluated by ELISA. Morphological and morphometrical analysis and evaluation of the content of Ki-67+ cells demonstrated epithelium hyperplasia in the prostate ventral lobe after sulpiride treatment for 30 or 60 days and in 10 days after withdrawal, but serum level of prostate-specific antigen did not differ from the control. After 60-day sulpiride treatment and in 30 days after withdrawal, pronounced hyperplastic changes of prostate and elevated concentrations of prostate-specific antigen (but not prolactin) were observed. Thus, administration of sulpiride (40 mg/kg) to Sprague-Dawley rats for 60 days allows, by morphological criteria and serum level of prostate-specific antigen, to model stable hyperplastic changes in the prostate corresponding to benign prostatic hyperplasia in humans.

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.

[Morphology of the thymus and the specific features of its cellular composition in experimental acute and chronic ulcerative colitis]. / Morfologiia timusa i osobennosti ego kletochnogo sostava pri éksperimental'nom ostrom i khronicheskom iazvennom kolite.

OBJECTIVE: To investigate morphological changes in the thymus, the subpopulation composition of lymphocytes and its non-lymphoid cells in dextran-induced experimental acute ulcerative colitis and in different periods of chronic ulcerative colitis. MATERIAL AND METHODS: Acute and chronic ulcerative colitis was simulated in C57BL/6 mice, by replacing drinking water with a 1% aqueous dextran sulfate sodium solution. Thymic changes were morphometrically assessed; the number and absolute area of thymic corpuscles and epithelial cells were calculated; and the subpopulation composition of lymphocytes and thymic stromal cells was determined using flow cytofluorimetry; the Kruskal-Wallis test and the Mann-Whitney test were used to compare the groups. RESULTS: In acute catarrhal and ulcerative colitis, there was acute accidental thymic involution with devastation of the cortical substance and with a decline in its volume fraction, with an increase in the levels of cells dying through the mechanism of apoptosis, and with a decrease in the absolute number of lymphocytes, T-helper cells, cytotoxic T-cells, regulatory T-lymphocytes, B-lymphocytes, and dendritic cells, with a rise in the index of the area of thymic corpuscles and in the content of late-phase corpuscles among them, and with the appearance of thymic corpuscles as cyst-like cavities. In chronic ulcerative colitis, the cortex was expanded and the area of thymic corpuscles and the count of medullary epithelial cells increased. The cyst-like thymic corpuscles formed clusters, the count of dendritic cells increased in early-stage chronic ulcerative colitis, but the levels of macrophages decreased in both periods of its development. CONCLUSION: There is acute accidental involution and thymic hyperplasia with an increase in medullary epithelial cells and thymic corpuscles consisting of cytokeratin 19+ in the epithelial cells in experimental acute and chronic ulcerative colitis, respectively. The more pronounced epithelial cell response found in end-stage experimental chronic ulcerative colitis reflects the enhanced differentiation of regulatory T-lymphocytes and the larger number of which is observed in peripheral blood and in the focus of inflammation in patients with ulcerative colitis, according to the literature.

Expression of Hif-1α, Nf-κb, and Vegf Genes in the Liver and Blood Serum Levels of HIF-1α, Erythropoietin, VEGF, TGF-ß, 8-Isoprostane, and Corticosterone in Wistar Rats with High and Low Resistance to Hypoxia.

We studied the expression of Hif-1α, Nf-κb, and Vegf genes in the liver and serum levels of HIF-1α, erythropoietin, VEGF, TGF-ß, 8-isoprostane, and corticosterone in Wistar rats with different resistance to hypoxia in 5 and 90 min after acute exposure to hypobaric hypoxia. In 5 min after hypoxic exposure, Hif-1α expression in the liver and serum levels of erythropoietin, VEGF, and TGF-ß in high-resistant rats were higher than in low-resistant animals. In highresistant rats, the increment in expression of Nf-κb gene responsible for the control over the inflammatory processes was more pronounced than in low-resistant animals. In 90 min after hypoxic exposure, the serum levels of HIF-1α, erythropoietin, VEGF, and TGF-ß returned to normal in high-resistant rats, while in low-resistant animals, an increase in 8-isoprostane and TGF-ß concentrations was observed. The rats with different resistance to hypoxia were characterized by different changes in biomolecular parameters determining predilection to inflammatory diseases.

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.

URINARY MALONDIALDEHYDE AS A PREDICTIVE AND DIAGNOSTIC MARKER FOR NEONATAL ACUTE KIDNEY INJURY.

Newborns are especially prone to oxidative stress (OS). Many free radical-mediated diseases have been described in newborns including bronchopulmonary dysplasia, respiratory distress syndrome, encephalopathy, and kidney injury. Objective of this work was to determine predictive and diagnostic value of urinary malondialdehyde (UMDA) as a marker for acute kidney injury (AKI) in critically sick full-term newborns. 67 critically sick full-term neonates were enrolled in the study including 31 newborns with AKI (group I) and 36 newborns without AKI (group II). The control group included 40 healthy full-term neonates (group III). The level of UMDA was measured by means of the test based on the reaction of MDA to thiobarbituric acid. The mean and 95% confidence interval (CI) for UMDA was 12.7 (11.5; 13.9) µmol/l in group I, 10.2 (9.61; 10.8) µmol/l in group II, 9.01 (8.16; 9.93) µmol/l in group III (pI-II<0.05; pI-III<0.05; pII-III<0.05). ROC curve analysis showed that UMDA had AUC 0.80 (95% CI 0.68; 0.93, p=0.0014) for AKI. The optimum UMDA cut-point was 12.0µmol/l. For AKI sensitivity and specificity of UMDA were determined to be 68.2% (95% CI 45.1; 86.1%) and 85.7% (95% CI 69.7; 95.1%) respectively, with positive predictive value of 75.0% (95% CI 55.9; 87.6%), and negative predictive value of 81.1% (95% CI 69.7; 88.9%), positive likelihood ratio of 4.77 (95% CI 2.02; 11.3), and negative likelihood ratio of 0.37 (95% CI 0.20; 0.69). This data support studies to evaluate UMDA as an immediate biomarker for AKI in critically sick newborns. However, a larger study should be conducted to assess the diagnostic accuracy of other serum and urinary markers for OS and renal dysfunction, which would enable us to formulate a mathematical model for the prognosis and diagnosis of AKI in newborns.

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.

Antiproliferative Potential of Officinal Forms and Nanoparticles of Lithium Salts.

We studied the effect of officinal forms and nanoparticles of lithium carbonate and lithium citrate on proliferative activity of hepatoma-29 cells. Lithium carbonate nanoparticles suppressed proliferation of hepatoma-29 cells in lower concentrations than officinal form of this salt. The antiproliferative effect of lithium salts i activation of apoptosis and arrest of hepatoma-29 cells in the G2/M phase of the cell cycle.

Morphological Criteria of Cell Differentiation Stages in Experimental Hepatocarcinoma and Evaluation of Antitumor Drug Efficiency.

Structural polymorphism of 5 cell differentiation stages of hepatocarcinoma-29 from ascitic fluid is detected and the morphological criteria for identification of these stages are defined on the base of optic and electron microscopy findings, cytofluorometry, and DNA cytometry. The percentage of cells at differentiation stages 4 and 5 in the tumor structure increases after hepatocarcinoma cell inoculation into the hip. Injection of a cell cycle-modulating substance to animals with tumor growth shifts the proportion of cells with various differentiation stages. The morphological criteria of 5 stages of hepatocarcinoma-29 cell differentiation can be used for prospective drug testing.

[Effects of lithium carbonate nanosized particles on oxidant-antioxidant status in tumor tissue of hepatocarcinoma-29].

Oxidant-antioxidant status in tumor tissue of male-mice CBA at spontaneous course of hepatocarcinoma-29 and after repeated injections of lithium carbonate nanosized particles was evaluated on changes of lipid peroxidation (LPO) products level reacted with 2-thiobarbituric acid (TBA) as indicator of oxidative stress and activity of superoxide dismutase (SOD) and catalase enzymes as indicators of antioxidant defense by spectrophotometer <> (Bio-Rad, USA). Tumor development after hepatocarcinoma-29 cells injection into muscle right leg changed the levels of LPO activity in two-phase manner. TBA-active products content were decreased in 2,4 times in comparison with the control indicates after invasion of tumor cells, it was raised in 2,1 times at excessive tumor growth and diminished at terminal stage. Catalase activity was significantly elevated, but SOD activity was reduced in tumor tissue samples at active growth of hepatocarcinoma. The repeated injections of lithium carbonate nanosized particles at hepatocarcinoma inhibited processes of lipid peroxidation in tumor tissue in 2,4 times, but didn't influence on activities of catalase and SOD. Thus the effects lithium carbonate nanosized particles injections referred on maintenance of balance between the oxidant and antioxidants may be of some help to limit the progression of precancerous condition toward malignancy and tumor growth.

Effects of Nanosized Lithium Carbonate Particles on the Functional Activity of Macrophages During Development of Hepatocarcinoma 29.

The functional activity of macrophages in response to injection of nanosized lithium carbonate particles after initiation of hepatocarcinoma 29 in male CBA mice was evaluated by the production of NO, arginase activity, and absorption of zymosan granules. In intact animals, NO production by peritoneal macrophages increased by 4 times and arginase activity 3.1 times in response to a single injection of nanosized particles into the hip muscle. The level of NO production by macrophages remained high after 4 and 5 injections, while arginase activity returned to normal. The level of phagocytic peritoneal macrophages increased by 1.4 times after 5 injections of the particles. The level of NO production by macrophages gradually increased in animals with hepatocarcinoma developing in the hip muscle: by 1.6 times on day 3, 3.2 times on day 7, and by 2.6 times on day 13 in comparison with the corresponding parameters in intact animals. The increase of NO production by peritoneal macrophages after tumor process initiation was not paralleled by changes in arginase activity and absorption of zymosan granules. The results indicated that injection of nanosized lithium carbonate particles after inoculation of hepatocarcinoma 29 cells in the right hip muscle tissue was inessential for the function of peritoneal macrophages by the studied parameters.

[Effects of lithium carbonate nanosized particles on nitric oxide production and arginase activity in tumor and peritoneal macrophages in hepatocellular carcinoma 29].

Three groups of male CBA mice were used. Group 1 consisted of intact mice. Hepatocarcinoma cells 29 (HCC-29) were transplanted into the right thigh muscle of animals group 2. Group 3 mice were injected 0.1 ml of lithium carbonate nanosized particles (NPs Li2CO3) at a dose of 0,058 mg on periphery of tumor growth one fold (3 day) and 5-fold (7 and 13 days). On day 7, numerical density of macrophages was raised in 5.8 times, the NO levels were increased by 1.9 times, and arginase activity was decreased in HCC tissue as compared with those values in normal liver. Tumor growth led to an increase in NO production by peritoneal macrophages (pMf) in 2.8 and 2.2-fold on day 7 and 13 days, respectively, and hadn't effect on arginase activity. A single injection of NPs Li2CO3 after inoculation tumor cells (3 days) didn't alter the NO levels rise in the tumor but five injections (7 days) increased it in 1.7 times as compared with values of mice group 2. The treatment of NPs Li2CO3 influenced the increasing the number density of macrophages in the tumor. Numerical density of macrophages in the tumor of mice group 3 was increased 9.6 and 1.6 times as compared with similar values in groups 1 and 2, respectively on 7 day after 5 injections of NPs Li2CO3. Treatment of NPs Li2CO3 after tumor cell transplantation didn't affect on the rise of NO levels and arginase activity in pMf. Thus, the effects of NPs Li2CO3, administered after HCC-29 cells transplantation, aimed at increasing activity of the NO-synthase way in HCC tumors and pMf, which can reduce the arginine levels required for tumor growth.