Computer Morphometry Research Articles

Синдром избыточного бактериального роста у больных с хроническими диффузными заболеваниями печени в зависимости от этиологии и морфологических особенностей

Актуальность. Ряд исследований указывают на повышение риска появления нарушений со стороны печени при дисбиотических нарушениях в кишечнике. В свою очередь, изменение физиологических процессов в печени может стать толчком для возникновения синдрома избыточного бактериального роста (СИБР). Доказано, что СИБР в тонком кишечнике может играть определенную роль в патогенезе алкогольной болезни печени и осложнений, связанных с хроническим гепатитом С. Целью исследования было оценить частоту возникновения синдрома избыточного бактериального роста у больных хроническими диффузными заболеваниями печени (ХДЗП) с учетом степени стеатоза, стадии фиброза и степени активности воспалительного процесса в печени. Материалы и методы. 27 обследованных пациентов были распределены на 2 группы: I — 12 больных с хроническим гепатитом, ассоциированным с вирусом С (ХГС); II — 15 больных с алкогольной болезнью печени (АБП) в зависимости от этиологии при формировании и прогрессировании стеатоза и фиброза печени с использованием данных гистологических методов исследования, компьютерной морфометрии, транзиентной эластометрии и показателя степени стеатоза САР на аппарате FibroScan 502. Синдром избыточного бактериального роста определяли с помощью водородного дыхательного теста (ВДТ) с глюкозой. Результаты. СИБР был выявлен у 51,8 % больных с ХДЗП, достоверно почти в 1,5 раза преобладал у больных с АБП (60,0 %) по сравнению с ХГС, у которых только в 41,7 % случаев выявлен СИБР. Выявлено превалирование положительных случаев ВДТ в группе S3 (70,0 %), что достоверно отличалось от группы S1 (37,5 %). У больных ХГС с прогрессированием стеатоза увеличивается количество случаев СИБР, и наоборот, у большинства больных с АБП, у которых был выявлен гистологически подтвержденный микровезикулярный и мелкокапельный стеатоз печени, выявляли при ВДТ нарушения микробиоты в тонком кишечнике. Выводы. Между показателями водородного дыхательного теста и индексом гистологической активности, гистологическими показателями прогрессирования стеатоза печени у больных хроническим гепатитом С выявлена четкая взаимо­связь: с увеличением активности воспалительного процесса (9 баллов) и степени стеатоза печени (S3) повышается уровень нарушения микробиоты в тонком кишечнике (> 20 ppm), то есть при ХГС уровень выделения водорода адекватно отображает степень активности воспалительного процесса в печени.

Blood and cell infiltrate neutrophilic leucocytes As inflammation markers in chronic endometritis: A prospective non-randomised controlled trial

Background. Inflammation declares itself with the presence of cellular tissue infiltrate, which composition reflects the inflammation type. Chronic inflammation is predominated by mononuclear cell infiltration with a certain amount of neutrophils, which role and significance are not fully understood to date.Objectives. Assessment of the infiltrated neutrophil count at various chronic endometritis severity and its dependency on the functional and metabolic activity in neutrophilic leucocytes in peripheral blood.Methods. This prospective non-randomised controlled trial estimated the CD45+ leucocyte and activated CD16b+ neutrophil counts in inflammation infiltrate using immunohistochemistry protocols. Cell counts per section 1 mm2 were measured with computer morphometry. The content of and NADPH oxidase activity in activated neutrophilic leucocytes in venous blood were estimated with a nitroblue tetrazolium reduction test.Results. The study included 40 women with a history of chronic endometritis (CE) divided in two cohorts by endometrial biopsy data, with inactive (n = 25) and active CE (n = 15). A control cohort comprised 20 women with no signs of CE. The inactive CE cohort had higher counts of CD45+ leucocytes and activated CD16b+ neutrophils in infiltrate compared to control. Higher content of activated neutrophilic leucocytes with higher NADPH oxidase activity were found in peripheral blood. Morphological exacerbation markers of EC were associated with sharper peaks of CD45+ and CD16b+ cell counts in infiltrate and an elevated functional metabolic activity in circulating neutrophilic leucocytes. A strong direct correlation was revealed between blood activated neutrophil and endometrial CD16b+ neutrophil counts, as well as NADPH oxidase activity in blood neutrophils and infiltrate CD16b+ cell counts.Conclusion. Even minor morphological markers of exacerbated endometrial inflammation are accompanied by the elevated infiltrate counts of both total CD45+ leucocytes and activated CD16b+ neutrophils. The functional metabolic activity of peripheral blood neutrophilic leucocytes is interlinked with the inflammatory infiltrate cell composition and reflects severity of chronic endometrial inflammation.

ДИАГНОСТИЧЕСКАЯ РОЛЬ МОРФОМЕТРИЧЕСКОЙ ОЦЕНКИ КОМПОНЕНТОВ ОПУХОЛЬ-АССОЦИИРОВАННОЙ СТРОМЫ СЕРОЗНЫХ КАРЦИНОМ ПРИДАТКОВ МАТКИ В ПРОГНОЗЕ ОПУХОЛЕВОЙ ПРОГРЕССИИ

The method of computer morphometry of stroma components and evaluation of stromal-parenchymal relationships in the primary tumor node of serous ovarian cancer was used. A high potential for malignancy of tumor tissue, characterized by the resumption of tumor growth after radical treatment, is observed with the predominance of the epithelial component over the stroma, with a small number of necrosis in the tumor node, low density of microcirculatory vessels in the tumor, and the predominance of fi broblastic elements in the cellular microenvironment of the tumorassociated stroma.

Ligature induced periodontitis causes atherosclerosis in rat descending aorta: an experimental study.

IntroductionIn recent years a link between periodontitis and cardiovascular diseases has been proposed. Oxidative stress has been found to be involved in their pathophysiology.ObjectivesThe aim of this study was to demonstrate that a wire ligature placed around the first mandibular molars of the rat causes inflammation in the periodontium and lipid deposits in the aorta, via oxidative stress.MethodsTwenty male Wistar rats were divided randomly into two groups of 10: Control group and Periodontitis group. An orthodontic wire ligature was placed around the cervix of the first mandibular molar and was kept in place for 4 weeks. Clinical attachment loss was determined to assess the gingival recession. Aorta was harvested after 4 weeks and the malondialdehyde (MDA) levels in the aorta tissue homogenate were determined. Lipid deposits were evaluated using Oil Red staining and immunofluorescence under confocal microscopy. Total lipid deposits in the aorta were assessed using a computer morphometry system.ResultsSignificant gingival recession was observed in the Periodontitis group in comparison with the Control group. Increased MDA levels were present in the aorta in the ligatured rats in comparison with the Control group. Significant lipid deposits were observed in the aorta of the Periodontitis group as opposed to the Control group.ConclusionsThe affixing of an orthodontic wire ligature produces gingival recession, increased oxidative stress and lipid deposits in the rat descending aorta.

STRUCTURAL CHANGES OF THE ORAL MUCOSA IN RATS WITH INSULIN RESISTANCE, IODINE DEFICIENCY AND UNDER CONDITIONS OF THEIR COMBINATION

Relevance. The relationship between endocrinopathies and the condition of the oral cavity is associated with disorders of metabolism, hemodynamics, immunological and neuroregulatory changes. Since the first line of contact with the environment of the oral cavity is the mucous membrane, changes in its properties adversely affect the functional state of other periodontal tissues.
 Objective: to examine the structural changes of the oral mucosa in rats with combined endocrinopathy.
 Materials and methods. The studies were carried on 120 rats, which were divided into three experimental (animals with iodine deficiency – ID, insulin resistance – IR, IR on the background of ID) and a control group of animals. Histological examination of the oral mucosa, computer morphometry, and densitometry of objects was performed.
 Results. In rats with ID, the epithelium of the mucous membrane was characterized by the development of acanthosis, expansion of the granular layer, enhanced keratin formation. The development of IR caused hyperplastic changes in the epithelium with the formation of unexpressed cords of acanthosis, epithelial basal hypercellular infiltration, which was confirmed by morphometric data. Under conditions of combined endocrinopathy, more pronounced changes in the histological structure of oral mucosa were observed. In particular, the development of mucosal edema in connective tissue, with predominantly macrophage hypercellularity at the periphery, was noted. Densitometric examination of the samples indicated a decrease in the optical density of connective tissue. At the same time, hyperplastic changes in most layers of the epithelium and pronounced changes in the vascular wall were observed.
 Conclusions. Combined endocrinopathy leads to pronounced changes of the oral mucosa, which can lead to disruption of the structure of periodontal tissues and the entire tooth-alveolar complex.

A role of endothelial dysfunction in development of maternal, perinatal and pediatric morbidity

Aim : to analyze an effect of endothelial dysfunction on development of obstetric, perinatal and pediatric pathology by using morphometric studies of peripheral blood desquamated endothelial cells (DECs). Materials and Methods . A prospective cohort clinical study included 163 female patients with singleton pregnancy. All pregnant women in the first trimester underwent DECs computer morphometry together with clinical and statistical examining course of pregnancy, labor as well as follow-up observation of born children under the age of 1 year. Results . The study demonstrated that endothelial damage in the first trimester of pregnancy profoundly affected maternal, perinatal and pediatric morbidity: detection of DECs in the first trimester with mean diameter less than 30 mkm increased probability of developing placental insufficiency by 52.0 %, preeclampsia - by 48.8 %, threatened preterm labor - by 32.4 %, premature detachment of the normally situated placenta - by 6.0 %. Identifying in DECs in the first trimester with mean diameter less than 40 mkm was associated with increased risk of acute fetal hypoxia in labor by 7.9 %, pediatric central nervous system disorders - by 37.9 %, increased rate of acute respiratory viral infections in children under 1 - by 17.3 %. Conclusion . Examining state of vascular endothelium in the first trimester of pregnancy represents an important component of prenatal diagnostics and opens up new avenue for prevention and treatment of obstetric, perinatal and pediatric pathology.

Morphological and immunohistochemical predictors of renal response to therapy patients with myeloma cast nephropathy and dialysis-dependent acute kidney injury

Reveal morphological and immunohistochemical predictors of reversibility of dialysis-dependent acute kidney injury (AKI) in patients with myeloma cast nephropathy (MCN) based on the study of kidney biopsy. Renal pathological findings were studied in 36 patients with MCN and dialysis-dependent stage 3 AKI (AKIN, 2012). The study of biopsy samples was performed by a semi-quantitative and quantitative analysis using computer morphometry. The expression of E-cadherin, vimentin and-smooth muscle actin was determined immunohistochemically in the tubular cells and interstitium. Induction therapy for 26 patients was carried out to bortezomib-based programs; in 10 patients other schemes were used. A comparative analysis of morphological changes in nephrobiopathy depending on the renal response was performed in patients with achieved hematologic remission. Improved renal function was observed only in patients with hematologic response to therapy. There were no differences in the number of sclerotic glomeruli, protein casts, the area of inflammatory interstitial infiltration, and the degree of acute tubular damage in patients with and without renal response. In patients with renal response compared with patients without improving renal function, the area of interstitial fibrosis was less (24.9% and 45.9%, respectively;p=0.001), and the area of E-cadherin expression was larger (15.9% and 7.1%, respectively;p=0.006). Interstitial fibrosis of 40% or more and/or the area of expression of E-cadherin less than 10% of the area of tubulo-interstitium have an unfavorable prognostic value in achieving a renal response in MCN. If the interstitial fibrosis area is 40% or more and the expression area of E-cadherin is less than 10%, the probability of the absence of a renal response is 93.3% (OR=24.5) even when a hematological response to induction therapy is achieved. The number of protein casts, the prevalence of acute tubular damage and inflammatory interstitial infiltration have not prognostic value.

Dry Eye Epidemiology in Patients before Cataract Surgery

Purpose. To evaluate the prevalence, clinical and pathogenetic variants and statistically significant risk factors of dry eye (DE) in patients before cataract surgery. Patients and Methods. 600 age-related and complicated cataract patients (70.6 ± 7.8 years old; 269 men, 331 women) were examined. Visometry and biomicroscopy with photographic recording of the lens state and assessment of its opacities according to the LOCS III classification were used to cataract revealing. OSDI testing, lipid interferential test, TBUT, Shirmer-1, -2 tests, visual and OCT meniscometry, evaluation of epitheliopaty and microerosion (with vital staining), compression Norn test in Korb modification, visual and OCT LIPCOF assessment, lid viper epitheliopathy evaluation, anterior segment of the eye photoregistration with computer morphometry were performed to assess the ocular surface condition. The structure of the DE risk factors, was studied both in DE patients and non-DE subjects with calculation of the Pearson xi-square test. To estimate the strength of the connection between the etiological condition and the DE, the normalized value of the Pearson coefficient (C´) was used. Results. The dry eye prevalence was 53.2 % (of them, 25.5 % was mild, 27.7 % — moderate, by Brzhesky). Subclinical DE prevalence was 27 %. Clinical and pathogenetic variants of mild DE included: isolated lipid deficiency (71.9 %), lipid-mucin deficiency (28.1 %). In patients with moderate DE were identified: aqueous-lipid-mucin deficiency (54.8 %), aqueous-lipid deficiency (37.95 %), isolated aqueous deficiency (7.2 %). The most significant systemic-organ DE risk factors were: diabetes mellitus (C´ = 0.302; in 18.8 % DE patients), female sex (C´ = 0.240; in 62.1 % de patients), allergy (С´ = 0.233; in 23.2 % DE patients). Local risk factors most significantly associated with DE were: meibomian glands dysfunction (С´ = 0.58; in 77.7 % DE patients), chronic blepharitis (С´ = 0.233; in 23.2 % DE patients), pterygium (С´ = 0.276; in 13.2 % DE patients), allergic conjunctivitis (С´ = 0.21; in 21.3 % DE patients). Among the exogenous risk factors, the most significant were: the use of medication affecting tear production or tear film stability (С´ = 0.485; in 89.03 % DE patients), preservative eye drops instillation (С´ = 0.2975; in 56.1 % DE patients). Conclusion. In our opinion, data on the high DE prevalence in cataract patients (53.2 %) should be taken into account when planning cataract surgery, since perioperative correction of the ocular surface condition can increase the surgical treatment efficiency, predictability and safety. The data on the structure of significant risk factors and clinical and pathogenetic DE variants in cataract patients can be the basis for the development of measures to ensure timely DE detection and implementation of reasonable therapy. The risk factor most significantly associated with DE in cataract patients was the meibomian gland dysfunction (MGD) (Pearson xi-square test = 88.542, p < 0.001, С´ = 0.58; “relatively strong” strength). Considering the modifiability of this risk factor (MGD), as well as its high prevalence (77.7 %), it can be considered that eye lids hygiene may be a reasonable therapy in most DE and cataract patients before phacoemulsification.

Immunohistochemical profile of mononuclear infiltrate in the myocardium of transplanted heart. Computer morphometry data

Aim. To carry out a quantitative immunophenotypic characterisation of cellular corporations in a mononuclear inflammatory myocardial infiltrate in the cell and humoral forms of heart transplant rejection using the computer morphometry of endomyocardial biopsy samples.Materials and methods. Endomyocardial biopsy samples (n = 226) were obtained from 56 heart recipients who underwent transplantation in 2018–2019. Sections with a thickness of 5 μm after the paraffin infiltration were stained with hematoxylin and eosin according to the standard procedure. The expression of CD3 T-lymphocyte, CD20 B-lymphocyte and CD68 macrophage markers was determined by the immunohistochemical streptavidin-biotin method. Using computer morphometry, the staining area coefficient (SAC) was calculated as the percentage of the total area of the stained objects to the area of the biopsy. The statistical processing of the results included verification of the distribution nature by the Kolmogorov-Smirnov method and the calculation of the Cramer — Welch criterion.Results. Lymphocytes and macrophages were found in the inflammatory infiltrate of all heart transplants. The expression of CD3 T-lymphocyte marker in the absence of rejection (0R) was at SAC = 0.99 ± 0.02%. In comparison with 0R cases, this coefficient increased 2.1 times (p <0.05), 3.4 times (p <0.05), 5.5 times (p <0.05) and 4.8 times (p <0.05) in 1R, 2R, 3R and humoral rejection, respectively. The expression of the CD20 B-lymphocyte marker in 0R cases was characterised by SAC = 0.19 ± 0.01%. In comparison with 0R cases, this coefficient increased 2.7 times (p <0.05), 3.4 times (p < 0.05), 4.4 times (p <0.05) and 9.5 times (p <0.05) in 1R, 2R, 3R and humoral rejection, respectively. The value of the CD68-positive macrophage region for 0R was only 0.34 ± 0.01%. This parameter increased 2.7 times (p <0.05), 4.0 times (p <0.05), 9.6 times (p <0.05) and 4.1 times (p <0.05) in 1R, 2R, 3R and humoral rejection, respectively.Conclusion. Cellular corporations in the mononuclear inflammatory infiltrate of transplanted heart are characterised by the predominance of T-lymphocytes in the cases of both cellular and humoral rejection. The expression of the B-lymphocyte marker is most pronounced in an antibody-mediated form. The maximum presence of macrophages in the infiltrate characterises severe cell rejection. An increase in the severity of cell rejection leads to an increase in the relative content of B-lymphocytes and macrophages in the infiltrate.

Morphological and Cytophysiological Changes in the Adult Rat Adrenal Medulla after Prenatal and Postnatal Exposure to Endocrine-Disrupting DDT.

The aim of the investigation was to study morphological and cytophysiological changes in the adult rat adrenal medulla after prenatal and postnatal exposure to endocrine-disrupting chemical dichlorodiphenyltrichloroethane (DDT).Materials and MethodsThe experiment was carried out on male Wistar rats (n=30). The rats were exposed to low doses of DDT during prenatal and postnatal development (n=10) and during postnatal development (n=10) only. The average daily intake of DDT by rats was 3.30±0.14 μg/kg. The control (n=10) and experimental animals were sacrificed at the age of 10 weeks (post-puberty), when rat adrenal glands reached their maximum development. Histological samples of the equatorial sections of the adrenal glands were prepared. The adrenal medulla was studied using light microscopy and computer morphometry. Immunohistochemistry was performed to evaluate tyrosine hydroxylase expression in chromaffin cells. Plasma epinephrine content was measured using enzyme-linked immunosorbent assay.ResultsPostnatal exposure of rats to low doses of the endocrine disruptor was found to interfere with adrenal medulla development and cytophysiology of chromaffin cells. Prenatal and postnatal exposure resulted in more profound delay in adrenal medulla development of adult rats as compared to the values of rats exposed postnatally, which was manifested by reduced area of the adrenal medulla and reduced total area of chromaffin cells in its section. Postnatal exposure is manifested by more significant functional disorders including decreased synthesis of tyrosine hydroxylase enzyme in the cytoplasm of chromaffin cells and elevated number of tyrosine-hydroxylase-negative cells in the medulla section, leading to a decrease in plasma epinephrine concentration.ConclusionExposure to low doses of DDT in the early stages of development leads to morphological and cytophysiological changes in the adult rat adrenal medulla and, therefore, reduced functional activity of chromaffin cells, which allows considering the endocrine disruptor DDT a potential risk factor disrupting the function of the neuroendocrine system.

The Influence of the Xymedon Сonjugate with l-Methionine on the Regeneration of Schmidtea mediterranea Planarians

The effect of the Xymedon conjugate with methionine on blastema regeneration in Schmidtea mediterranea planarians was investigated. It was established that the preparation has low toxicity towards planarians, which is evidenced by its CL50 values (lethal concentration). The results of the vital computer morphometry and flow cytofluorometry confirm that Xymedon with methionine induces regeneration at high concentrations only: 0.01 g/100 mL, 0.05 g/100 mL, and 0.1 mg/100 mL. Low concentrations of the preparation have no influence on blastema regeneration in planarians.

Expression of vascular endothelial growth factor a in the endometrium in uterine factor infertility

Aim. To assess the expression of vascular endothelial growth factor A for evaluating the functional state of the endometrium in women suffering from infertility and chronic endometritis.Materials and methods. Endometrium biopsy specimens obtained from 41 patients with uterine factor infertility (experimental group) were examined. A comparison group was composed of 39 women diagnosed with the “male factor” infertility exhibiting no infl ammatory processes of the reproductive organs. An endometrial biopsy was performed in both phases of the menstrual cycle: on days 8–10 of the follicular phase and on days 21–24 of the luteal phase. The expression of vascular endothelial growth factor receptors was evaluated on the basis of immunohistochemical staining of the biopsy specimens using monoclonal antibodies. The results of the immunohistochemical reaction were quantifi ed using the ImageJ software. A quantitative criterion — a stained area coeffi cient (per cent) representing the ratio of the immunohistochemical staining area to that of a biopsy specimen — was proposed for assessing the level of VEGF-A expression.Results. In the chronic endometritis group, the intensity of VEGF-A expression was found to be 1.4 times higher than that in the comparison group during the proliferation phase (p < 0.001). Expression values of the secretion phase were 1.9 times higher than those in the comparison group (p < 0.01).Conclusion. Chronic endometritis is characterized by an overexpression of VEGF-A by various endometrium cellular components. Interpretation of immunohistochemical stains using the method of computer morphometry allows quantitative indicators characterizing the intensity of angiogenesis marker expression in the endometrium during the menstrual cycle to be obtained. The level of VEGF-A expression can be used as an additional marker improving the quality of biopsy diagnostics in patients with infertility.

The key role of pancreatic fibrosis severity in the surgical treatment algorithm of patients with chronic pancreatitis

The aim of the work was to improve the results of surgical treatment of complicated forms of chronic pancreatitis. Materials and methods: The results of surgical treatment of 181 patients with complicated forms of chronic pancreatitis have been analyzed. All these patients were treated in surgical clinic of 2 nd medical faculty of National Pyrogov Memorial Medical University in Vinnytsya. Results: It is possible preoperative indirectly assess the severity of fibro- degenerative changes in pancreas, that includes 1 - the definition of pain type; 2 - CT or MRI (Marseilles-Rome classification); 3 - assessment of the stage of chronic pancreatitis (Büchler classification); 4 - patient's nutrition status and preoperative differential diagnosis with pancreatic cancer; 5 - assessment of the fibrosis severity (elastomers). The key point in treatment depended on intraoperative examination: detection of strictures of the main pancreatic duct (pacemaker of chronic pancreatitis); the tissue pressure resistance to the liquid, which is the maximum value> 200 mmHg, in the region of stricture and falls in other parts of the pancreas; pressure in the main pancreas duct, which rises only in 59.5% of patients. The head of the pancreas was involved in the pathological process in 83.8%, in 16.2% it was isolated in the isthmus or the body and tail of the pancreas. In general, the distal pancreas was involved in 37.8%. At computer morphometry of histological samples, the area of connective tissue fields reached 81.4 ± 6.62%, preserved exocrine part in 4.87 ± 1.62%, endocrine - 1.92 ± 0.12%, total area of ducts - 6 , 47 ± 1.12%. Conclusions: The combined Frey-Izbickiy local resection provides a wide excision of the pacemaker (stricture) of chronic pancreatitis. In case of extrapancreatic complications or repeated surgical interventions on the pancreas due to chronic pancreatitis, this effect can be achieved by pancreatoduodenal resection.

Structural Transformations in Neural Clusters of the Cerebral and Cerebellar Cortex in Children

Objective – the study of age-related changes in neural groups of the cerebral cortex and cerebellar cortex in the process of upward ontogenesis. Material and methods . The work was done on post-mortal material (75 left cerebral hemispheres, 62 cerebellum) obtained from children from birth to 12 years old who died as a result of injuries without damage to the brain. Using computer morphometry on the Nissl stained frontal histological sections of the cerebral cortex taken in layer III of fields 8, 6o р and 37ac, as well as in the granular layer of the paramedian lobule of the cerebellar cortex (H VII B), the area of the neural group on the section and the total area of neurons were measured as part of a grouping. Quantitative data analysis was carried out using variation statistics in annual intervals. Results . In children from birth to 12 years, in the dynamics of developmental changes in the microstructure of the cerebral and cerebellar cortical zones associated with visual-spatial perception, 4 stages of quantitative changes in the cluster (ensemble) organization of the cortex are distinguished: I - from birth to the end of 1 year of life, II – from 1 to the end of 2 years, III – from 3 to 5–6 years, IV – from 5–6 to 8–9 years. In the cerebellum cortex, significant changes in the structure of neural clusters are observed by the end of 1 year of life, by 3 and by 5–6 years. Structural changes in neural groups differ in terms and intensity in each of the studied fields. Conclusion . The sizes of neural clusters in the microstructure of the cerebral and cerebellar cortex are informative indicators for identifying the stages of significant age-related transformations in different zones of the human cortical formations in postnatal ontogenesis, and can be recommended for use in comparative anatomical and clinical studies.

Cytoprotective effect of non-opioid leu-enkephalin analogue in primary culture of pulmonary fibroblasts in oxidative stress

Aim. Analysis of the cytoprotective effect of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the primary culture of pulmonary fibroblasts in conditions of oxidative stress.
 Methods. Pulmonary fibroblasts were incubated with the peptide of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the concentration 0.1 μM for 6 hours. To simulate oxidative stress, 60 μM H2O2 was added to the culture medium for 2 hours. Experimental series included (1) «control»; (2) «non-opiate leu-enkephalin analogue» (the peptide was added to the culture medium 44 hours after the final passage); (3) «oxidative stress» (H2O2 was added to the culture medium 48 hours after the final passage); (4) «non-opiate leu-enkephalin analogue + oxidative stress» (the peptide and H2O2 were added to the culture medium 44 and 48 hours respectively after the final passage). In order to evaluate the generation of superoxide anion by pulmonary fibroblasts, the method of lucigenin-dependent chemiluminescence was used. Computer morphometry of the nucleo-nucleolar apparatus of fibroblasts stained with silver nitrate by the AgNOR method was used to assess the cell state: the area of fibroblast nuclei, the total nucleoli area in the nucleus, and the number of nucleoli in the nucleus were measured. These parameters correlate with the activity of anabolic processes in the cells.
 Results. The effect of H2O2 on the primary culture of pulmonary fibroblasts caused an increase of superoxide anion generation by the fibroblasts, reduction of fibroblast nuclei size, decrease of nucleoli amount and size. Pre-incubation of pulmonary fibroblasts with a non-opioid leu-enkephalin analogue reduced the H2O2-induced generation of superoxide anion, corrected changes in the nucleo-nucleolar apparatus of fibroblasts caused by oxidative stress. In our previous studies, similar effect in the same model was shown for non-selective μ/δ-opioid receptor agonist peptide sedatin (dermorphin analogue). The mechanism of cytoprotective effect of non-opioid leu-enkephalin analogue may include the affinity of this peptide to nociceptin receptors (NOR receptors) that requires further studies.
 Conclusion. The results of the study indicate a direct cytoprotective effect of the peptide Phe-D-Ala-Gly-Phe-Leu-Arg (non-opioid leu-enkephalin analogue) in oxidative stress.

Ligature induced periodontitis causes atherosclerosis in rat descending aorta: an experimental study.

IntroductionIn recent years a link between periodontitis and cardiovascular diseases has been proposed. Oxidative stress has been found to be involved in their pathophysiology.ObjectivesThe aim of this study was to demonstrate that a wire ligature placed around the first mandibular molars of the rat causes inflammation in the periodontium and lipid deposits in the aorta, via oxidative stress.MethodsTwenty male Wistar rats were divided randomly into two groups of 10: Control group and Periodontitis group. An orthodontic wire ligature was placed around the cervix of the first mandibular molar and was kept in place for 4 weeks. Clinical attachment loss was determined to assess the gingival recession. Aorta was harvested after 4 weeks and the malondialdehyde (MDA) levels in the aorta tissue homogenate were determined. Lipid deposits were evaluated using Oil Red staining and immunofluorescence under confocal microscopy. Total lipid deposits in the aorta were assessed using a computer morphometry system.ResultsSignificant gingival recession was observed in the Periodontitis group in comparison with the Control group. Increased MDA levels were present in the aorta in the ligatured rats in comparison with the Control group. Significant lipid deposits were observed in the aorta of the Periodontitis group as opposed to the Control group.ConclusionsThe affixing of an orthodontic wire ligature produces gingival recession, increased oxidative stress and lipid deposits in the rat descending aorta.

Structural changes in the wall of the abdominal aorta in modeling of long-term cannabinoid receptors CB1 blockade in laboratory animals

The purpose of the work is to detect the effect of long-term cannabinoid receptors CB1 blockade on the abdominal aorta wall structure in experimental rats. Materials and methods. The study was performed on two groups of 100-day-old male Wistar rats. The first group was made up of intact animals. The second group consisted of animals that were administrated with rimonabant hydrochloride solution (10 mg/kg -1 of body weight) per os with drinking water per day. The experiment duration was 10 days, and then the abdominal aorta of each animal was isolated for the histological measurements. Computer morphometry, the study of the subendothelial layer thickness with the internal elastic lamina and media, the ratio of the lumen diameter to the vessel wall were conducted in the abdominal aorta wall structure assessment. Results. It has been established that long-term cannabinoid CB1 receptors blockade in 100-day-old male Wistar rats resulted in morphological changes in all layers of the abdominal aorta wall that manifested in alterations of endothelial cells structure and their desquamation in the intima. The inner elastic lamina became thin and discontinuous. The thickness of the media was not uniform and there was a decrease in the number of elastic laminae as well as an increase in the number of chaotically oriented smooth muscle cells. Changes in adventitia manifested in swelling and loosely packed fibers with their increased dispersion. The number of collapsed vasa vasorum was decreased to single and hypertrophy of the nerve ganglia was observed. There was a thinning of the internal lamina and a decreased percentage of the lumen and vessel components of the wall at the expense of other tissues. Conclusions . The study results indicate that the long-term cannabinoid CB1 receptors blockade is a factor that damages endothelium of the abdominal aorta and leads to a decrease in elastin and proliferation of smooth muscle cells in the media, as well as a decrease in the percentage of the lumen and wall of the abdominal aorta at the expense of other tissues.

The Protective Effects of Phosphodiesterase 5 Inhibitors "Sildenafil & Ordonafil” on Ischemia‐Reperfusion Injury

Phosphodiesterase type‐5 (PDE‐5) inhibitors, Sildenafil (SILD), Vardenafil (VAR) and Ordonafil (ORD), have been developed for erectile dysfunction. Recently SILD and VAR were shown to have cardioprotective effect by reducing the infarct size in ischemic/reperfusion injury. To support the suggestion that this effect is a generalized "class" effect we decided to test the cardioprotective effect of ORD and to compare it to that of SILD. Isolated rabbit's hearts, perfused using Langendorff system, were subjected to 30 min of global ischemia followed by 2 hrs of reperfusion. At end of perfusion the heart was sliced into 5–6 cuts, 2 mm thick and incubated in 1 % triphenyltetrazolium chloride. The area of infarction and the whole left ventricle (LV) were measured by computer morphometry using image tool software. The infarct size was expressed as % of LV. Creatine kinase enzyme (CK) as a marker of infarction, was determined in the perfusate pre‐ and post‐ischemia by enzyme bioassay. The drugs, SILD or ORD at 1 mg/L, perfused the heart for 5 min before induction of ischemia. The results of both drugs showed significant cardioprotective effect and the infarct size ( as % of LV) was reduced from 38 ± 12 % in the control group to 5±2 and 9±6 %, mean±SD, n=6–8, P<0.004 for ORD and SILD respectively). Similarly the increase in CK was attenuated from 32 times increase in the control group to 20 times and 26 times for ORD and SILD respectively. Theses results support the notation that the class of PDE‐5 inhibitors has protective effect in the ischemic heart beside their effect in the treatment of erectile dysfunction.Support or Funding InformationDeanship of researchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in thickness of cortex and its layers in the posterior lobe of the cerebellum in postnatal ontogenesis

Objective– the study of age-related changes in the thickness of the cortex and its layers in the posterior lobe of the cerebellum of children.Material and methods.The work is performed on postmortem material (62 cerebellum), obtained from children aged from birth to 12 years who died as a result of injuries without brain damage. Using computer morphometry on the painted method Nissle frontal histological sections of cortex, taken in region right and left posterior quadrangular lobules (H VI) on top of the folia of cerebellum, was measured the cortical thickness and the thickness of molecular and granular layers. Analysis of quantitative data was performed at annual intervals.Results. The posterior lobe of the cerebellum the increase in the thickness of the cortex in the left hemisphere occurs in two stages: from birth to 1 year and then to 2 years, in the right hemisphere in three stages: from birth to 1 year, and 2 and 7 years. Right-sided asymmetry of the thickness of the cerebellar cortex observed in infants and children 7 years of age, thickness of granular layer in infants and children 9 years. Left-hand asymmetry is specific to cortical thickness and its molecular layer in children 12 months.Conclusion. The thickness of the cerebellar cortex and its layers in the area of the lobule H VI increases during early childhood, and in the right hemisphere also during the first childhood. Reducing the diameter of the cortex and layer in the posterior lobe of the cerebellum of children from birth to 12 years of age are not detected.

Analyses of tumor cells by mathematical models.

In this contribution we present graph theoretical approach to image processing focus on biological data. We use the graph cut algorithms and extend them for obtaining segmentation of biological data. We deal with tumor brain cells and rats brain to show the existence and presence of inflammatory molecules. We introduce a completely new method for filtering of data (Tab. 3, Schema 4, Fig. 7, Ref. 13). Keywords: graph cuts, segmentation, tumore analyses of cells, computer morphometry.