Mayer's Hematoxylin Research Articles

Experimental and Histological Validation of Nanostructured Products Use in Maxillofacial Surgery

The aim of the study is using experimental histological techniques to validate the beneficial potential of specific medical devices made of nanostructured titanium, and compare their effect with factorymade products currently applied in maxillofacial surgery. Material and methods. The study involved 50 sexually mature male Chinchilla rabbits. An open fracture of the lower jaw was simulated in the animals. In the 1st series of the experiment, the fragments were fixed using nanostructured miniplates and miniscrews made of Grade 4 titanium. In the 2nd series, they were fixed with standard miniplates and miniscrews from Konmet LLC. The material for the study was taken on the 7th, 14th, 21st, 28th and 40th days after the operation. In all series the object of the study was the extirpated lower jaw in the fracture area. The prepared histological sections were stained with Mayer's hematoxylin and eosin. The immunohistochemistry technique was used to identify cells with signs of proliferation (synthesizing the Ki67 protein), to assess the expression of the synthesis of proteins p53, caspasa 3 and the anti-apoptotic protein bcl-2. The surfaces of titanium structures were studied by scanning electron microscopy. The results were processed statistically. Results. The study investigated the effect of miniplates and miniscrews made of nanostructured and standard titanium on rabbits. The data obtained revealed the nature of the proliferative and apoptotic potentials of osteoblasts in the regenerate, and also indicated an increase in the ratio of the number of cambial cells and cells with an apoptotic dominant in the zone contacting with the nanostructured surface of the titanium screw compared to the titanium screw manufactured by Konmet LLC. Taken together, the obtained data indicate that the use of nanostructured titanium Grade 4 for the production of miniplates and miniscrews has an optimizing effect on reparative osteogenesis. The mechanism of this effect is determined by the presence of osseointegration properties of titanium. Conclusion. Nanostructured titanium is advisable to use for the production of implants, reconstructive titanium plates and other products for bone grafting.

Different types of tumor microvessels in stage I-IIIA squamous cell lung cancer and their clinical significance.

Lung cancer (LC) is the leading cause of morbidity and mortality among malignant neoplasms. Improving the diagnosis and treatment of LC remains an urgent task of modern oncology. Previously, we established that in gastric, breast and cervical cancer, tumor microvessels (MVs) differ in morphology and have different prognostic significance. The connection between different types of tumor MVs and the progression of LC is not well understood. To evaluate the morphological features and clinical significance of tumor MVs in lung squamous cell carcinoma (LUSC). A single-center retrospective cohort study examined medical records and archival paraffin blocks of 62 and 180 patients with stage I-IIIA LUSC in the training and main cohorts, respectively. All patients underwent radical surgery (R0) at the Orenburg Regional Cancer Clinic from May/20/2009 to December/14/2021. Tumor sections were routinely processed, and routine Mayer's hematoxylin and eosin staining and immunohistochemical staining for cluster of differentiation 34 (CD34), podoplanin, Snail and hypoxia-inducible factor-1 alpha were performed. The morphological features of different types of tumor MVs, tumor parenchyma and stroma were studied according to clinicopathological characteristics and LUSC prognosis. Statistical analysis was performed using Statistica 10.0 software. Univariate and multivariate logistic regression analyses were performed to identify potential risk factors for LUSC metastasis to regional lymph nodes (RLNs) and disease recurrence. Receiver operating characteristic curves were constructed to discriminate between patients with and without metastases in RLNs and those with and without disease recurrence. The effectiveness of the predictive models was assessed by the area under the curve. Survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between patient subgroups. A value of P < 0.05 was considered to indicate statistical significance. Depending on the morphology, we classified tumor vessels into the following types: normal MVs, dilated capillaries (DCs), atypical DCs, DCs with weak expression of CD34, "contact-type" DCs, structures with partial endothelial linings, capillaries in the tumor solid component and lymphatic vessels in lymphoid and polymorphocellular infiltrates. We also evaluated the presence of loose, fine fibrous connective tissue (LFFCT) and retraction clefts in the tumor stroma, tumor spread into the alveolar air spaces (AASs) and fragmentation of the tumor solid component. According to multivariate analysis, the independent predictors of LUSC metastasis in RLNs were central tumor location (P < 0.00001), the presence of retraction clefts (P = 0.003), capillaries in the tumor solid component (P = 0.023) and fragmentation in the tumor solid component (P = 0.009), whereas the independent predictors of LUSC recurrence were tumor grade 3 (G3) (P = 0.001), stage N2 (P = 0.016), the presence of LFFCT in the tumor stroma (P < 0.00001), fragmentation of the tumor solid component (P = 0.0001), and the absence of tumor spread through the AASs (P = 0.0083). The results obtained confirm the correctness of our previously proposed classification of different types of tumor vessels and may contribute to improving the diagnosis and treatment of LUSC.

Morphogenetic Promoters in the Development of Provisional and Definitive Urinary Organs in Humans and Vertebrates

The aim is to show the regulatory significance of promoter organs of morphogenesis in a stable state of the kidneys of vertebrates (fish, birds) and humans. Material and methods. 118 embryos at stages 12-23 of Carnegie (SC) and 28 human fetuses of 9-12 weeks of fetogenesis, 268 embryos of meat chickens (cross "Hibro PG +") from the stage of 48 hours to 20 days of broiler egg incubation, 50 fry of peled Coregonus peled were studied (Gmelin, 1788) at the stage of yolk sac atrophy (stage 36-37 according to the classification of Detlaf, 1975). To study the method of light microscopy, the material fixed in a 10% neutral form, embedded in paraffin. Sections stained with Mayer's hematoxylin and eosin, McManus CHIC method. Ki-67, CD31, CD34, positive cells are detected by immunohistochemistry. Primary kidney examination by low-vacuum electron microscopy. Embryos and human fetuses are observed in the Tyumen health care facility during medical abortion on demand in anamnestic healthy women with their warning about emergency work. Observation chicken embryos at the poultry farm “Tyumensky Broiler”, incubation workshop (Kaskara village, Tyumen region). Morphometric analysis and statistical processing of the actual material were carried out. Results. It was shown that the organotypic differentiation of the intermediate mesenchyme and the formation of nephrons of the provisional and definitive urinary organs control the state of the mesonephric duct and metanephritic diverticulum. The mesonephric duct initiates the organotypic antibiotic of the perifocal intermediate mesonephric mesenchyme and detects the proximodistal vector of saltatory mesonephronogenesis. The metanephritic diverticulum and its daughter branches initiate the organotypic differentiation of the intermediate mesenchyme and control the fan mechanism of metanephronogenesis. Activity of nephronogenesis Determination of the dynamics of convergence of immunocompetent cells. The absence of ventrodorsal generation of mesonephrons in humans is associated with a significant vital cycle of a normal kidney, atrophy of the mesonephric duct, and a decrease in the activity of immunocompetent cells. An increase in the expression of CD31, CD34, Bcl-2, Ki-67 positive cells is realized in combination with the formation of nephron rudiments as the promoter organs move in the developing organ of urinary formation. Conclusion. Prenatal organogenesis monitors and constantly monitors the formation of promoter organs of morphogenesis - the mesonephric duct and the metanephric disserticulum. Organotypic infection of the intermediate mesenchyme during the development of fish, birds and humans correlates with the chronovector of the convergence of immunocompetent cells.

Predictors of disease recurrence after radical resection and adjuvant chemotherapy in patients with stage IIb-IIIa squamous cell lung cancer: A retrospective analysis.

Lung cancer (LC) is a global medical, social and economic problem and is one of the most common cancers and the leading cause of mortality from malignant neoplasms. LC is characterized by an aggressive course, and in the presence of disease recurrence risk factors, patients, even at an early stage, may be indicated for adjuvant therapy to improve survival. However, combined treatment does not always guarantee a favorable prognosis. In this regard, establishing predictors of LC recurrence is highly important both for determining the optimal treatment plan for the patients and for evaluating its effectiveness. To establish predictors of disease recurrence after radical resection and adjuvant chemotherapy in patients with stage IIb-IIIa lung squamous cell carcinoma (LSCC). A retrospective case-control cohort study included 69 patients with LSCC who underwent radical surgery at the Orenburg Regional Clinical Oncology Center from 2009 to 2018. Postoperatively, all patients received adjuvant chemotherapy. Histological samples of the resected lung were stained with Mayer's hematoxylin and eosin and examined under a light microscope. Univariate and multivariate analyses were used to identify predictors associated with the risk of disease recurrence. Receiver operating characteristic curves were constructed to discriminate between patients with a high risk of disease recurrence and those with a low risk of disease recurrence. Survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between patient subgroups. Differences were considered to be significant at P < 0.05. The following predictors of a high risk of disease recurrence in patients with stage IIb-IIa LSCC were established: a low degree of tumor differentiation [odds ratio (OR) = 7.94, 95%CI = 1.08-135.81, P = 0.049]; metastases in regional lymph nodes (OR = 5.67, 95%CI = 1.09-36.54, P = 0.048); the presence of loose, fine-fiber connective tissue in the tumor stroma (OR = 21.70, 95%CI = 4.27-110.38, P = 0.0002); and fragmentation of the tumor solid component (OR = 2.53, 95%CI = 1.01-12.23, P = 0.049). The area under the curve of the predictive model was 0.846 (95%CI = 0.73-0.96, P < 0.0001). The sensitivity, accuracy and specificity of the method were 91.8%, 86.9% and 75.0%, respectively. In the group of patients with a low risk of LSCC recurrence, the 1-, 2- and 5-year disease-free survival (DFS) rates were 84.2%, 84.2% and 75.8%, respectively, while in the group with a high risk of LSCC recurrence the DFS rates were 71.7%, 40.1% and 8.2%, respectively (P < 0.00001). Accordingly, in the first group of patients, the 1-, 2- and 5-year overall survival (OS) rates were 94.7%, 82.5% and 82.5%, respectively, while in the second group of patients, the OS rates were 89.8%, 80.1% and 10.3%, respectively (P < 0.00001). The developed method allows us to identify a group of patients at high risk of disease recurrence and to adjust to ongoing treatment.

MORPHOGENESIS OF SKIN REGENERATE AND LOCAL FACTORS OF ITS REGULATION

Regeneration of the skin and its derivatives after injuries occurs in one of two ways: cutaneous (restitution) or dermal (substitution). However, there are three healing mechanisms: primary intention, secondary intention and healing by a scab. Regeneration activity correlates with environmental factors, stimulants, compliance with the chronovector of involvement in the cell regenerate of all differon derivatives, involved in histo- and organogenesis in the affected area.&#x0D; The aim of the study is to identify the importance of promoter cells, temperature and Eikovit gel at different stages of thermal burn wound healing, contact dermatitis and wound skin defects and to determine the stage of provisional regenerate transformation into the definitive state.&#x0D; Materials and Methods. The formation of reparative skin regenerate was studied on outbred male mice weighing 25±5 g (n=126). Different injuries to the skin of the back were modelled. All mice were divided into 4 groups: Full-thickness wound, Thermal burn, Contact dermatitis, and Control. Thermal damage was carried out with Tertsik RS232C device (Russia), module area – 1 cm2, exposure time – 3 minutes, and temperature – 80 °C. Contact dermatitis was modeled by rubbing a 0.5 % alcohol-acetone solution of 2,4-dinitrochlorobenzene (2,4-DNCB) into the skin. The skin wound was cut out using an oval stencil (3×4 mm). We used three temperature modes to influence the developing regenerate: +8 °C (cold), +42 °C (heat), and +33 °C (control). Sampling was conducted on days 3, 7, 10, 14, 20, 30 of the experiment, fixed in 10 % neutral formalin, and embedded in paraffin. Sections were stained with Mayer's hematoxylin and eosin. Immunohistochemistry detected CD1-alpha, CD3, and CD31. Proliferative activity was monitored by Ki-67-positive cells.&#x0D; Results. The convergence chronovector of immunocompetent cells during skin wound healing ensures the restitution of skin components and its derivatives. One of the signs of the correct chronovector direction is the formation of a provisional level substrate and its further transformation into a definitive state. Convergence desynchronosis can lead to dermal healing and substitution.

Histomorphological and histochemical Study of the jejunum in moorhen (Gallinula chloropus) in Iraq

The goal of the current investigation was to gather information on the histology and histochemical composition of the jejunum in moorhens (Gallinula chloropus). In order to conduct the current investigation, six moorhens were gathered. They were purchased from regional vendors in particular markets in Baghdad, Iraq. To collect the intestine, birds were put down after being dissected, and the specimens were then fixed in 10% neutral buffered formalin. The blocks were sectioned at 6 m thickness and stained with Mayer's hematoxylin and eosin, Masson trichrome stain, periodic acid Schiff reagent (PAS), and Alcian blue stain (AB)PH-2.5 ,after being thoroughly fixed. The specimens were then cleared in xylene and embedded in paraffin wax. According to the Gross findings, the small intestine is made up of the duodenum, the jejunum, and the ileum. Histologically, the mucous membrane was thrown into large numerous villi in the jejunum, showing blunt apical part and wide basal part with thin layer of muscularis mucosa. The submucosa was thin layer of connective tissue. Below the submucosa was thick layer arranged in a thick circular and thin longitudinal. According to histochemical results, the PAS stain had a negative reaction on columnar cells while having a positive reaction on goblet cells. When the AB (pH 2.5) was applied, the wall displayed epithelial cells that stained negatively while the goblet cells responded favorably to their acidic mucopolysaccharides.

REMODELING OF THE AERO-HEMATIC LUNG BARRIER COMPONENTS UNDER HIGH ALTITUDES

The aim of the study is to examine structural and ultrastructural aero-hematic barrier (AHB) changes in rats during adaptation to high-altitude hypoxia.&#x0D; Materials and Methods. The experiments were carried out on 60 sexually mature outbred laboratory male rats weighing 140–160 g at high altitude (3200 m above sea level). The authors followed Mayer's hematoxylin and eosin staining protocol, and used transmission and scanning electron microscopy on the 7th, 15th and 30th days of the animals' life in the mountains. Statistical data processing was carried out using Statistica 6.0 (USA) and StatTech v. 2.8.4 (Russia) and Student's t-test. Differences were considered statistically significant at p&lt;0.05.&#x0D; Results. At early stages of high-altitude adaptation, dystrophic changes in the epithelial layer of lung capillaries, hypertrophy and hyperplasia of histiocytes, fibroblasts and lipofibroblasts were noted. The authors also revealed proliferation of type II alveolocytes, thickening of the interalveolar septa (IAS) and cytoplasmic processes of endotheliocytes, destruction of type II alveolocytes (apocrine method, with the release of surfactant into the alveoli lumen), multivesicular lamellar bodies in the cytoplasm of type II alveolocytes; interstitial edema with transudate in the alveoli lumen. In later periods, surfactant system activity decreases due to a reduced release of osmiophilic lamellated corpuscles (OLC) into the alveoli lumen. The main AHB components are next to control data, while the IAS thickness exceeds the norm. In the alveoli wall, the number of fibroblasts increases, causing an increase in the number of Kohn's pores and providing collateral breathing.&#x0D; Conclusion. The study of AHD components in experimental animals at different periods of high-altitude adaptation revealed dystrophic processes of AHD in IAS, compensated by type I alveolocytes and proliferation of type II alveolocytes, confirming the adaptive mechanisms of ultrastructural alteration of all epithelial and endothelial components of the pulmonary alveoli.

Pathomorphological changes in the liver of broiler chickens caused by adeno-virus infection. Description of a spontaneous case in a private farm of the Lviv region

The article covers the results of pathomorphological studies of broiler chickens' liver (Cobb 500 cross) spontaneously damaged by Group I fowl adenovirus. Clinical signs of the disease in broiler chickens appeared on the 15th day of life and were manifested by a sharp decrease in water and feed consumption. The fowl was lethargic for several days, breathing hard, mostly sitting with its head down and eyes closed. During the breeding period, the mortality rate reached 13.2 %, accompanied by significant growth retardation. A peak of mortality was observed within 20–24 days of life. Antibody titers monitored by immunoenzymatic method using BioChek ELISA tests for infectious bursal disease (IBD), infectious bronchitis (IBV), and Newcastle disease (ND) virus were within normal post-vaccination levels. Pathological autopsy of broiler chickens revealed hepatomegaly, diffuse punctate and petechial hemorrhages under the Glisson's capsule, and military necrosis in the liver parenchyma. Hydropericarditis, pulmonary edema, renal dystrophy, and catarrhal enteritis were also detected in some cases. Liver fragments were selected for histological examination, fixed in a 10 % aqueous solution of neutral formalin, washed in water, and dehydrated in an ascending series of alcohols, followed by sealing and embedding in paraffin. Deparaffinized sections were stained with Mayer's hematoxylin and eosin. Optical examination of hepatocytes revealed large, round basophilic intranuclear inclusion bodies, multifocal necrosis of hepatocytes, stasis, and fatty dystrophy. According to the molecular genetic test results of broiler chickens' liver fragments, the DNA of Group I fowl adenovirus, serotype FAdV-11, was isolated.

Good Practices for Histological Analysis of the Annual Killifish Nothobranchius furzeri (Nothobranchiidae).

Paraffin histology is one of the most important and commonly used laboratory techniques enabling the study of the microscopic structure of animal and plant tissues. This technique uses paraffin wax, which in liquid form impregnates fixed and dehydrated tissues and allows the preparation of thin sections when solidified in blocks. This protocol on good practices in paraffin histology of Nothobranchius furzeri (Nothobranchiidae) summarizes the authors' current experience in terms of technique, evaluation, and interpretation of sectioned tissues. The steps that precede paraffin block preparation are also presented as they play a key role in maximizing the quality of examined sections. The paraffin technique as described only requires basic laboratory conditions to produce good-quality results. The description of staining methods is limited to Mayer's hematoxylin and eosin (H&E), the routinely used histological dye staining cell nuclei in blue-black (hematein) and cell cytoplasm and connective tissue fibers in shades of pink-red (eosin). Killifish specialists are encouraged to engage in the study of histology and histopathology, taking advantage of interdisciplinary cooperation.

Inhibitory effect of ginsenglactone A from Panax ginseng on the tube formation of human umbilical vein endothelial cells and migration of human ovarian cancer cells

BackgroundHere, we aimed to assess the inhibitory effect of a new compound from Panax ginseng on the migration of human ovarian cancer cells and tube formation of human umbilical vein endothelial cells (HUVECs). MethodsA new compound, ginsenglactone A (1), was isolated from ginseng roots, together with seven known compounds (2-8). Spectroscopic data were used to elucidate the chemical structure of 1. The tubular structure formation in HUVECs was assessed by Mayer's hematoxylin staining. The migration of A2780 cells was evaluated using the scratch wound healing assay. ResultsHUVECs treated with 1 had the statistically significant decrease in tubular structure formation compared to the HUVECs treated with compounds 2-8. This effect was enhanced by co-treatment with inhibitors for phosphatidylinositol 3-kinase (PI3K) (LY294002) and extracellular signal-regulated kinase (ERK) (U0126). Treatment with 1 decreased the expression of phosphorylation of ERK, PI3K, vascular endothelial growth factor receptor2 (VEGFR2), Akt, and mammalian target of rapamycin (mTOR). In addition, the ability of A2780 cells to cover the scratched area were also decreased. This effect was enhanced by co-treatment with U0126. Lastly, treatment with 1 decreased the phosphorylation of ERK, matrix metalloproteinase-9 (MMP-9), and MMP-2. ConclusionThese results suggest that ginsenglactone A is a potential inhibitor of HUVEC tubular structure formation and A2780 cellular migration, which may be helpful for understanding its anticancer mechanism.

P-227 Overexpression of survivin-1, TAG-72 and HERC5 in patients diagnosed with hepatocellular carcinoma in the Black Sea Coast geographical area

Epidemiological data regarding hepatocellular carcinoma (HCC) report unsatisfactory morbimortality rates despite the global efforts to decrease the incidence and prolong patient survival. Current guidelines lack diagnostic biomarkers to better characterize patients with HCC. We aimed to validate the overexpression of Survivin-1, tumor-associated glyocoprotein 72 (Tag-72), and HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5) as tissue biomarkers for HCC characterization in patients from our geographical area and to standardize a local biomarker panel to be introduced in the current management guideline. Thirty liver specimens of HCC and a similar number of liver tissue specimens of benign liver tumors were selected from the Gastroenterology file database and Pathology Clinic registries from St. Apostle Andrew Emergency Clinical Hospital, Constanta and Fundeni Institute, Bucharest and compared in terms of Survivin-1, Tag-72, and HERC5 overexpression. The morphological features of the tumors were noted based on the WHO Histological Classification for digestive tumors. Demographic data of all patients providing the liver specimens, as well as liver diseases background like chronic viral infections, other co-morbidities and laboratory parameters recorded at the time of hospital admission were noted. For the IHC assessment, the representative samples were chosen, and 4-μm sections of formalin-fixed, paraffin-embedded tissue blocks were obtained for each case enrolled. Epitope retrieval was conducted prior to incubation of tissue sections with a panel of three primary antibodies (ready-to-use) from Novus Biological: survivin-1 (NB100-911 clone), Tag-72 (CC49 clone), and HERC5 (NBP-91985 clone). The immunostaining protocol for each antibody used was provided by the manufacturer. As chromogen, we used 3,3'diaminobenzidine (DAB), and brown staining was obtained. The final step was represented by counterstaining all slides with Mayer's Hematoxylin. Comparisons of the studied biomarker overexpression from HCC tissue samples with a matched non-HCC group of normal liver tissue specimens were made.Student t-test, Mann-Whitney U-test and Chi-square test were used to find differences between the two studied groups and to compare the categorical variables. The discriminative power of Survivin -1, Tag-72, and HERC5 overexpression was assessed using ROC curves. The multivariate linear regression analysis revealed that Survivin-1, Tag-72, and HERC5 were significantly overexpressed upon IHC analysis in HCC samples in patients older than 50 years (P=0.003, P=0.006, P=0.004), male gender (P=0.031, P=0.004, P=0.020), patients with increased AFP over 180 ng/dl (P=0.012, P=0.004, P=0.029), with low serum albumin < 3 mg/dl (P=0.031, P=0.021, P=0.003, respectively), with imaging features of portal thrombosis (P=0.004, P=0.020, P=0.004, respectively), ascites (P=0.002, P=0.004, P=0.019, respectively) and in BCLC B and C classes patients (P=0.045, P=0.036, P=0.045, and P=0.033, P=0.001, P=0.027, respectively). The diagnostic performance of Survivin-1, Tag-72 and HER-C5 tissue biomarkers for HCC characterization was superior to that of AFP, considered the gold standard biomarker used in clinical guidelines (Survivin-1: Z statistic=2.911, P=0.0039; Tag-72: Z statistic=2.789, P=0.0049, respectively; HERC5: Z statistic=2.844, P=0.0043) and AFP assay alone (Z statistic=5.022, P < 0.0001). Our study results validate the overexpression of Survivin-1, Tag-72, and HERC5 as tissue biomarkers for HCC characterization in patients from our geographical region and could be standardized in the current HCC management guideline.

Evaluation of New Immunohistochemical Approaches for the Study of Kidney Tumors in Geriatric.

Kidney malignancies are among the most deadly genitourinary tumors. It is more common in males and is often seen in people aged 60-70 years old. The incidence rate of kidney cancer seems to be increasing. One reason for this may be the fact that imaging techniques, such as computed tomography scans are more commonly used. These tests may lead to the accidental detection of more kidney cancers. Fortunately, kidney cancer is often detected in the early stages, when the tumor is small and confined to the kidney. The objective of this study was the development of new diagnostic immunohistochemical methods. Clinical examination material of 134 people, including 94 (70%) males and 40 (30%) females, were used in this study. Immunohistochemical staining of tryptase was carried out in compliance with the requirements using Anti-Mast Cell Tryptase antibodies. Goat anti-mouse antibodies #AS-M1-HRP were used as secondary antibodies, visualized with ImmPACTTM DAB Peroxidase Substrate Kit (#SK-4105) according to the instructions of the manufacturer. The nuclei were counterstained with Mayer's hematoxylin, and the sections were embedded in a permanent mounting medium.The immunohistochemical study showed an increase in both tryptase- and chymase-positive mast cells in the renal parenchyma, compared with the control group. The number of mast cells with tryptase expression directly in the tumor was significantly less than the peritumoral localization. A similar pattern was observed for chymase-positive mast cells as the content of the tumor was more than 10 times higher than the intratumoral arrangement. The histological and immunological characteristics did not differ in different age groups. The immunohistochemical method of research in the diagnosis of renal tumors plays an important diagnostic and prognostic value. It can assist pathologists in difficult and ambiguous cases to correctly diagnose renal tumors. This will make it possible to prescribe the correct treatment and predict the course of malignant tumor growth in patients.

Changes in cytoplasmic and extracellular neuromelanin in human substantia nigra with normal aging.

Neuromelanin (NM) is a dark polymer pigment produced in certain populations of catecholaminergic neurons in the brain. It is present in various areas of the human brain, most often in the substantia nigra (SN) pars compacta and the locus coeruleus, the main centers of dopaminergic and noradrenergic innervation, respectively. Interest in NM has revived in recent years due to the alleged link between NM and the particular vulnerability of NM-containing neurons to neurodegeneration. The aim of this work was to study the structural, cytochemical, and localization features of cytoplasmic and extracellular NM (eNM) in the human SN pars compacta during normal aging. Sections of human SN from young/middle-aged adults (25 to 51 years old, n=7) and older adults (60 to 78 years old, n=5), all of which had no neurological disorders, were stained histochemically for metals (Perls’ reaction, Mayer's hematoxylin) and immunohistochemically for tyrosine hydroxylase (TH), Iba- 1, and CD68. It was shown that dopaminergic neurons in SN pars compacta differ in the amount of NM and the intensity of TH-immunoreactivity. The number of NM-containing neurons with decreased TH-immunoreactivity positively correlates with age. eNM is present in SN pars compacta in both young/middle-aged and older adults. The number of eNM accumulations increases with aging. Cytoplasmic and eNM are predominantly not stained using histochemical methods for detecting metals in people of all ages. We did not detect the appearance of amoeboid microglia in human SN pars compacta with aging, but we found an age-related increase in microglial phagocytic activity. The absence of pronounced microgliosis, as well as a pronounced loss of NM-containing neurons, indicate the absence of neuroinflammation in human SN pars compacta during normal aging.

Neoangiogenesis in the Econiche of Opisthorchis Felineus After Partial Hepatectomy Accompanied by Superinvasive Opisthorchiasis

Research goal: revealing the liver vessels formation mechanisms after partial hepatectomy accompanied by superinvasive opisthorchiasis (SO).Material and methods. The study was carried out on Syrian hamsters, weighing 98.0–110.0 g, mature animals of both genders. The animals were divided into three groups. Group I (n=168): a model of SO. Group II (n=40): partial liver resection in healthy animals. Group III (n=86): partial hepatectomy accompanied by SO. The histological specimens were stained with Mayer's hematoxylin and eosin by the methods of Van Gieson, Slinchenko, V.V. Kupriyanov. Immunohistochemical reactions were performed using primary antibodies to CD31, CD34, CD117, Oct4, and α-fetoprotein. The results were statistically processed, the differences were considered significant at p&lt;0.05.Results. In animals of group I accompanied by SO, proliferation of progenitor cells (CD31+, CD34+, CD117+, Oct4+) was observed in the portal tracts and perivascular space with the development of vascular formations, cholangiocellular and hepatocellular differnеntiations. Liver regeneration in healthy hamsters during 1– 3 days consisted in an active reaction of mitotic and amitotic division of hepatocytes with the development of avascular islets. In the subsequent periods (the 7th day), the cells of preexisting vascular structures (sinusoids, capillaries) multiplied. After partial hepatectomy accompanied by SO (group III), there was an intensive proliferation of stem and progenitor cells with differentiation into endothelial cells, elements of cholangio- and hepatocellular differnеntiations; endotheliocytes and capillary pericytes multiplied.Conclusion. Neoangiogenesis during regeneration after partial hepatectomy in healthy animals is implemented like angiogenesis; in case of partial liver resection accompanied by SO, it is followed by the vessels formation mainly from progenitor cells (vasculogenesis), advancing the development of elements of cholangio- and hepatocellular differnеntiations.

Impact of acute fonofos exposure on skeletal muscle of zebrafish: Histopathological and biometric analyses

It is widely known that pesticides generally do not show target specificity, and off-target species are strikingly affected by these chemicals. In the current work, histological changes in skeletal muscles of zebrafish (Danio rerio) caused by fonofos, an acetylcholinesterase (AChE) inhibitor organophosphate insecticide, were examined. Zebrafish were treated with 1 mg/L, 2 mg/L and 4 mg/L of fonofos for 96 hours. Skeletal muscle samples were removed from the pectoral region and embedded in paraffin. Sections were stained with Mayer's Hematoxylin and Eosin, Gomori's Trichrome and Periodic Acid Schiff techniques. Histopathological alterations were investigated by light microscopy. Fibrosis, intramyofibrillar vacuoles, disintegrated myofibrils, splitting of myofibers, atrophic and disappeared fibers, histoarchitectural loss, necrosis and progressive decrement in glycogen content were noted. Muscle fiber diameter measurements were also performed. Statistical analysis showed that measured fiber diameters of all fonofos exposed groups were significantly different from the control group, and they decreased in a concentration-dependent manner. These results suggested that fonofos caused significant myoarchitectural impairments in non-target freshwater zebrafish.

Expression of collagen type IV in human kidney during prenatal development

Background/Aim. Type IV collagen belongs to the group of nonfibrillar collagens and is an important component of the basement membranes, where it accounts for approximately 50% of its structural elements. The aim of the study was to describe the expression and distribution of collagen type IV in the embryonic and fetal metanephric kidney and to determine the volume density of collagen type IV in kidney tissue in each trimester of development. Methods. The material consisted of 19 human embryos/fetuses, in the gestational age from 8th to 37th week. Kidney tissue specimens were routinely processed to paraffin molds, stained immunohistochemically using polyclonal anti-collagen IV antibody and counterstained with Mayer hematoxylin and eosin. Stained slides were examined using a light microscope, and images of the selected areas under different lens magnification were captured with a digital camera. Volume density of collagen type IV was determined using ImageJ 1.48v and a plugin of the software, which inserted a grid system with 336 points. For the data comparison, the One-Way Analysis of Variance (ANOVA) was used. Results. Strong collagen IV immunopositivity was seen in all specimens, with a distribution in the basement membranes of urinary bud, parietal leaf of Bowman?s capsule, glomerular basement membrane, basement membrane of interstitial blood vessels, and basement membranes of nephron tubules and collecting ducts. No statistically significant difference in the volume density of type IV collagen was found among the different trimesters of the embryonic and fetal development. Conclusion. The synthesis and secretion of collagen type IV simultaneously follow the development of nephron structures, collecting system and blood vessels. The volume density of collagen type IV remains constant throughout all the trimesters of metanephric kidney development, indicating that it plays a crucial role in the normal development of nephron and collecting system structures, as well as in maintaining the normal kidney function.

Morphological Characteristics and Clinical Significance of Different Types of Tumor Vessels in Patients with Stages I-IIA of Squamous Cervical Cancer.

The determination of factors associated with progression of cervical cancer is important, both for a recurrence risk assessment and for determining optimal treatment tactics. Previously, we showed the prognostic value of different types of tumor microvessels (MVs) in gastric and breast cancer. The object of this research was to study the morphology and clinical significance of different tumor microvessels in early cervical cancer. A total of 65 archived paraffin blocks of patients with I-IIA stages of squamous cervical cancer were investigated. Samples were stained with Mayer hematoxylin and immunohistochemically using antibodies to CD34, podoplanin, HIF-1a, and Snail. The eight types of tumor MVs differed in morphology were identified. It was established that only the dilated capillaries (DСs) with weak expression of CD34, the contact type DCs, the capillaries in tumor solid component, and the lymphatic vessels in the lymphoid and polymorphic cell infiltrates of tumor stroma are associated with clinical and pathological characteristics of early cervical cancer. Preliminary results also suggest that a combination of fragmentation in tumor solid component and the contact type DCs may predict a recurrence of early cervical cancer. Given the small number of cervical cancer recurrences, the predictive significance of the described markers requires a more thorough examination.

Expression of CD44 and IL-10 in normotensive and preeclamptic placental tissue.

We aimed to demonstrate cell-cell adhesion and apoptotic changes in preeclamptic placentas by examining the expression of CD44 and IL-10. Placenta samples of 15 preeclamptic and 15 healthy 35-38th week-pregnant women were involved in the study. Tissue samples were taken only from the maternal side of the placenta and fixed in 10% formaldehyde, then blocked in paraffin wax and 5 μm-thick sections were cut and stained with Masson Trichrome. Antigen retrieval was performed for sections, incubated with CD44 antibody and anti-IL-10 antibody. After the application of streptavidin peroxidase followed by AEC chromogen solution, sections were counterstained with Mayer hematoxylin. In the preeclampsia group, increased CD44 positive expression was observed in maternal decidua cells and fibroblast cells close to root villi. CD44 was positively expressed in muscle cells around the blood vessels, mucosal connective tissue areas, syncytial nodes, and syncytial bridges. In the preeclampsia group, significant increased IL-10 expression was seen in subendothelial layers of the medium-sized vessels in the maternal region. IL-10 was also positively expressed in decidua cells outside the vessels, and inflamed connective tissue areas, chorionic villus cells with intense inflammation in intervillous spaces. CD44 was found to be an essential molecule in the regulation of vascular permeability, inflammatory response, activation of the cells, cell-to-cell interaction, and the signaling pathways to which they are associated. Since IL-10 regulates appropriate pregnancy outcomes and contributes to the balance of anti-inflammatory signals via both paracrine and autocrine regulators of trophoblast activity, we proposed that it might be a key to elucidate the etiology of preeclampsia with CD44 receptor.

CIRCADIAN DYNAMICS OF OPTIC DENSITY OF MELATONIN RECEPTORS IN THE NEURONS OF HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RATS UNDER ALTERED PHOTOPERIOD

Introduction. Melatonin production is considerably suppressed by light and affects the ability to transfer daily rhythm information from the hypothalamus to other neural target sites and thus alters the expression of some biological rhythms. The hormone controls the state of the hypothalamic-pituitary system and endocrine gland activity through melatonin receptors (membrane, cytosolic and nuclear ones). In addition, using a mechanism of the feedback, it interferes with the activity of supraoptic nucleus of the hypothalamus, which regulates water-salt metabolism and responses to stress. Objective: to provide quantitative circadian characteristics of melatonin receptors density in the neurons of the hypothalamic supraoptic nucleus of rats being under light stimulation as well as the correction of changes after injecting exogenous melatonin. Material and methods. The experiments were conducted on 60 white mongrel mature male rats weighing 150 – 180 g. The test animals were divided into 3 parts each with 2 groups, kept under the conditions of standard light regime, hyperilluminated and the injection of exogenous melatonin and day-round lighting within 7 days. To perform immunohistochemical methods, we used polyclonal antibodies to melatonin 1A receptors produced by Abcam and streptavidin biotin visualization system LSAB2 (peroxidase mark + diaminobenzidine) produced by Chemicon International Inc. We adhered to protocol standardization of methods for all sections. Additional staining of nuclei was performed with Mayer hematoxylin. Results. The indices of optical density of specific melatonin 1A receptors of supraoptic neurocytes staining obtained in the intact group (at 02.00 AM- 0,488 ± 0,0024, at 02.00 P.M. - 0,464 ± 0,0023, p = 0.002) and in animals subjected to light stress (at 02.00 AM-0,295 ± 0, 0019, at 02.00 P.M.- 0,286 ± 0,0018, p = 0,012) had a probable value and were characterized by a clear diurnal periodicity. In the group of animals with pineal gland hypofunction modulation (at 02.00 A.M.- 0,216 ± 0,0017, at 02.00 P.M. - 0,214 ± 0,0021, p&gt; 0,05). Conclusions The density of 1A melatonin receptors in rat’s hypothalamic supraoptic neurons are normally characterized by an accurate circadian rhythm. The highest density of receptors is observed at 02.00 AM, and at 02.00 PM it is significantly lower (p = 0.002). Immunohistochemical studies revealed that under inhibition of pineal gland activity the circadian rhythm of melatonin receptors density in neurons of supraoptic nuclei of the hypothalamus gets disturbed, which is characterized by an incredible difference of indices in the tested periods of the day.

Abstract 4901: p53 expression is a useful predictive marker for recurrence of meningioma

Abstract Introduction: Meningiomas are common intracranial tumor originate from arachnoid cap cells. Meningiomas are classified into three grades (grade I, II, and III) according to World Health Organization (WHO) 2016 criteria. Meningiomas are generally benign tumors with slow growing, and most of them can be treated by one-time resection. However, some of them regrow rapidly and invade into the dura mater widely, which often require additional treatments such as re-operation and irradiation. No useful marker was found to predict the recurrence of meningioma, so far. Then, the aim of this study is to investigate the immunohistochemical (IHC) expression of p53 and to determine the association of this results with the factors which related to the retreatment. Materials and methods: This retrospective study included 389 patients with intracranial or intraspinal meningiomas which were surgically operated at Osaka City University from 2007 to 2017. The clinicopathological data of each patient was from medical records of Osaka City University hospital. IHC staining of p53 was performed on 389 meningiomas, as follows. The paraffin-embedded sections were de-paraffinized and heated for 10 min at 105°C by autoclave in Target Retrieval Solution. Then sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The specimens were then incubated with anti-p53 (DO-7) for 1 h at room temperature. The slides were treated with streptavidin-peroxidase reagent and incubated in PBS with 1% (vol/vol) hydrogen peroxide, followed by counterstaining with Mayer's hematoxylin. p53 staining is considered negative when no cell with brown nuclear staining exist. The SPSS software program (SPSS Japan, Tokyo, Japan) was used for the analyses. In all tests, a p-value of less than 0.05 was defined as being statistically significant. Results: Of the 389 meningiomas, 77cases (19.8%) were p53 positive. Univariate analysis revealed that recurrence of meningioma was significantly correlated with p53 positive (p=0.025), young age (p&amp;lt;0.001), large dural attachment (p&amp;lt;0.001), low height dural attachment ratio (p=0.016), large max diameter (p=0.020) and high Ki-67 index (p&amp;lt;0.001). Multivariate analysis indicated that p53 positive (p=0.037), young age (p=0.002), large dural attachment (p=0.002), low height dural attachment ratio (p=0.046), large max diameter (p=0.001) and high Ki-67 index (p&amp;lt;0.001) were significantly associated with recurrence of meningioma. Conclusion: p53 expression was correlate with the proliferative activity of meningiomas. p53 might be a useful independent predictive marker for recurrence of meningioma. Citation Format: Atsufumi Nagahama, Masakazu Yashiro, Yuichiro Miki, Hiroki Morisako, Takehiro Uda, Takeo Goto, Takehiro Takami, Kenji Ohata. p53 expression is a useful predictive marker for recurrence of meningioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4901.