Expression patterns of male germ cell markers in cryptorchid pig testes Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Hyun-Jung Park, Hyuk Song, Jae-Seok Woo, Hak-Jae Chung, Jin-Ki Park, Kwang-Hyun Cho, Joon Mo Yeo, Won-Young Lee Abstract Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

Expression patterns of natriuretic peptides in pre-hibernating and hibernating anatolian ground squirrel (Spermophilus xanthoprymnus) lung Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Mustafa Öztop, Mehmet Özbek, Narin Liman, Feyzullah Beyaz, Emel Ergün, Levent Ergün, Ural Kemal Kavraal, Ergül Ergen Abstract Anatolian ground squirrel (Spermophilus xanthoprymnus) is a true hibernator. This animal transiently reduces pulmonary function during hibernation. Continuance of pulmonary function is very important to survive ground squirrels during the hibernation. Natriuretic peptides may be key players in the modulation of pulmonary hemostasis. However, NPs’ role in pulmonary function during hibernation remains unclear. We aimed to investigate the localization and distribution of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) in squirrel lungs during pre-hibernation and hibernation periods using immunohistochemistry. Our immunohistochemical data indicate that ANP, BNP, and CNP were produced by the mucosal epithelium of terminal and respiratory bronchioles, smooth muscle cells in the lamina propria of terminal bronchioles and vascular smooth muscle cells, alveolar type II cells, and macrophages. ANP immunoreactivity was weaker than BNP and CNP immunoreactivities in these cells. The results also demonstrate that the number of ANP, BNP and CNP positive alveolar type II cells tended to increase, although statistically non-significant, during the hibernation period, but the expression of NPs in other pulmonary cells is unaffected by hibernation. This study firstly investigates ANP, BNP and CNP distribution in the Anatolian ground squirrel lung. However, further studies are required to dissect their functional roles during the hibernation.

Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Billel Chaouad, Elara N. Moudilou, Adel Ghoul, Fouzia Zerrouk, Anissa Moulahoum, Khira Othmani-Mecif, Mohamed El Hadi Cherifi, Jean-Marie Exbrayat, Yasmina Benazzoug Abstract Objective Numerous studies have shown that a methionine-rich diet induces hyperhomocysteinemia (Hhcy), a risk factor for cardiovascular diseases. The objective of the present study was to determine the involvement of Hhcy in cardiac remodeling in the sand rat Psammomys obesus. Materials and methods An experimental Hhcy was induced, in the sand rat Psammomys obesus, by intraperitoneal injection of 300 mg/kg of body weight/day of methionine for 1 month. The impact of Hhcy on the cellular and matricial structures of the myocardium was analyzed with histological techniques (Masson trichrome and Sirius red staining). Immunohistochemistry allowed us to analyze several factors involved in myocardial remodeling, such as fibrillar collagen I and III, metalloproteases (MMP-2 and -9) and their inhibitors (TIMP-1 and -2), TGF-β1 and activated caspase 3. Results Our results show that Hhcy induced by an excess of methionine causes, in the myocardium of Psammomys obesus, a significant accumulation of fibrillar collagens I and III at the interstitial and perivascular scales, indicating the appearance of fibrosis, which is associated with an immuno-expression increase of TGF-β1, MMP-9 and TIMP-2 and an immuno-expression decrease of MMP-2 and TIMP-1. Also, Hhcy induces apoptosis of some cardiomyocytes and cardiac fibroblasts by increasing of activated caspase 3 expression. These results highlight a remodeling of cardiac tissue in hyperhomocysteinemic Psammomys obesus.

Role of bone marrow-derived stem cells versus insulin on filiform and fungiform papillae of diabetic albino rats (light, fluorescent and scanning electron microscopic study) Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Rania Osama Mohamed Mohsen, Ahmed M. Halawa, Rabab Hassan Abstract Background Diabetes mellitus (DM) is a chronic metabolic disease characterized by high blood glucose levels. DM affects many body’s organs and caused by insulin production deficiency or by the ineffectiveness of the produced insulin. Administration of exogenous insulin is required for management of type I DM; however, it does not cure the disease. Bone marrow-mesenchymal stem cells (BM-MSCs) have been highlighted to offer a novel cell based approach for treatment of diabetes because of their anti-diabetic effect, direct differentiation into a variety of cell types, or release of paracrine factors. Aim To examine the effect of BM-MSCs versus insulin on true filiform and fungiform papillae of diabetic rats. Materials and methods Fifty six male Wistar albino rats weighing 200–250 g were equally divided into: Control group (Gp I): Rats did not receive any drug. Diabetic group (Gp II): Rats received a single intra-peritoneal injection of streptozotocin (40 mg/kg). BM-MSCs treated diabetic group (Gp III): After DM confirmation; rats received a single intravenous injection of BM-MSCs (million units) through tail vein. Insulin treated diabetic group (Gp IV): After DM confirmation; rats received a daily subcutaneous injection of insulin (5IU/kg). After four weeks, half of the tongue specimens were processed and stained by Hematoxyline & Eosin and Anti-proliferating cell nuclear antigen (Anti-PCNA) then examined by light microscope. Fluorescent microscope was used to detect homing of injected labeled BM-MSCs in rats’ filiform and fungiform papillae. While the other half were examined by scanning electron microscope. Results True filiform and fungiform papillae of Gp II showed significant histological and morphological alterations. In treated groups, Gp III and Gp IV, both papillae showed marked improvements, being more noticeable in Gp IV. There was a significant increase in the number of Anti-PCNA positive cells and a significant decrease in fasting blood glucose level in Gp III and Gp IV in comparison to Gp II. Conclusions DM had degenerative effects on true filiform and fungiform papillae. Administration of BM-MSCs reduced the deleterious effects of DM on both papillae. Insulin injection caused more obvious improvements in both papillae of diabetic rats than BM-MSCs.

Hyperoside protects against heart failure-induced liver fibrosis in rats Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Xiao Guo, Chengzhan Zhu, Xiaojun Liu, Yiping Ge, Xianyan Jiang, Wei Zhao Abstract Heart failure (HF) is an end-stage of various serious cardiovascular diseases, which causes liver injury. Hyperoside has been reported to exert protective effect on liver injury and fibrosis. However, the role and related mechanisms of hyperoside in HF-induced liver fibrosis are still unclear. In the current study, we established a model of HF via aortocaval fistula (ACF) in rats in vivo. Hyperoside treatment in ACF rats increased cardiac output, the maximum peak rate of rise/fall in left ventricular pressure (+dP/dt, -dP/dt) and LV ejection fraction (LVEF), decreased LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV), and reduced heart weight/body weight ratio in a dose-dependent manner. Moreover, hyperoside could attenuate liver fibrosis and injury in ACF rats, as evidenced by reduction of fibrosis area and hydroxyproline content, amelioration of edema and degeneration of liver cell vacuoles, and inhibition of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. Further, α-smooth-muscle actin (α-SMA), collagen I, profibrotic factor-connective tissue growth factor (CTGF), matrix metalloproteinase-2 (MMP2) and MMP9 levels were down-regulated in hyperoside-treated ACF rats. Additionally, hyperoside inhibited the activation of TGF-β1/Smad pathway. Finally, we confirmed that hyperoside suppressed TGF-β1-mediated hepatic stellate cell activation in vitro. Collectively, hyperoside showed a suppressive role in HF-induced liver fibrosis and injury.

High quality human sperm selection for IVF: A study on sperm chromatin condensation Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Aslihan Saylan, Sevilay Erimsah Abstract The study consisted of semen samples of 20 male individuals who applied to Abant Izzet Baysal University Faculty of Medicine and participated in a spermiogram. The aim of this study was to determine how to obtain the healthiest spermatozoa by employing a variety of swim-up methods over differing time periods and without the use of centrifuge. Ejaculate samples were taken from the 20 patients and each patient's homogenized semen sample was divided into 4 groups without centrifugation. Group 1 was taken as the sample of untreated semen. For the other 3 groups, 250 μl of medium was added in the semen samples. Afterwards, the samples were kept at 37 °C for different time periods, 30 min for Group 2, 60 min for Group 3 and 90 min for Group 4 in order for the spermatozoa to swim to the media in the upper layer. At the end of the periods, 10 μl of propagation preparations were prepared from the swim-up fluid. Using Aniline Blue for chromatin condensation analysis, two hundred cells were immunostained by Caspase 3 for apoptotic analysis. Subsequently, the result of the four groups were compared for each test. The spermatozoa obtained at the end of the 30 min. of swim-up was compared to the spermatozoa obtained from the swim-up of 60 min., the swim-up of 90 min. It was found that the control group had statistically significant lower rates of apoptosis and was healthier in terms of chromatin integrity. The swim-up method without centrifugation is the best suited sperm preparation, based on sperm DNA integrity and sperm chromatin condensation

Comparison of incremental concentrations of micron-sized superparamagnetic iron oxide for labelling articular cartilage derived chondroprogenitors Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Elizabeth Vinod, Jithu Varghese James, Upasana Kachroo, Solomon Sathishkumar, Abel Livingston, Boopalan Ramasamy Abstract Introduction In vivo tracking of labelled cells can provide valuable information about cellular behavior in the microenvironment, migration and contribution of transplanted cells toward tissue regeneration. Articular cartilage derived chondroprogenitors (CPs) show promise as a candidate for cell-based therapy as they have been classified as mesenchymal stem cells with inherent chondrogenic potential. Iron oxide labelling is known to withstand harsh processing techniques known to be associated with staining of osteochondral specimens. Aim and methods The aim of our study was to investigate the feasibility of labelling CPs with micron-sized super paramagnetic iron oxide (M-SPIO) particles and to study the effects of this approach on the labelling efficiency, viability, maintenance of phenotype and potential for differentiation. Human CPs were isolated using fibronectin adhesion assay, passage 2 cells were labelled using three concentrations of M-SPIO (12.75 μg/ml, 25.5 μg/ml and 38.25 μg/ml). At sub confluence, cells were assessed for a) iron uptake by Prussian blue stain and colorimetry b) viability using 7-amino actinomycin D, c) MSC marker expression by flow cytometric analysis and d) trilineage differentiation potential. Results and conclusion Iron uptake was higher with increase in M-SPIO concentration whereas CD73, CD90 marker expression significantly decreased and chondrogenic potential appreciably reduced with increase in M-SPIO concentration. In conclusion, 12.75 μg/ml M-SPIO can successfully label human articular cartilage derived chondroprogenitors with minimal effect on cellular viability, MSC marker expression and potential for differentiation.

The protective effect of Stevia rebaudiana Bertoni on serum hormone levels, key steroidogenesis enzymes, and testicular damage in testes of diabetic rats Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Fatemeh Gholizadeh, Sanaz Dastghaib, Farhad Koohpeyma, Elahe Bayat, Pooneh Mokarram Abstract Diabetes Mellitus (DM) is a kind of metabolic endocrine diseases, which has various effects on the gonadal system. The current study aimed to examine the effect of Stevia rebaudiana Bertoni extract on the mRNA expression involved in testosterone synthesis, and stereological parameters in rat testes, for improving diabetes complications. In this study, 48 rats were randomly classified into control, diabetic (streptozocin 60 mg/kg + nicotinamide 120 mg/kg), diabetic + Stevia (400 mg/kg), and diabetic + metformin (500 mg/kg) groups. Finally, Fasting Blood Sugar (FBS) level, the serum level of LH and testosterone, the Star, Cyp11a1, and Hsd17b3 gene expressions, and changes in the testis histology were evaluated. The results indicated a decrease in body weight, serum LH and testosterone level, the star gene expression, stereological changes of testes, and an increase in the FBS level in diabetic group, compared with the control group (P<0.05). Nonetheless, Stevia significantly reduced the FBS and increased the serum LH level, in comparison with diabetic rats (P<0.05), but no significant differences in the serum testosterone level and the Star gene expression has been found. Stevia also resulted in an increase in weight, testis volume, the number of sexual lineage cells, and sperm count and motility, compared to diabetic rats (P<0.05). Due to its antioxidant properties, Stevia enhanced the alteration in spermatogenesis and stereological characteristics in diabetic rat testes. Hence, Stevia could diminish the reproductive system problems and improve infertility in diabetic male rats. Graphical abstract

Chronic valproate treatment influences folliculogenesis and reproductive hormones with possible ameliorating role for folic acid in adult albino rats Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Ibrahim Hassan Ibrahim, Adel Mohamed Aboregela, Rehab Hassan Elbanna Gouda, Khaled Adel Eid Abstract Sodium Valproate (VPA) is known to have deleterious consequences on ovarian function and folliculogenesis. Folic acid (FA) is associated with the quality of many parameters in folliculogenesis. Therefore, we aimed to investigate the effects of chronic Valproate administration on ovarian morphology, folliculogenesis, reproductive hormones, and the possible protective effect of Folic acid supplementation. Forty adult female albino rats were divided into four groups and treated orally for 90 days as follows: Control group received distilled water; FA group received (folic acid 400 μg/day); VPA group received (Na Valproate 200 mg/kg/day) and VPA + FA group received (Na Valproate 200 mg/kg/day + folic acid 400 μg/day). In addition, ovaries were processed for routine histology and immunohistochemistry (TGFβ1 and PCNA) and reproductive hormones levels were measured. Results showed a significant decrease in number of follicles in VPA group, while atretic follicles increased compared with control group (P < 0.001). Interestingly, the number of follicles significantly increased in VPA + FA group compared with VPA group (P < 0.001). Also, number of atretic follicles significantly decreased in the VPA + FA group compared to the VPA group. Histochemistry score decreased for TGFβ1 and PCNA staining in VPA group compared with control group (P < 0.01). Moreover, Valproate demonstrated a significant increase in testosterone levels in VPA group than control group (P < 0.001). However, VPA group demonstrated a significant decrease in levels of estradiol, progesterone, FSH and LH levels compared with control group. These changes were partially improved in VPA + FA group. In conclusion, FA co-treatment can modulate ovarian follicular and hormonal disturbances induced by valproate, which needs further investigations to identify the precise mechanisms.

3D analysis of morphological alterations of the fibroblastic reticular cells in reactive and neoplastic human lymph nodes Publication date: October 2019 Source: Acta Histochemica, Volume 121, Issue 7 Author(s): Marvin Siegfried Oswald, Patrick Wurzel, Martin-Leo Hansmann Abstract Histopathological methods based on 2 μm thin sections are routinely used in pathological anatomical diagnosis. Many medical disciplines already rely on a 3D representation, regarding visualization and imaging techniques. Pathology in particular uses different tissue visualizations to make the final diagnosis. Thereby, a standard 2D histological section only represents a flat snapshot of a three-dimensional complex cell system. Despite that, 3D cell analysis is not yet standardly used in clinical routine. This work used 3D analysis systems to investigate the morphological alterations of the fibroblastic reticular cell network inside human lymph nodes during neoplastic transformation and evaluates the added value of 3D visualizations in tissue interpretation. We investigated the surface and volume quotient, cell cross-linking and percentage cell volume of the fibroblastic reticular cell (FRC) network inside Lymphadenopathy (follicular hyperplasia) (LAD), Follicular Lymphoma Grade 1 (FL1), Nodular Sclerosis classical Hodgkin Lymphoma (NScHL) and Angioimmunoblastic T-Cell Lymphoma (AITL). We found that the average quotient of LAD and FL1 differed from those of NScHL and AITL, indicating that the surface and volume quotient changes in the course of neoplastic transformation. This is probably due to an increased network convolution, while the total cell volume remains the same at about 2%. In conclusion, this paper describes the tumor-related morphological changes of the FRC network, which would have been difficult to achieve without the use of 3D analysis systems.