Acta Histochemica

Comparison of incremental concentrations of micron-sized superparamagnetic iron oxide for labelling articular cartilage derived chondroprogenitors Publication date: Available online 18 July 2019 Source: Acta Histochemica 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.

Expression patterns of male germ cell markers in cryptorchid pig testes Publication date: Available online 17 July 2019 Source: Acta Histochemica 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.

Chronic valproate treatment influences folliculogenesis and reproductive hormones with possible ameliorating role for folic acid in adult albino rats Publication date: Available online 12 July 2019 Source: Acta Histochemica 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: Available online 5 July 2019 Source: Acta Histochemica 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.

Effects of short and long-term alcohol-based fixation on Sprague-Dawley rat tissue morphology, protein and nucleic acid preservation Publication date: Available online 3 July 2019 Source: Acta Histochemica Author(s): Simona Panzacchi, Federica Gnudi, Daniele Mandrioli, Rita Montella, Valentina Strollo, Bruce Alexander Merrick, Fiorella Belpoggi, Eva Tibaldi Abstract Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory’s trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation. Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.

Expression and localization analyses of the cholinergic anti-inflammatory pathway and α7nAchR in different tissues of rats with rheumatoid arthritis Publication date: Available online 3 July 2019 Source: Acta Histochemica Author(s): Zhen Li, Huiqin Hao, Yuting Gao, Ze Wang, Wenjing Lu, Jin Liu Abstract Rheumatoid arthritis (RA) is a complicated chronic multisystem autoimmune disease, wherein the inflammatory cascade leads to vasospasm and osteoclastogenesis, which ultimately results in bone and cartilage destruction. In this study, we investigated the expression and localization of the alpha-7 nicotinic receptor (α7nAchR) gene CHRNA7 in the heart, liver, spleen, lung, kidney, and joints of the collagen-induced arthritis (CIA) rat model. The CHRNA7 mRNA and protein expression levels in these tissues of rats from CIA and normal groups were analyzed via real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The cellular localization of CHRNA7 protein was determined via immunohistochemistry (IHC) assays. CHRNA7 was expressed at varying levels in different tissues of rats from the groups, among which joints showed significantly higher CHRNA7 expression levels than other tissues (P < 0.05). CIA rats had significantly higher CHRNA7 expression levels in the spleen and joints than the control group rats (P < 0.05). Positive expression signals for CHRNA7 were detected in various tissues of CIA and control group rats, among which strong positive signals were detected in joint fibroblast-like synoviocytes (FLSs), endothelial cells, stromal cells, and macrophages. Our results further confirmed the involvement of the CAP in the onset and development of inflammatory responses in RA, suggesting that CHRNA7 may be a new therapeutic target for RA. This study is of great clinical and theoretical significance for understanding the differential expression of CHRNA7in various tissues and cholinergic anti-inflammatory pathway (CAP)-targeted treatment of RA. Graphical abstract CHRNA7 was expressed at varying levels in different tissues of rats from the groups, among which joints showed significantly higher CHRNA7 expression levels than other tissues (P < 0.05). CIA rats had significantly higher CHRNA7expression levels in the spleen and joints than the control group rats (P < 0.05). Positive expression signals for CHRNA7 were detected in various tissues of CIA and control group rats, among which strong positive signals were detected in joint fibroblast-like synoviocytes (FLSs), endothelial cells, stromal cells, and macrophages. Our results further confirmed the involvement of the CAP in the onset and development of inflammatory responses in RA, suggesting that CHRNA7 is a new therapeutic target for RA. This study is of great clinical and theoretical significance for understanding the differential expression of CHRNA7 in various tissues and cholinergic anti-inflammatory pathway (CAP)-targeted treatment of RA.

Effect of acrylamide on BEAS-2B normal human lung cells: Cytotoxic, oxidative, apoptotic and morphometric analysis Publication date: July 2019 Source: Acta Histochemica, Volume 121, Issue 5 Author(s): Sedat Kacar, Varol Sahinturk, Hatice Mehtap Kutlu Abstract Due to the broad toxic relevance of acrylamide, many measures have been taken since the 1900s. These measures increased day by day when acrylamide was discovered in foods in 2002, and its toxic spectrum was found to be wider than expected. Therefore, in some countries, the products with higher acrylamide content were restricted. On the other hand, the effects of acrylamide on the respiratory system cells have yet to be well understood. In this study, we aimed at investigating the effect of acrylamide on lung epithelial BEAS-2B cells. Initially, the cytotoxic effect of acrylamide on BEAS-2B was determined by MTT assay. Then, cellular oxidative stress was measured. Flow cytometry analysis was conducted for Annexin-V and caspase 3/7. Furthermore, Bax, Bcl-2 and Nrf-2 proteins were evaluated by immunocytochemistry. Finally, acrylamide-induced cellular morphological changes were observed under confocal and TEM microscopes. According to MTT results, the IC50 concentration of acrylamide was 2.00 mM. After acrylamide treatment, oxidative stress increased dose-dependently. Annexin V-labelled apoptotic cells and caspase 3/7 activity were higher than untreated cells in acrylamide-treated cells. Immunocytochemical examination revealed a marked decrease in Bcl-2, an increase in Bax and Nrf-2 protein staining upon acrylamide treatment. Furthermore, in confocal and TEM microscopy, apoptotic hallmarks were pronounced. In the present study, acrylamide was suggested to display anti-proliferative activity, decrease viability, induce apoptosis and oxidative stress and cause morphological changes in BEAS-2B cells.

Molecular cloning and cellular expression of the cholesterol synthesizing enzymes during the prenatal development of the optic nerve in the dromedary camel (Camelus Dromedarius) Publication date: July 2019 Source: Acta Histochemica, Volume 121, Issue 5 Author(s): Elsayed Metwally, Sameh Mohamed Farouk, Abdel-Hamid Kamel Osman Abstract The Cholesterol-synthesizing proteins (HMGCS1 and HMGCS2) are mitochondrial enzymes that believed to catalyze the first reaction of ketogenesis, the process by which energy is provided from fats in the absence of carbohydrates. Typically, astrocytes developed from its progenitor cells in the embryonic optic nerve and enriched with HMGCS1 and 2. However, the detailed histomorphology of camel HMGCS1 and 2 remains to be clearly defined. Here, we investigated the changes that associate with astrocytes differentiation within the developing camel optic nerve. Firstly, we isolated cDNAs encoding HMGCS1 and 2 from the optic nerve. Then, we found that HMGCS1 shared high similarity to human, while HMGCS2 showed a lower similarity and was more diverse. Immunohistochemical studies revealed that distinct correlation of astrocytes differentiation with HMGCS1 and 2 expressions in the developing camel optic nerve. Both encoded proteins were localized throughout the cytoplasm, as well as the nuclei of the astrocytes. In addition, semi-quantitative PCR analysis and western analysis confirmed that both HMGCS1 and 2 were highly expressed in camel optic nerve as well as other tissue, but they were lower in both skeletal and heart muscles. Moreover, various stains such as Sudan black and florescence filipin stains were used to visualize the free cholesterol in the astrocytes, indicating the enzymatic activity of HMGCS1 and 2. Together, our study reported the first comprehensive investigation of the molecular cloning and cellular expression of HMGCS1 and 2 in the optic nerve of dromedary camel.

Impact of renal ischemia/reperfusion injury on the rat Kupffer cell as a remote cell: A biochemical, histological, immunohistochemical, and electron microscopic study Publication date: July 2019 Source: Acta Histochemica, Volume 121, Issue 5 Author(s): Sara Mohamed Naguib Abdel Hafez, Rehab Ahmed Rifaai, Asmaa M.A. Bayoumi Abstract Almost all transplanted solid organs are exposed to some degree of ischemia-reperfusion (IR) damage. It is interesting to know that this IR damage affects various remote tissues including the liver and resulted in serious adverse effects. Liver injury triggers different responses of liver tissue especially Kupffer cells (KCs). The goal of this current study is to assess the biochemical and morphological changes of hepatic KCs after the induction of renal ischemia-reperfusion (RIR) and point out their role in remote liver injury after RIR. Sixteen male Sprague-Dawley rats were randomly divided into two equal groups: Group I; sham group. Group II; renal ischemia reperfusion (IR) group in which rats were exposed to renal ischemia for 45 min followed by renal reperfusion for 48 h. Three rats from each group were subjected to charcoal injection to evaluate KCs activity. Specimens of rat liver from each group were obtained and processed for biochemical, light microscopic and ultramicroscopic examination. The current results showed elevated serum levels of AST and ALT. The liver HGF-α protein expression increased in IR group compared to the sham group. In IR group, numerous charcoal labeled KCs were observed mainly localized around the central vein. Scanning electron micrographs showed complex primary and secondary foot process of the KCs. Ultrastructural study showed KCs with multiple cytoplasmic vacuoles, lysosomes and mitochondria, rough endoplasmic reticulum and ribosomes. Immuno-histochemical study showed more tumor necrosis factor-α (TNF-α) expression in KCs than the sham group. These results collectively demonstrated that renal IR produced biochemical and morphological changes in the liver KCs and theses cells might have a role in the remote liver injury after renal IR. This might be one of the mechanisms through which RIR affects the liver.

Histopathological, biochemical and molecular studies on the toxic effect of used engine oil on the health status of Oreochromis niloticus Publication date: July 2019 Source: Acta Histochemica, Volume 121, Issue 5 Author(s): Yasmine H. Ahmed, Dina W. Bashir, Dalia A. Abdel-moneam, Rehab A. Azouz, Mona K Galal Abstract The accidental spilling of petroleum oils into natural water resources expose fishes in the effluent area to serious problems.. Oreochromis niloticus were used in the current study as a model to investigate the toxicity of used engine oil and to evaluate the protective role of vitamin C against this toxicity. The oil concentration used in this study was previously determined to be 0.25 ml/l by 96 h-LC50. After 21 days of engine oil exposure, haematological and biochemical analyses revealed significant reduction in RBCs counts, haemoglobin concentrations and total proteins. However, ALT, AST and glucose levels were significantly increased by the end of the experiment indicating the damaging effects of the oil on fish tissues. Oxidative stress biomarkers were also measured; liver CAT activity was significantly decreased in the oil exposed group compared to control group, while MDA levels were significantly elevated. Histopathological examination showed the presence of several alterations in hepatic and branchial tissues in exposed group compared to the control group. Significant elevations in CYP1 A1 mRNA expression levels in hepatic tissue were also detected in the group exposed to used engine oil compared to the control group. However, supplementation of fishexposed to used engine oil with vitamin Csignificantly enhance the biochemical, oxidative and histological parameters.