European Journal of Histochemistry <p>The <strong>European Journal of Histochemistry&nbsp;</strong>has been an influential cytology journal for over 60 years, publishing research articles on functional cytology and histology in animals and plants. The&nbsp;<strong>European Journal of Histochemistry&nbsp;</strong>offers original research articles investigating on structural and molecular components performed by histochemical and immunohistochemical methods, at light and electron microscopy, cytometry and imaging techniques.</p> <p>Areas of particular interest include cell differentiation, senescence and death, and cell-cell interactions in normal and pathological tissues; attention is also given to articles on newly developed or originally applied histochemical and microscopical techniques.</p> <p>Since its foundation in 1954,&nbsp;the <strong>European Journal of Histochemistry&nbsp;</strong>is the official organ of the Italian Society of Histochemistry.</p> <h3>The Proceedings of the XXXIX National Meeting of the Italian Society for the Study of Connective Tissues (SISC) are available <a href="">here</a>.</h3> en-US <p><strong>PAGEPress</strong> has chosen to apply the&nbsp;<a href="" target="_blank" rel="noopener"><strong>Creative Commons Attribution NonCommercial 4.0 International License</strong></a>&nbsp;(CC BY-NC 4.0) to all manuscripts to be published.<br><br>An Open Access Publication is one that meets the following two conditions:</p> <ol> <li class="show">the author(s) and copyright holder(s) grant(s) to all users a free, irrevocable, worldwide, perpetual right of access to, and a license to copy, use, distribute, transmit and display the work publicly and to make and distribute derivative works, in any digital medium for any responsible purpose, subject to proper attribution of authorship, as well as the right to make small numbers of printed copies for their personal use.</li> <li class="show">a complete version of the work and all supplemental materials, including a copy of the permission as stated above, in a suitable standard electronic format is deposited immediately upon initial publication in at least one online repository that is supported by an academic institution, scholarly society, government agency, or other well-established organization that seeks to enable open access, unrestricted distribution, interoperability, and long-term archiving.</li> </ol> <p>Authors who publish with this journal agree to the following terms:</p> <ol> <li class="show">Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.</li> <li class="show">Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.</li> <li class="show">Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.</li> </ol> (Nadia Moscato) (Tiziano Taccini) Fri, 10 Jan 2020 10:14:59 +0000 OJS 60 Immuno- and glyco-histochemistry as a tool to evaluate the oregano supplemented feed effects in pig gut <p>Among oregano properties, its antioxidant and antibacterial effects are particularly interesting. Oregano is also able to induce a higher glycoconjugate production in gut, creating a physical barrier against microorganisms. This study evaluated the effects of adding an aqueous extract of oregano (OAE) to the diet of two homogenous groups of pigs during the finisher phase. The diets were as follows: control commercial diet (CTR group) and CTR diet supplemented (2 g/kg) with OAE (O group). Samples of ileum and caecum from the two groups were examined by conventional histochemistry to analyze complex carbohydrates and by immunohistochemistry to detect Bcl-2 Associate X protein (BAX), an indicator of oxidative stress. Glyco-histochemistry showed significant differences between the two groups. Immunohistochemistry revealed a lower presence of BAX in O group. The OAE supplementation improved the production of glycoconjugates, able to enhance in pig the protection of intestinal mucosa by means of direct and indirect defense actions. The reduced BAX immunostaining observed in O group may be an indicator of enhanced antioxidant action promoted by oregano. The results of this study can be used in further research to identify ways to improve endogenous defence ability, with the aim of reducing antibiotic use and preventing antimicrobial resistance.</p> Cecilia Dall'Aglio, Francesca Mercati, Valerio Faeti, Gabriele Acuti, Massimo Trabalza Marinucci, Elena De Felice, Federico Maria Tardella, Maria Pia Franciosini, Patrizia Casagrande Proietti, Daniele Catorci, Paolo Stacchini, Augusto Pastorelli, Paola Scocco Copyright (c) 2020 The Author(s) Thu, 20 Feb 2020 09:51:57 +0000 Expression, localization and synthesis of small leucine-rich proteoglycans in developing mouse molar tooth germ <p>The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. <em>In situ</em> hybridization, immunohistochemistry, and organ culture with metabolic labeling of [<sup>35</sup>S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, <em>decorin</em> mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. <em>Biglycan </em>mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both <em>decorin </em>and<em> biglycan </em>mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. <em>Decorin </em>mRNA was expressed earlier in newly differentiating odontoblasts than <em>biglycan</em>. With odontoblast maturation and dentin formation, <em>decorin </em>mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, <em>biglycan </em>mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of <em>fibromodulin </em>and <em>lumican</em> mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.</p> Angammana Randilini, Kaoru Fujikawa, Shunichi Shibata Copyright (c) 2020 The Author(s) Mon, 10 Feb 2020 13:16:17 +0000 Can the AGE/RAGE/ERK signalling pathway and the epithelial-to-mesenchymal transition interact in the pathogenesis of chronic rhinosinusitis with nasal polyps? <p>Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent sinonasal mucosa inflammatory disease with still unclear pathophysiologic mechanisms that imply events of tissue repair and structural remodelling. Several cascades seem to have a considerable role in the onset and progression of mucosa hyperproliferation in nasal polyps including transforming growth factor β/Small mother against decapentaplegic (TGFβ/Smads), mitogenactivated protein kinases (MAPKs), advanced glycosylation end-products (AGEs) together with epithelial-tomesenchymal transition (EMT). Since many inflammatory mediators are reported to play important roles in the development of nasal polyps (NP) disease, this study aimed to analyse the correlation between the AGEs/receptor of advanced glycosylation end-products (RAGE)/extracellular signal-regulated kinase (ERK) signalling pathway and the main markers of EMT to better understand the influence that they exert on the remodelling of nasal mucous membranes in patients affected by CRSwNP vs normal controls. A total of 30 patients were enrolled in this study. Immunohistochemical analysis, using AGE, RAGE, p-ERK, MMP-3, TGF-β1, Smad2/3, Collagen I-III, α-SMA, E-cadherin, IL-6 and Vimentin antibodies, was performed. AGE, RAGE, ERK, p-ERK and MMP3 were also evaluated using western blot analysis. We observed an overexpression of the AGE/RAGE/p-ERK and the main mesenchymal markers of EMT (Vimentin and IL-6) in CRSwNP <em>vs</em> controls whereas the TGF-β/Smad3 pathway did not show any significant differences between the two groups of patients. These observations suggest a complex network of processes in the pathogenesis of NP, and the AGE/RAGE/ERK pathway and EMT might work together in promoting tissue remodelling in the formation of CRSwNP.</p> Antonella Vetuschi, Simona Pompili, Gian Piero Di Marco, Federico Calvaruso, Enzo Iacomino, Lidia Angelosante, Claudio Festuccia, Alessandro Colapietro, Roberta Sferra Copyright (c) 2020 The Author(s) Thu, 23 Jan 2020 10:40:47 +0000 Immunolocalization of aromatase P450 in the epididymis of Podarcis sicula and Rattus rattus <p>The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard <em>Podarcis sicula</em>, a seasonal breeder, and <em>Rattus rattus</em>, a continuous breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both <em>P. sicula</em> and <em>R. rattus</em>. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of <em>P. sicula</em> epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of <em>P. sicula</em> epididymis, whereas in <em>R. rattus</em> it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.</p> Luigi Rosati, Marina Prisco, Mariana Di Lorenzo, Maria De Falco, Piero Andreuccetti Copyright (c) 2020 The Author(s) Mon, 27 Jan 2020 14:05:34 +0000 Nuclear factor-kB and nitric oxide synthases in red blood cells: good or bad in obesity? A preliminary study <p>Emerging evidence suggests that red blood cells (RBCs) are involved in many functions essential for life. Nuclear factor-kB (NF-kB), nitric oxide synthases (inducible nitric oxide synthase -iNOS-, endothelial nitric oxide synthase -eNOS-) and interleukin-1β (-IL-1β-) are all proteins that have been identified in RBCs. In nucleated cells, such as white blood cells (WBCs), these proteins have well investigated roles, linked to stress and inflammation. It is not the same in erythrocytes, for this reason, we considered obese patients for studying the morphology of RBCs. We studied a possible correlation between their morphological changes and several protein expressions. Moreover, we compared the results about the aforementioned proteins and antioxidant markers with those obtained in WBCs from healthy and obese patients before and after omega-3 polyunsaturated fatty acid supplementation. This latter scientific point is important in order to determine whether there are differences in the expression of nucleated and anucleated cells. The morphology of RBCs changed in obese patients, but it is significantly restored after six weeks of supplementation. The expression of antioxidant enzymes changed in RBCs and WBCs in obesity but all proteins restore their positivity after supplementation. We found that: the presence of NF-kB, antioxidant enzymes and eNOS in healthy RBCs could indicate a role of these proteins as regulators of cellular metabolism; obese WBCs showed a higher NF-kB, iNOS and IL-1β positivity, whereas eNOS presence did not significantly change in these cells. We tried to explain the different positivity of NF-kB, proposing a dual role for this protein, as prolifespan and as proinflammatory processes, depending on examined cells. In conclusion, we have considered the literature that focuses on the omega-6/omega-3 ratio. The ratio changed from the past, especially in people whose diet is strongly westernized worsening the state of health of the patient and leading to an higher incidence of obesity. Our study hypothesizes that the supplementation could help to restore the correct ratio<strong>.</strong></p> Monika Široká, Caterina Franco, Zuzana Guľašová, Zdenka Hertelyová, Vladimíra Tomečková, Luigi F. Rodella, Rita Rezzani Copyright (c) 2020 The Author(s) Tue, 21 Jan 2020 08:56:01 +0000 Anastomotic healing in a rat model of peritonitis after non-steroidal anti-inflammatory drug administration <p>The tissue inflammatory response can influence the outcome of anastomotic healing. Anastomotic leakage represents a dreadful complication after gastrointestinal surgery, in particular sepsis and intra-abdominal infections impair the restorative process of colic anastomoses. It has been debated whether the administration of non-steroidal anti-inflammatory drugs (NSAIDs) is a risk factor for dehiscence, since many patients receive NSAIDs in the early postoperative period. Our aim was, for the first time, to analyze the morpho-functional effects of postoperative administration of two commonly used NSAIDs, Diclofenac and Ketorolac, on the healing process of colo-colic anastomoses constructed under condition of fecal peritonitis in a rat model. Sixty adult male rats underwent two surgical procedures: peritonitis induction and colo-colic anastomosis, and were divided into three groups: 20 rats received saline; 20 rats 4 mg/kg Diclofenac and 20 rats 5 mg/kg Ketorolac. We assessed anastomosis strength, morphological features of tissue wound healing, immunohistochemical metalloproteinase 9 (MMP9) expression and collagen deposition and content by Sirius red staining and hydroxyproline level. We found no significant difference in bursting pressure, collagen content and organization and morphological features between the groups, except a significantly reduced presence of inflammatory cells and MMP9 expression in the groups treated with NSAIDs. Our findings showed that Diclofenac and Ketorolac administration did not affect post-surgical healing and did not increase the leakage risk of colo-colic anastomoses during peritonitis.</p> Roberto Ghiselli, Guendalina Lucarini, Monica Ortenzi, Eleonora Salvolini, Stefania Saccomanno, Fiorenza Orlando, Mauro Provinciali, Fabio Casciani, Mario Guerrieri Copyright (c) 2020 The Author(s) Fri, 10 Jan 2020 10:14:07 +0000 Morphological analysis of the seeds of three pseudocereals by using light microscopy and ESEM-EDS <p>The seed morphology of three Pseudocereal Grains (PSCg), <em>i.e.</em> quinoa (<em>Chenopodium quinoa </em>Willd<em>, </em>Chenopodiaceae), buckwheat (<em>Fagopyrum esculentum </em>Moench<em>, </em>Polygonaceae) and amaranth (<em>Amaranthus caudatus </em>L.<em>, </em>Amaranthaceae) was studied by light microscopy (LM) and Environmental Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy (ESEM-EDS). LM was used with visible light to evaluate either unstained sections or sections stained with Azan mixture and with fluorescent light. The aim of the study was to compare the architecture of the three seeds in order to connect their morphology with nutrient localization. The Azan staining allowed for the visualization of the seed coat, the embryo - with its shoot apical meristem - and the radicle cell layers, whereas the use of fluorescent microscopy identified the cells rich in phenolic compounds. Finally, the ESEM-EDS analysis revealed that the seed coat of the quinoa was thinner than that of amaranth or buckwheat. In all PSCg, starch granules appeared to be located in large polygonal cells, surrounded by a thin cell wall. Several globoids of proteins were observed in the embryo cells. In the radicle section, the vascular bundles of the procambium were evident, while Amaranth only showed a consistent layer of calcium crystals, located between the embryo and the perysperm. The morphological differences of the three PSCg were discussed in the context of their structural resistance to processing technologies which impact on nutritional value of derived foods.</p> Paolino Ninfali, Anna Panato, Federica Bortolotti, Laura Valentini, Pietro Gobbi Copyright (c) 2020 The Author(s) Fri, 10 Jan 2020 10:00:09 +0000 Chronic cypermethrin exposure alters mouse embryonic stem cell growth kinetics, induces Phase II detoxification response and affects pluripotency and differentiation gene expression <p>Worldwide uncontrolled use of synthetic pyrethroids contaminates water and soil leading to health hazards. Cypermethrin (CYP), the most used pyrethroid, induces detrimental effects on adults and embryos at different stages of development of several vertebrate species. In Mammals, CYP-induced alterations have been previously described in adult somatic cells and in post-implantation embryos. It remains unknown whether CYP has effects during pre-implantation development. Studies to access pre-implantation embryo toxicity are complicated by the restricted number of blastocysts that may be obtained, either <em>in vivo</em> or <em>in vitro</em>. Embryonic stem cells (ESCs) are an <em>in</em> <em>vitro</em> model study that overcomes these limitations, as millions of pluripotent cells are available to the analysis. Also, ESCs maintain the same pluripotency characteristics and differentiation capacity of the inner cell mass (ICM) present in the blastocyst, from which they derive. In this work, using mouse R1 ESCs, we studied CYP-induced cell death, ROS production, the activation of oxidative stress-related and detoxification responses and the population growth kinetics following 72 h exposure at the 0.3 mM LD50 dose. Also, the expression levels of pluripotency genes in exposed ESCs and of markers of the three germ layers after their differentiation into embryoid bodies (EBs) were determined. Two apoptotic waves were observed at 12-24 h and at 72 h. The increase of ROS production, at 24 h until the end of the culture period, was accompanied by the induction, at 48 h, of redox-related <em>Cat</em>, <em>Sod1</em>, <em>Sod2</em>, <em>Gpx1</em> and <em>Gpx4</em> genes. Up-regulation of <em>Cyp1b1</em>, but not of <em>Cyp1a1</em>, phase I gene was detected at 72 h and induction of <em>Nqo1</em>, <em>Gsta1</em> and <em>Ugt1a6</em> phase II genes began at 24 h exposure. The results show that exposed R1 ESCs activate oxidative stress-related and detoxification responses, although not sufficient, during the culture period tested, to warrant recovery of the growth rate observed in untreated cells. Also, CYP exposure altered the expression of <em>Oct-4</em> and <em>Nanog</em> pluripotency genes in ESCs and, when differentiated into EBs, the expression of <em>Fgf5</em>, <em>Brachyury</em> and <em>Foxa2</em>, early markers of the ectoderm, mesoderm and endoderm germ layers, respectively. NIH/3T3 cells, a differentiated cell line of embryonic origin, were used for comparison.</p> Paola Rebuzzini, Cinzia Civello, Edouard Nantia Akono, Lorenzo Fassina, Maurizio Zuccotti, Silvia Garagna Copyright (c) 2020 The Author(s) Mon, 17 Feb 2020 10:26:38 +0000