European Journal of Histochemistry https://ejh.it/index.php/ejh The Journal publishes Original papers, Technical reports, Reviews, Brief Reports, Letters to the Editor, Book reviews, Views and Comments, concerning investigations performed with the aid of biophysical, biochemical, molecular-biological, enzymatic, immunohistochemical, cytometric, and image analysis techniques. PAGEPress Scientific Publications, Pavia, Italy en-US European Journal of Histochemistry 1121-760X <p><strong>PAGEPress</strong> has chosen to apply the&nbsp;<a href="http://creativecommons.org/licenses/by-nc/4.0/" 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>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>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>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>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>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> Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost Cyprinus carpio https://ejh.it/index.php/ejh/article/view/2932 <p>Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid <em>Cyprinus carpio</em>, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. &nbsp;In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the <em>Cyprinus carpio</em> central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.</p> Arianna Casini Rosa Vaccaro Mattia Toni Carla Cioni ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-07-24 2018-07-24 10.4081/ejh.2018.2932 Activation of the renin-angiotensin system in mice aggravates mechanical loading-induced knee osteoarthritis https://ejh.it/index.php/ejh/article/view/2930 <p>Epidemiological studies have shown an association between hypertension and knee osteoarthritis (OA). The purpose of this study was to investigate whether activation of the renin–angiotensin system (RAS) can aggravate mechanical loading-induced knee OA in mice. Eight-week-old male Tsukuba hypertensive mice (THM) and C57BL/6 mice were divided into running and non-running groups. Mice in the running group were forced to run (25 m/min, 30 min/day, 5 days/week) on a treadmill. All mice in the four groups (n=10 in each group) were euthanized after 0, 2, 4, 6, or 8 weeks of running or natural breeding. Cartilage degeneration in the left knees was histologically evaluated using the modified Mankin score. Expression of Col X, MMP-13, angiotensin type 1 receptor (AT1R), and AT2R was examined immunohistochemically. To study the effects of stimulation of the AT1R in chondrocytes by mechanical loading and/or Angiotensin II (AngII) on transduction of intracellular signals, phosphorylation levels of JNK and Src were measured in bovine articular chondrocytes cultured in three-dimensional agarose scaffolds. After 4 weeks, the mean Mankin score for the lateral femoral condylar cartilage was significantly higher in the THM running group than in the C57BL/6 running group and non-running groups. AT1R and AT2R expression was not detected at 0 weeks in any group but was noted after 4 weeks in the THM running group. AT1R expression was also noted at 8 weeks in the C57BL/6 running group. The expression levels of AT1R, COL X, and MMP-13 in chondrocytes were significantly higher in the THM running group than in the control groups. Positive significant correlations were noted between the Mankin score and the rate of AT1R-immunopositive cells, between the rates of AT1R- and Col X-positive cells, and between the rates of AT1R- and AT2R-positive cells. The phosphorylation level of JNK was increased by cyclic compression loading or addition of AngII to the cultured chondrocytes and was reversed by pretreatment with an AT1R blocker. A synergistic effect on JNK phosphorylation was observed between compression loading and AngII addition. Transgene activation of renin and angiotensinogen aggravated mechanical load-induced knee OA in mice. These findings suggest that AT1R expression in chondrocytes is associated with early knee OA and plays a role in the progression of cartilage degeneration. The RAS may be a common molecular mechanism involved in the pathogenesis of hypertension and knee OA.</p> Kotaro Yamagishi Ichiro Tsukamoto Fumihisa Nakamura Kazuhiko Hashimoto Kazuhiro Ohtani Masao Akagi ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-07-24 2018-07-24 10.4081/ejh.2018.2930 Effect of high-fat mixed lipid diet and swimming on fibre types in skeletal muscles of rats with colon tumours https://ejh.it/index.php/ejh/article/view/2945 <p>Skeletal muscle fibre types, whose characteristics are determined by myosin heavy chain (MyHC) isoforms, can adapt to changed physiological demands with changed MyHC isoform expression resulting in the fibre type transitions. The endurance training is known to induce fast-to-slow transitions and has beneficial effect in carcinogenesis, whereas the effect of an excessive fat intake and its interaction with the effect of swimming are less conclusive<em>.</em> Therefore, we studied the effect of high-fat mixed lipid (HFML) diet and long-term (21-week) swimming on fibre type transitions and their average diameters by immunohistochemical demonstration of MyHC isoforms in slow soleus (SOL), fast extensor digitorum longus (EDL), and mixed gastrocnemius medialis and lateralis (GM, GL) muscles, divided to deep and superficial portions (GMd, GMs, GLd, GLs), of sedentary and swimming Wistar rats with experimentally (dimethylhydrazine) induced colon tumours and fed either with HFML or low-fat corn oil (LFCO) diet. HFML diet induced only a trend for fast-to-slow transitions in SOL and in the opposite direction in GMd. Swimming triggered significant transitions in unexpected slow-to-fast direction in SOL, whereas in GMs the transitions had tendency to proceed in the expected fast-to-slow direction. The average diameters of fibre types were mostly unaffected. Hence, it can be concluded that if present, the effects of HFML diet and swimming on fibre type transitions were counteractive and muscle-specific implying that each muscle possesses its own adaptive range of response to changed physiological conditions.</p> Vika Smerdu Martina Perše ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-07-25 2018-07-25 10.4081/ejh.2018.2945 Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study https://ejh.it/index.php/ejh/article/view/2956 <p>A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis.&nbsp; These results, if confirmed by <em>in vitro</em> studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.</p> Roberta Sferra Simona Pompili Luca Ventura Caroline Dubuquoy Silvia Speca Eugenio Gaudio Giovanni Latella Antonella Vetuschi ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-07-31 2018-07-31 10.4081/ejh.2018.2956 Expression of acetylated tubulin in the postnatal developing mouse cochlea https://ejh.it/index.php/ejh/article/view/2942 <p>Acetylation tubulin is one of the major post-translational modifications of microtubules. Stable microtubules are well known to contain acetylated tubulin. Here, we examined the spatiotemporal expression of acetylated tubulin in the mouse cochlea during postnatal development. At postnatal day 1 (P1), acetylated tubulin was localized primarily to the auditory nerve inside the cochlea and their synaptic contacts with the inner and outer hair cells (IHCs and OHCs). In the organ of Corti, acetylated tubulin occurred first at the apex of pillar cells. At P5, acetylated tubulin first appeared in the phalangeal processes of Deiters’ cells. At P8, staining was maintained in the phalangeal processes of Deiters’ cells. At P10, labeling in Deiters’ cells extended from the apices of OHCs to the basilar membrane. Labeling was expressed throughout the cytoplasm of pillar cells. At P12, acetylated tubulin displayed prominent and homogeneous labeling along the full length of the pillar cells. Linear labeling was present mainly in the Deiters’ cell bodies underlying OHCs. Between P14 and P17, acetylated tubulin was strongly expressed in inner and outer pillar cells and Deiters’ cells in a similar pattern as observed in the adult, and labeling in these cells were arranged in bundles. In addition, acetylated tubulin was intensely expressed in stria vascularis, root cell bodies, and a small number of fibrocytes of the spiral ligament until the adult. In the adult mouse cochlea, immunostaining continued to predominate in Deiters’ cells and pillar cells. Immunolabeling formed cups securing OHCs basal portions, and continued presence of acetylated tubulin-labeled nerve terminals below IHCs was shown. Our results presented here underscored the essential role played by acetylated tubulin in postnatal cochlear development, auditory neurotransmission and cochlear mechanics.</p> Wenjing Liu Chuanxi Wang Hao Yu Shaofeng Liu Jun Yang ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-08-08 2018-08-08 10.4081/ejh.2018.2942 Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation https://ejh.it/index.php/ejh/article/view/2947 <p>Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and <em>in vivo</em> electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bone-staining revealed that MAR of alveolar bone was as similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining non-viral BMP-2/7 gene expression vector and <em>in vivo</em> electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.</p> Mariko Kawai Yohei Kataoka Junya Sonobe Hiromitsu Yamamoto Hiroki Maruyama Toshio Yamamoto Kazuhisa Bessho Kiyoshi Ohura ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-08-09 2018-08-09 10.4081/ejh.2018.2947 Cancer stem cell markers ALDH1 and CD44+/CD24- phenotype and their prognosis impact in invasive ductal carcinoma https://ejh.it/index.php/ejh/article/view/2943 <p>Breast cancer is a very heterogeneous disease. The intrinsic molecular subtypes can explain the intertumoral heterogeneity and the cancer stem cell (CSC) hypothesis can explain the intratumoral heterogeneity of this kind of tumor. CD44<sup>+</sup>/CD24<sup>-</sup> phenotype and ALDH1 expression are the major CSC markers described in invasive breast cancer. In the present study, 144 samples of invasive breast carcinoma, no special type were distributed in 15 tissue microarrays (TMA) and then evaluated for expression of the CD44<sup>+</sup>/CD24<sup>-</sup> phenotype and ALDH1 to understand the importance of these CSC markers and the clinical aspects of breast cancer. The samples were classified into four molecular subtypes according to clinicopathological criteria: Luminal A, Luminal B, HER2, and Basal-like. A statistical association was found between the molecular subtypes and the CSC markers, with HER2 the most frequent subtype for both markers. ALDH1 was also associated with other poor prognostic variables, such as a high histological grade and larger tumors, but it was not associated with the patients’ prognosis in this sample and nor was the CD44<sup>+</sup>/CD24<sup>- </sup>phenotype in a multivariate analysis. There are still many controversies about the role of these markers in breast cancer molecular subtypes. The identification of these populations of cells, through immunohistochemical markers, can help to better understand the CSC theory in clinical practice and, in the near future, contribute to developing new target therapies.</p> <p>&nbsp;&nbsp;&nbsp;</p> Iris Rabinovich Ana Paula Martins Sebastião Rubens Silveira Lima Cícero de Andrade Urban Eduardo Schunemann Junior Karina Furlan Anselmi Selene Elifio-Esposito Lucia de Noronha Andréa Novais Moreno-Amaral ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-09-03 2018-09-03 10.4081/ejh.2018.2943 Study of liver in HBV-related hepatocellular carcinoma: Stereology shows quantitative differences in liver structure https://ejh.it/index.php/ejh/article/view/2950 <p>Hepatocellular carcinoma is one of the main consequences of liver chronic disease. Hepatocellular carcinoma-related changes may be seen in patients with chronic hepatitis B. The aim of the current study was to quantitate liver tissue elements by stereological technique in patients with hepatitis B-related cancer and compare the results with control and only hepatitis B group. Needle liver biopsies from 40 patients with only chronic hepatitis B infection, from 41 patients with only early hepatocellular carcinoma, from 40 patients with early hepatitis B-related cancer and 30 healthy subjects (control group) were analyzed by stereological method using systematic uniform random sampling method. Haematoxylin and eosin stained sections were used. The numerical density of hepatocytes, hepatocyte volume, numerical density of Kupffer cells, volume density of the connective tissue in the portal space, and volume density of the connective tissue were assessed. Quantitative analysis of liver samples indicated that there were statistically significant differences in the numerical density of hepatocytes, hepatocyte volume, numerical density of Kupffer cells, volume density of the connective tissue in the portal space, and volume density of the connective tissue between control and hepatitis B-related cancer and hepatitis B groups. Quantitative, stereological technique is simple and reliable for evaluating HCC in chronic hepatitis B. It is useful for assessing the liver tissue parameters. Stereology is recommended for the diagnosis of people prone to cancer in patients with chronic hepatitis B.</p> Bita Moudi Zahra Heidari Hamidreza Mahmoudzadeh-Sagheb ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-09-18 2018-09-18 10.4081/ejh.2018.2950 Damaged muscle fibers might masquerade as hybrid fibers – a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies https://ejh.it/index.php/ejh/article/view/2896 <p>We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+).&nbsp; We demonstrate that such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serial-section-labeling, to identify damaged fibers. The quadriceps femoris muscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21 ± 0.21%, 98.34 ± 1.06%, 0.07 ± 0.01%, and 0.53 ± 0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively.&nbsp; All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.</p> Morium Begam Joseph A. Roche ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-07-24 2018-07-24 10.4081/ejh.2018.2896 Low ozone concentrations promote adipogenesis in human adipose-derived adult stem cells https://ejh.it/index.php/ejh/article/view/2969 <p>Ozone is a strong oxidant, highly unstable atmospheric gas. Its medical use at low concentrations has been progressively increasing as an alternative/adjuvant treatment for several diseases. In this study, we investigated the effects of mild ozonisation on human adipose-derived adult stem (hADAS) cells i.e., mesenchymal stem cells occurring in the stromal-vascular fraction of the fat tissue and involved in the tissue regeneration processes. hADAS cells were induced to differentiate into the adipoblastic lineage, and the effect of low ozone concentrations on the adipogenic process was studied by combining histochemical, morphometric and ultrastructural analyses. Our results demonstrate that ozone treatment promotes lipid accumulation in hADAS without inducing deleterious effects, thus paving the way to future studies aimed at elucidating the effect of mild ozonisation on adipose tissue for tissue regeneration and engineering.</p> Manuela Costanzo Federico Boschi Flavia Carton Giamaica Conti Viviana Covi Gabriele Tabaracci Andrea Sbarbati Manuela Malatesta ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-09-03 2018-09-03 10.4081/ejh.2018.2969 From fix to fit into the autoptic human brains https://ejh.it/index.php/ejh/article/view/2944 <p>Formalin-fixed, paraffinembedded (FFPE) human brain tissues are very often stored in formalin for long time. Formalin fixation reduces immunostaining, and the DNA/RNA extraction from FFPE brain tissue becomes suboptimal. At present, there are different protocols of fixation and several procedures and kits to extract DNA/RNA from paraffin embedding tissue, but a gold standard protocol remains distant. In this study, we analyzed four types of fixation systems and compared histo and immuno-staining. Based on our results, we propose a modified method of combined fixation in formalin and formic acid for the autoptic adult brain to obtain easy, fast, safe and efficient immunolabelling of long-stored FFPE tissue. In particular, we have achieved an improved preservation of cellular morphology and obtained success in postmortem immunostaining for NeuN. This nuclear antigen is an important marker for mapping neurons, for example, to evaluate the histopathology of temporal lobe epilepsy or to draw the topography of cardiorespiratory brainstem nuclei in sudden infant death syndrome (SIDS). However, NeuN staining is frequently faint or lost in postmortem human brain tissues. In addition, we attained Fluoro Jade C staining, a marker of neurodegeneration, and immunofluorescent staining for stem cell antigens in the postnatal human brain, utilizing custom fit fixation procedures.</p> Beatrice Paradiso Michele Simonato Gaetano Thiene Anna Lavezzi ##submission.copyrightStatement## http://creativecommons.org/licenses/by-nc/4.0 2018-08-27 2018-08-27 10.4081/ejh.2018.2944