Histology and histopathology Vol.35, nº1 (2020)

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http://hdl.handle.net/10201/179774

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    Bone biology in postnatal Wistar rats following hypoxia-reoxygenation
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Hameister, Rita; Lohmann, Christoph H.; Dheen, Thameem; Singh, Gurpal; Kaur, Charanjit
    Hypoxia response pathways have a central role in normal and abnormal bone biology but the effect of systemic hypoxia-reoxygenation on bone is not clear. Following hypoxic exposure, aberrant synthesis, folding and trafficking of proteins has been reported to occur, which can result in endoplasmic reticulum (ER) stress and may finally cause cell death. This study aimed to examine the effect of systemic hypoxia-reoxygenation injury on bone biology in postnatal rats. Immunoexpression of HIF-1α and VEGF was upregulated in femurs of newborn Wistar rats in response to systemic hypoxia-reoxygenation. Along with that, increased apoptosis of osteoblast precursors, osteoblasts, osteocytes and endothelial cells was observed in comparison to femurs of control animals by transmission electron microscopy, TUNEL staining and immunoexpression of cleaved caspase-3. The viability of osteoclasts was not affected. After hypoxia- reoxygenation, ER stress was observed in the osteoblasts and osteocytes as indicated by dilatation of the ER and enhanced immunoexpression of the ER stress marker GRP78. Localisation of collagen α1 immunoreaction was widespread in the bone matrix of control femurs but was confined to the osteoblasts and osteocytes in response to hypoxia-reoxygenation. In support of these findings, in vitro work showed reduced viability of osteoblast-like SaOs-2 cells and upregulation of GRP78 protein expression in them by western blotting following exposure to hypoxia. This suggests that systemic hypoxia-reoxygenation may disturb bone biology in postnatal Wistar rats by inducing ER stress and apoptosis in osteoblasts and osteocytes, without affecting the viability of osteoclasts. More in-depth research is needed to confirm causality between ER stress and apoptosis of osteoblasts and osteocytes
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    Morphological changes during the formation of amoebic liver abscess in vagotomized hamsters.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Sánchez Alemán, Esperanza; Lili Carrillo, Leticia María; Muñoz Ortega, Martin Humberto; Martínez Saldaña, Ma. Consolación; Ventura Juárez, Javier
    Amoebic liver abscess (ALA) is the main extra-intestinal complication caused by Entamoeba histolytica. Given the histological features of ALA in hamsters and the importance of the vagus nerve in the immune response, the aim of this study was to identify and analyze the major changes in ALA that are caused by a vagotomy. The changes found are related to inflammatory foci and abscess size, the type of collagen formed, and the number of trophozoites in lesions. Male hamsters were divided into three groups: Intact animals (IA) and those undergoing a false operation (SHAM) or a subdiaphragmatic vagotomy (VAG). In each group, E. histolytica trophozoites or culture medium (CM) were inoculated in hamsters by the intrahepatic route, and then euthanized at 6h, 12h, 24h, 48h, 4d or 7d post- infection. Initially the growth of the abscess was more rapid in the VAG group, but at day 7 it was faster in the IA and SHAM groups. VAG animals showed a higher quantity of type III collagen than the IA and SHAM groups. A larger number of amoebic trophozoites/mm 2 was observed up to day 4 in VAG hamsters (23.3±2.19) compared to IA (14.6±0.23) and SHAM (6.13±0.87) animals. This parameter decreased by day 7 in VAG (13.4±0.87) with respect to IA (24.7±1.47) and SHAM (21.7±1.48). The results show that a subdiaphragmatic vagotomy influenced the development of ALA in hamsters, suggesting a modification of the morphological structure of damaged hepatic tissue.
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    Heat shock proteins in cancer stem cell maintenance: a potential therapeutic target?
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Lettini, Giacomo; Lepore, Silvia; Crispo, Fabiana; Sisinni, Lorenza; Esposito, Franca; Landriscina, Matteo
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    Hypertension accelerates age-related intrarenal small artery (IRSA) remodelling and stiffness in rats with possible involvement of AGEs and RAGE.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Ba, Yajing; Shi, Xiaoyun; Ke, Yilang; Lin, Xiaohong; Hong, Huashan
    Objectives. To study changes in morphology, advanced glycation end products (AGEs) and the AGEs receptor, RAGE, that occur with ageing in intrarenal small arteries (IRSAs) of spontaneously hypertensive rats (SHRs) and to investigate the possible roles of hypertension, AGEs and RAGE in the progression of IRSA remodelling and stiffness with ageing in rats. METHODS: Ageing SHRs and ageing normotensive Wistar Kyoto (WKY) rats were studied. The minimal renal vascular resistance (minRVR) was measured. Renal arcuate arteries (RAAs) and interlobular arteries (RILAs), the expression of α-smooth muscle actin, proliferating cell nuclear antigen, AGEs, RAGE and the plasma concentrations of AGEs were also examined. RESULTS: The IRSA minRVR, wall thickening, cell proliferation and collagen deposition in RILAs and RAAs gradually increased with age in SHRs and were much higher in 24-week-old SHRs than in age-matched WKY rats (p<0.05); these indexes in WKY rats were only elevated in the 72-week group (p<0.05). The expression of RAGE in the RAA and RILA tunica media in SHRs was upregulated by 24 weeks and 12 weeks (p<0.05), respectively, while AGEs levels in the plasma and in the IRSA tunica media were increased by 48 weeks (p<0.05) and increased gradually with age. The levels of both RAGE and AGEs in WKY rats were increased only at 72 weeks (p<0.05). CONCLUSION: Hypertension accelerates the development of age-related IRSA remodelling and stiffness in rats, which may be related to upregulation of RAGE in the IRSA tunica media and increased expression of AGEs at the late stage.
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    Age- and sex-associated changes in prosaposin and its receptors in the lacrimal glands of rats.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Islam, Farzana; Khan, Md. Sakirul Islam; Nabeka, Hiroaki; Shimokawa, Tetsuya; Yamamiya, Kimiko; Matsuda, Seiji
    Prosaposin, a saposin precursor, is a potent neurotrophic factor found in several tissues and various biological fluids. Saposin-deficient patients have different ophthalmic disorders, indicating a relationship between ocular health and prosaposin. However, there is little information about prosaposin on the ocular surface. Because ocular functions are diverse and depend on age and sex, we examined whether prosaposin and its receptors, G protein-coupled receptor 37 (GPR37) and GPR37L1, are expressed in the major ocular glands, the extra orbital lacrimal gland (ELG), and harderian gland (HG) of rats and whether sex and aging affect their expression. Immunohistochemical analyses revealed that prosaposin and its receptors were expressed in the ELGs and HGs of rats, although their expression varied based on the type of gland, age, and sex. Prosaposin, GPR37, and GPR37L1 were expressed in the basolateral membranes and cytoplasm of acinar cells of the ELGs, and their immunoreactivities were higher in female rats of menopausal age than age-matched male rats. However, such age- and sex-related differences in the immunoreactivities of prosaposin, GPR37, and GPR37L1 were not observed in the HGs. Triple immunofluorescence labelling revealed that prosaposin, GPR37, and GPR37L1 were co-localised in the acinar and ductal cells in the ELGs, although the degrees of colocalization varied according to the age and sex of the rats. Together, the present results showed that prosaposin and its receptors were expressed in the major ocular glands of rats, and their immunoreactivities to the ELGs differed considerably with age and sex.
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    Optimization of RNA extraction from laser captured microdissected glomeruli from formalin-fixed paraffin-embedded mouse kidney samples for Nanostring analysis.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Hay, Abigail; Lapointe, Jean-Martin; Lewis, Arthur; Moreno Quinn, Carol; Miranda, Elena
    Optimized protocols for the microdissection of specific areas from archival tissues and the subsequent RNA analysis are needed but challenging due to RNA degradation and chemical modifications. The aim of this study was to present the most appropriate protocol for utilizing mouse FFPE kidney for laser capture microdissection and Nanostring gene expression analysis. We evaluated different section thicknesses (3, 5, 10 μm), 2 RNA extraction kits (Qiagen and Roche) and different H&E staining methods to optimize microdissection and RNA extraction from glomeruli and cortical tubules samples from FFPE mouse kidney. RNA quality and quantity were assessed via Nanodrop and Qubit. The protocol providing the best results consisted of 5 μm sections, a shorter protocol for H&E staining, and RNA extracted with the Roche kit. Higher Nanostring gene counts and lower qPCR cT significantly correlated with RNA concentrations measured with the Qubit, but not with measures obtained with the Nanodrop. The Nanostring data showed that none of the genes included in the panel was differentially expressed in the cortical tubule compartment compared to the whole kidney. However, 25 genes were differentially expressed in the glomerular compartment compared to the whole kidney. Our data showed that sufficient RNA can be extracted from small compartments like mouse renal glomeruli from archival FFPE tissue, and that whole kidney analysis does not accurately represent the transcriptome state of the glomeruli, which comprise only a small proportion of the overall kidney volume
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    Advances in understanding mechanisms of long-term sperm storage-the soft-shelled turtle model
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Chen, Hong; Liu, Tengfei; Holt, William V.; Yang, Ping; Zhang, Linli; Zhang, Li; Han, Xiangkun; Bian, Xunguang; Chen, Qiusheng
    Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species
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    Microvascular pathology in Friedreich cardiomyopathy.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Koeppen, Arnulf H.; Qian, Jiang; Travis, Alicia M.; Sossei, Alyssa B.; Feustel, Paul J.; Mazurkiewic, Joseph E.
    Heart disease is an integral part of Friedreich ataxia (FA). In addition to cardiomyocyte hypertrophy, fiber necrosis, and inflammatory infiltration, sections show fibrosis and disorganized capillaries. We examined the left ventricular wall (LVW) of 41 homozygous and 2 compound heterozygous FA patients aged 10-87 and 21 controls aged 2-69. Immunohistochemistry with an antibody to CD34 allowed quantitative counts of capillary profiles for a comparison with cardiomyocyte counts in the same field. Capillary counts (mean±standard deviation [SD]) in normal controls were 1926±341/mm2, while mean cardiomyocyte counts were 2003±686/mm 2 . The median ratio of capillaries to cardiomyocytes was 1.0 (interquartile range [IQR]: 0.9- 1.2). In FA, the number of cardiomyocytes/mm 2 was significantly less (704±361; p<0.001), and the median ratio of capillaries to heart fibers was 2.0 (IQR:1.4-2.4). There was a significant correlation of the expanded guanine-adenine-adenine trinucleotides (shorter allele, GAA1) with a younger age of onset, shorter disease duration, and lower cardiomyocyte counts. The ratio of capillaries to heart fibers was higher in patients with long GAA1 repeat expansions (e.g., 3.31 in GAA1 of 1200). Double-label immunofluorescence for CD34 and the fibroblast marker S100A4 revealed co-expression in endothelial cells, supporting endothelial-to- mesenchymal transition in the pathogenesis of cardiac fibrosis in FA. We propose that the pathogenesis of FA heart disease includes primary fibrosis.
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    The evaluation of the distribution of CD133, CXCR1 and the tumor associated macrophages in different molecular subtypes of breast cancer
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, ) Ilgın, Can; Çomut, Erdem; Sarıgül, Çağlar; Korkmaz, Selçuk; Vardar, Enver; Müftüoğlu, Sevda Fatma
    Breast cancer has different molecular subtypes, which determine the prognosis and response to the treatment. CD133 is a marker for cancer stem cells in tumor microenvironment with diagnostic/therapeutic importance. The tumor associated macrophages (TAMs) interact with the cancer stem cells through the CXCR1 receptor. In this study, we wanted to investigate the expression of these markers in patients with different molecular subtypes, in order to detect pathophysio- logical mechanisms and new molecular targets for the prospective targeted therapies. In this study we hypothesized a difference in expression of these antigens among different subtypes. We investigated expression of antigens in breast cancer patients with luminal A (LA), luminal B (LB), HER2 overexpressing (HER2OE), triple negative (TN) subtypes (n=70) and control patients (n=10) without cancer diagnosis. We applied indirect immunohistochemistry and evaluated immunostaining. CD133 expression was at the periphery and CXCR1 expression was at the central area of the tumor. The cytoplasmic CXCR1, CD133 expressions and nuclear CD133 expression, which is prominent in the TN subtype, were observed in patients. There was a statistically significant difference between the groups for CD133 (p=0.004), CXCR1 (p=0.002) H-Score values and M2 macrophages/whole TAM ratios (p=0.022). Between the CD133 and CXCR1 H-scores, there was a weak positive correlation (r=0.249, p=0.035). This study showed the compartment specific expression of the CD133 and CXCR1 antigens in neoplastic cells. The use of CD133 as a stem cell marker may be limited to TN subtype, due to its heterogeneous expression