Histology and histopathology Vol.35, nº3 (2020)
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- PublicationOpen AccessStorage of blood clots for histological analysis: how long is too long in saline and paraformaldehyde?(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Douglas, Andrew; Fitzgerald, Seán; Pandit, Abhay; Doyle, Karen M.To investigate the composition of blood clots following mechanical thrombectomy, it is essential to ensure optimum storage for highest quality histological and immunofluorescence analysis. We investigated for how long clots can be stored in paraformaldehyde (PFA), saline and heparinised saline before the tissue integrity is compromised. Whole blood and fibrin-rich clot analogues were made under dynamic flow conditions. Clots were stored in 4% PFA, saline or heparinised saline for timepoints ranging from 1 hour to two months. Five μm sections were stained with Martius Scarlet Blue to visualise red blood cells (RBCs), white blood cells (WBCs) and fibrin. Semi-quantitative analysis of the integrity of clot components used a scoring system (0: Poor; 1: Sub-par; 2: High). Quantitative analysis used Orbit Image Analysis software. Autofluorescence was assessed using a relative scale. Clots stored in PFA for up to two months were qualitatively similar to those stored for all shorter periods (median score: 2 per component). Clots stored in saline/heparinised saline for one week showed degradation of RBCs and WBCs, but fibrin remained intact (median score: 1, 1, 2 respectively). Degradation of the samples stored in saline/heparinised saline made accurate quantification using Image Analysis software difficult from 24h. Samples stored in PFA for up to two weeks showed an edging autofluorescence effect, which became more evident with prolonged storage. For optimum histology, ideally clots should not be stored in saline before fixation and should ideally be stored in formalin for less than one month to minimise the impact of autofluorescence on immunofluorescence
- PublicationOpen AccessAdipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Gonçalves Felix, Renato; Carvalho Bovolato, Ana Livia; Cotrim, Ondina Silvia; Leão, Patricia dos Santos; Batah, Sabrina Setembre; Golim, Márjorie de Assis; Velosa, Ana Paula P.; Teodoro, Walcy; Martins, Vanessa; Ferreira Cruz, Fernanda; Deffune, Elenice; Fabro, Alexandre Todorovic; Capelozzi, Vera LuizaThe immunogenic collagen V (Col V) and the proinflammatory cytokine interleukin (IL)-17 have been implicated in the pathogenesis of multiple autoimmune diseases. Col V is also up-regulated during adipogenesis and can stimulate adipocyte differentiation in vitro. Conditioned medium (CM) generated from adipose- derived mesenchymal stem cells (MSCs) reduces bleomycin (BLM)-induced lung injury in rats, suggesting a crucial role in situ of immunomodulatory factors secreted by MSCs in these beneficial effects. In the present work, we investigated this hypothesis, analyzing levels of plasma inflammatory mediators and inflammatory and fibrotic mediators in the lung tissue of BLM-injured rats after treatment with MSCs and CM. Pulmonary fibrosis was intratracheally induced by BLM. After 10 days, BLM animals were further randomized into subgroups receiving saline, MSCs, or CM intravenously. On days 14 and 21, the animals were euthanized, and the lungs were examined through protein expression of nitric oxide synthase (NOS), IL-17, transforming growth factor-β (TGF-β), vascular endothelial growth factor, endothelin-1, and the immunogenic Col V through histological quantitative evaluation and plasma levels of fibrinogen, Von Willebrand factor, and platelet-derived growth factor (PDGF). Rats that had been injected with MSCs and CM showed a significant increase in weight and significant improvements at 14 and 21 days after intravenous injection at both time points of analysis of plasma fibrinogen, PDGF, and Von Willebrand factor and NOS-2 expression, supporting an early anti-inflammatory action, thus reducing TGF-β and collagen I fibers. In contrast, intravenous injection of CM was able to significantly increase the deposition of Col V fibers and IL-17 on both day 14 and day 21 as compared with the amount observed in rats from the BLM group and MSC groups. In conclusion, this study reinforces previous observations on the therapeutic properties of MSCs and CM and is the first report to demonstrate the association of its actions with immunomodulatory biomarkers on lung tissue. We concluded that adipose-derived stem cells and adipose- derived stem cells-CM modulate an in situ imbalance between collagen I- and Col V-mediated IL-17 immune response, emerging as a promising therapeutic option for recovering from BLM pulmonary fibrosis
- PublicationOpen AccessSilencing of synaptotagmin 7 regulates osteosarcoma cell proliferation, apoptosis, and migration(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Wu, Zhiqiang; Sun, Zhengwang; Huang, Rui; Zang, Ding; Wang, Chunmeng; Yan, Xu; Yan, WangjunBackground. Synaptotagmin 7 (SYT7) is a component of the synaptotagmin family, which is essential in many physiological and pathological processes. In this study, we aimed to investigate the role of SYT7 in osteosarcoma. Methods. We defined the expression levels of SYT7 in osteosarcoma tissues and para-sarcoma tissues by immunohistochemistry and analyzed the possible correlation between SYT7 expression and pathological characteristics via Mann-Whitney U analysis and Spearman correlation analysis. The effects of SYT7 silencing in vitro on cell growth were assessed by MTT assay. Cell cycle and cell apoptosis were assessed by flow cytometry analysis. Wound healing assay and transwell assay were applied to assess the migration and invasion capacity. Results. The results showed that the expression levels of SYT7 were upregulated in osteosarcoma tissues compared with para-sarcoma tissues and positively correlated with the pathological characteristics of osteosarcoma. Functional experiments demonstrated that SYT7 silencing significantly inhibited cell proliferation and colony formation capacity (P<0.001), induced cell cycle arrest which increased the proportion of G2 phase and decreased the proportion of S phase, enhanced cell apoptosis (P<0.01), and limited the capacity of migration and invasion (P<0.01), compared with shCtrl group. Conclusion. The results indicated that SYT7 plays a crucial role in the development of osteosarcoma. SYT7 can be applied as a new diagnostic and therapeutic target in osteosarcoma.
- PublicationOpen AccessRoles of microRNAs as non-invasive biomarker and therapeutic target in colorectal cancer.(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Wan, Timothy Ming-Hun; Iyer, Deepak Narayanan; Ng, LuiMicroRNAs are endogenous, short non- coding RNA molecules that function as critical regulators of various biological processes. There is a strong functional evidence linking the involvement of dysregulated miRNAs to the occurrence, development and progression of colorectal cancer. Studies indicate that while overexpression of oncomiRs, and repression of tumor suppressor miRNAs tends to drive the overall tumorigenic process, the global picture of aberrant miRNA expression in colorectal cancer can classify the disease into multiple molecular phenotypes. Moreover, the expression pattern of miRNAs in colorectal cancer makes them viable disease determinants as well as potential therapeutic targets. Through this review, we will summarize the importance of miRNAs in the etiology and progression of colorectal cancer. Specifically, we will explore the key role played by these RNA molecules as likely therapeutic avenues and the strategies presently available to target them. Finally, we will investigate the role of miRNAs as potential non- invasive diagnostic and prognostic biomarkers in colorectal cancer.
- PublicationOpen AccessRegulation of DNA methylation levels in the process of oral mucosal regeneration of oral ulcer model.(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Akiyama, Naotaro; Fukuda, Tomomi Yamamoto; Yoshikawa, Mamoru; Kojima, HiromiDNA methylation is an important epigenetic mechanism for cellular maintenance. However, the methylation pattern and the key molecule regulated epigenetically in oral mucosal regeneration is unclear. In this study, we generated a rat oral ulcer model and investigated the cell proliferative activities and DNA methylation patterns immunohistochemically. We also performed immunohistochemical analysis of a regulator of epithelial stem/progenitor cell differentiation in the rat model. We demonstrated immunohistochemistry using antibodies for the molecules as follows: Ki-67, a marker of cellular proliferation; 5-methylcytosine (5-mC), a marker of DNA methylation; 5-hydroxymethylcytosine (5-hmC), a marker of DNA demethylation; Dnmt1, a maintenance DNA methyltransferase; Dnmt3a and Dnmt3b, de novo DNA methyltransferases; and Wnt5a, a regulator of stem/progenitor cell differentiation. In this model, re-epithelialization was completed at Day 4 after ulceration. Regenerating mucosal hypertrophy reached a peak at Day 5 and appeared normal at Day 14. Ki-67-positive cells increased at Day 2 and returned to normal at Day 6 after ulceration. The ratio of the expression level of 5-mC to 5-hmC declined at Day 5 and returned to normal at Day 6. The expression level of Dnmt1 had not changed compared to the normal control at every time point. On the other hand, the expression levels of Dnmt3a and Dnmt3b had decreased significantly at Day 5 and returned to normal at Day 6. Moreover, Wnt5a-positive cells increased at Day 5. In conclusion, oral mucosal regeneration was strictly regulated by DNA methylation. Moreover, Wnt5a might play a critical role in oral mucosal regeneration.