Biotechnic & Histochemistry最新文献

Reactive oxygen species (ROS) can cause a lot of pathophysiological consequences, a phenomenon referred to as oxidative stress. With regard to carcinogenesis, ROS is described as a "double-edged sword" due to its condition specific role as a tumor promoter or a tumor suppressor. The current work aims to delineate the mechanistic aspect of this dual role of oxidative stress during hematopoietic malignancy, i.e., leukemia. The study involves N-N' Ethylnitrosourea (ENU) based induction of leukemia in experimental mice followed by the characterization of the disease by investigating the peripheral blood scenario, bone marrow smear study, cytochemistry and histopathology of marrow, flow cytometry based measurement of ROS, and expressional analysis of signaling molecules viz; PCNA, histone-3, CXCR-4, CXCL-12, cyclin-D1, Rb, survivin, and nestin. The result showed that the increased ROS level in leukemic marrow is associated with pathological angiogenesis along with the alteration of CXCR-4/CXCL-12/Cyclin-D axis which was found to be correlated with the hyper-proliferation of the malignant clones. On the other hand, the negation of the tumor suppressive activity of ROS in the hematopoietic compartment of leukemic marrow can be related with the up-regulation of nestin and survivin. The mechanistic study regarding oxidative stress and leukemogenesis may certainly potentiate the development of new anti-leukemic therapeutic strategies.

Infertility affects around 15% of couples worldwide, with male factors being responsible for nearly half of these cases. Oxidative stress is a significant contributor to male infertility, leading to damaged sperm. This research examines the protective effects of Nacetylcysteine (NAC) on sperm exposed to hydrogen peroxide (H₂O₂) induced oxidative stress in rats. Sperm samples from adult male Wistar rats were divided into four groups: control, H₂O₂, NAC, and H₂O₂+NAC. Various parameters, including sperm viability, abnormal morphology, chromatin condensation, and plasma membrane integrity were evaluated after incubation using established assays. Exposure to H₂O₂ significantly decreased sperm viability, increased the rate of abnormal morphology, heightened chromatin condensation abnormalities, and compromised plasma membrane integrity. Treatment with NAC significantly ameliorated these effects, demonstrating its protective role against oxidative damage. NAC effectively counteracts oxidative damage in sperm, improving viability, morphology, chromatin integrity, and membrane integrity. These findings demonstrate the protective effects of NAC against oxidative stress-induced sperm damage under in vitro conditions, underscoring its potential as a subject for further investigation in the context of oxidative stress-related male infertility.

We investigated the effects of nobiletin, an antioxidant, in rats with testicular damage induced by chronic water avoidance stress. Thirty male Sprague-Dawley rats were divided into five groups: Control; Nobiletin (NOB); Chronic Stress (CS); Chronic Stress+DMSO (CS+DMSO); and Chronic Stress+Nobiletin (CS+NOB). Healthy testicular morphology was seen in the Control and NOB groups. In the CS group, seminiferous tubule diameters and germinal epithelial thicknesses decreased, and basement membranes were thicker. This morphological damage was reduced in the CS+NOB group. Immunohistochemical analysis showed that anti-ZO-1 expression, which was intense in the Control and NOB groups, decreased in the CS group, and increased in CS+NOB, similar to Control and NOB. Electron microscopy indicated that the blood-testis barrier (BTB) maintained its integrity in the Control and NOB groups, lost its integrity in the CS group, and was largely preserved in CS+NOB. Biochemical analyses showed that oxidative stress markers, significantly increased in the CS group, decreased significantly in the CS+NOB group. These findings underscore the protective effect of NOB on the male reproductive system against chronic stress, suggesting that nobiletin might be a valuable supportive agent in the treatment of male infertility.

Paraffin section is an important technique in histology research, and transparency is the key step connecting the procedures of dehydration and wax immersion in paraffin sectioning technology. However, the commonly used transparent reagent xylene is toxic to the human body and the environment and included in the list of carcinogens. Few biosafe and environmentally friendly transparent reagents have been widely used to replace xylene. In this study, we found a green transparent reagent, dimethyl carbonate (DMC). Physical and chemical analysis showed that DMC was nontoxic and biodegradable. The feasibility analysis of DMC as a substitute for xylene showed that DMC improved the quality of wax band, staining quality, cell boundary, cell morphology, and section integrity in the sections of plant and animal tissues. The toxicological analysis showed that DMC had no significant effect on the key organs of mice, did not induce obvious inflammation in vivo, and showed higher biosafety than xylene. DMC can be used as a transparent reagent to replace xylene in paraffin section, based on its better physical properties, better paraffin sectioning effect, and higher biosafety. DMC is expected to be widely used in the technical fields of tissue section through further optimization and improvement of the method.

The Nile red is a fluorescent and metachromatic dye, for hydrophobic and nonpolar materials such as lipids and plastics. However, when the microplastics (MP) are contained in a natural matrix composed of roots, plant and insect fragments rich in hydrophobic substances such as cuticles, chitin, and autofluorescence materials (cell wall, lignin, polyphenols) a false fluorescence could generate. In the present study, we explore the use of Methylene blue (C.I. 52015) in combination with Nile red, to stain MP in an intact organic complex composed of roots and rhizome of Cyperus papyrus and Pontederia sagittata from Floating Treatment Wetlands (FTW) installed at an urban eutrophicated ponds in Xalapa Veracruz, México. First, the sample was stained with 0.5 % v/v Methylene blue in 0.5% v/v borax to reduce the nonspecific stain and autofluorescence. After a wash with distilled water, the sample was stained with Nile red (1 µg mL^-1) in ethanol/water. This double stain reduces the background fluorescence of the non-plastic materials, obtaining the best contrast under the green light (ex. 450-490 nm, em. 515 nm). This is the first report of an easy, fast, and non-destructive staining technique to detect MP in natural conditions, that uses Nile red, together with Methylene blue to reduce false positive background staining.

Testicular neoplasms have high incidence in dogs and, despite curative surgical treatment, reported cases have shown aggressive behavior and metastatic dissemination in malignant versions of these tumors. Vimentin is a cytoplasmic protein characteristic of mesenchymal cells, but recently implicated in epithelial-mesenchymal cell transition. This reprogramming cellular event increases tumoral cell invasiveness and metastatic dissemination. Concerning such important oncological implications, vimentin immunohistochemical expression was analyzed in the three most frequent testicular tumors in dogs, as a predictor of local invasiveness. Sixty-eight samples retrieved from a pathological anatomy laboratory were evaluated and their histopathological diagnosis established. Immunohistochemical process followed lab protocol for anti-vimentin antibody. Immunolabeling was analyzed according to intensity criteria: absent, weak, moderate, or intense. In this case series, 39.7% were Leydig cell tumors, 33.8% were seminomas, and 26.4% were Sertoli cell tumors; the last two were further classified as intratubular or diffuse, according to seminiferous tubule location. Vimentin immunolabeling was observed in all three tumor types. Leydig cell tumors showed intense immunolabeling in samples totality, clearly differentiating tumoral from normal cells. Sertoli cell tumors presented marked immunolabeling but also did normal Sertoli cells. Seminoma vimentin-immunolabeling was variable. Diffuse Sertoli cell tumor and seminoma presented more intense vimentin immunolabeling when compared to intratubular counterparts, suggesting this protein role in expansive tumoral behavior and seminiferous tubule rupture. Vimentin overexpression in canine testicular tumors can contribute to tumoral local invasion and consequently impairment of the remaining normal testicular tissue in affected dogs.

The murine cell line MC3T3-E1 is used in many in vitro studies in bone-related research, but different protocols to induce its osteogenic differentiation have been reported. The aim of this study was to identify the best mixture of osteogenic supplements to induce osteogenic differentiation of MC3T3-E1 subclone 4 cells in a two-dimensional cell culture setup. As spheroids as three-dimensional cell aggregates are of increasing importance, we also present a simple method to generate osteogenic differ.entiated spheroids on this basis. Three different mixtures of osteogenic supplements were used to induce osteogenic differentiation for up to 28 days. Osteogenic differentiation was monitored by alizarin red and von Kossa staining, energy dispersive X-ray (EDX) analysis, and real-time quantitative PCR analysis of osteogenic marker genes. Spheroids were generated from osteogenic differentiated cells by liquid overlay technique. The use of 5 mM β-glycerophosphate, 10 nM dexamethasone, and 50 µg/mL ascorbic acid was able to induce osteogenic differentiation of MC3T3-E1 cells within 14 days, as shown by strong positive signals in both staining methods. Scanning electron microscopy revealed extracellular secretions on the membranes of differentiated cells with a significantly increased calcium content of 16.4 ± 2.4% and a phosphorus content of 10.1 ± 1.1%, as shown by energy dispersive X-ray analysis. Differentiated MC3T3-E1 cells could be detached by incubation in AccuMax for 10 min and spheroids were generated from this cell suspension on day 14. Significant upregulation of the osteogenic markers Sp7, osteocalcin, and bone sialoprotein was detected by real-time quantitative PCR analysis of these spheroids. In addition to other reports in the literature describing osteogenic differentiation of spheroids, we were able to show that it is also possible to generate spheroids from osteogenically differentiated two-dimensional cell cultures, which are easier to handle. Thus, there are indeed several ways to generate osteogenic differentiated MC3T3-E1 spheroids.

Age estimation is a critical aspect of forensic odontology for victim identification. Exfoliative cytology has frequently been investigated for this purpose with variable results, necessitating further exploration. This systematic review and meta-analysis aims to analyze the diagnostic utility of cytomorphometrically evaluated exfoliated buccal cells in living individuals for age estimation. A thorough search was conducted using PubMed, Google Scholar, and Cochrane Library databases. Original research articles that performed exfoliative cytology on healthy individuals and evaluated cytomorphometric parameters were included in this review. The risk of bias was analyzed for each study using the Joanna Briggs Institute criteria, and Review Manager was used for the meta-analysis. Twelve studies, and two subgroups included for qualitative and quantitative data synthesis, revealed a significant decrease in cellular parameters and an increase in nuclear parameters in healthy individuals as age progressed. The random-effects model also confirmed that significantly decreased cellular parameters were associated with an increased risk of advanced age. Mounting evidence confirms that cytomorphometric evaluation of exfoliated buccal cells, specifically cellular parameters, is a useful tool for age estimation. However, it still lacks the credibility to be used as a sole factor for predicting an individual's age. Therefore, a combination of factors should be considered for age estimation to provide more reliable results.