Biotechnic & Histochemistry最新文献

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.

Klebsiella pneumoniae frequently causes pneumonia; it is the eighth leading cause of death and one of the most common infectious causes of mortality. Artemisia judaica is well known for its various therapeutic effects. The goal of this study is to evaluate the efficacy of ciprofloxacin and A. judaica in treating pneumonia in K. pneumoniae infected rats. Transmission electron microscopy (TEM) demonstrated that ciprofloxacin and A. judaica extract had substantial antibacterial properties against K. pneumoniae. Five groups, each with ten rats, were studied: Group 1 (negative control), Group 2 (infected with 1 × 10⁵ CFU/mL of K. pneumoniae solution), Group 3 (infected and treated with 250 mg/kg of A. judaica extract), Group 4 (infected and treated with 500 mg/kg of A. judaica extract), and Group 5 (infected and treated with 500 mg/kg of ciprofloxacin). Animals were sacrificed after 24, 48, and 72 hours of treatment. We found that the A. judaica extract or ciprofloxacin treatment improved the rate of survival of infected rats and reduced bacterial spread in the lungs, liver, and spleen. Groups 3, 4, and 5 had substantial histological improvement in lung pathology, with lower TNF-α levels and elevated IL-4, SOD, and CAT levels relative to the positive controls. We conclude that A. judaica has antioxidant, anti-inflammatory, and antibacterial effects that can help combat pneumonia caused by K. pneumoniae in rats.

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.

Glial precursor cells are among the major types of glia in the dorsal root ganglias (DRGs) of the peripheral nervous system. Previous studies have shown that the transdifferentiation of DRGs-derived glial precursor cells contributes to peripheral neurogenesis. In the present study, we investigated the mRNA expression profiles and examined the effects of differential expression mRNAs (DEMs) during the differentiation of glial precursor cells derived from the rat DRGs. We characterized glial precursor cells derived from rat DRGs explants using immunofluorescence. Sequencing was subsequently conducted, followed by enrichment analysis utilizing gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The identified genes were subsequently subjected to protein-protein interaction (PPI) network analysis during the differentiation process of glial precursor cells derived from the rat DRGs. The establishment of a sciatic nerve injury (SNI) model was followed by the detection of the expression of key genes in the Wnt and Neurotrophin pathways in the DRGs of SNI rats via qRT-PCR. Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was employed to assess apoptosis in the DRGs. We detected the mRNA expression profiles during the neuronal differentiation of rat DRGs-derived glial precursor cells. More DEMs and GO terms were detected on the third day of DRGs-derived glial precursor cells transdifferentiation, accompanied by morphological alterations in the cells; that is, some cells presented neuronal-like phenotypic characteristics (the early neuronal marker Tuj1 was positive). KEGG enrichment and PPI network analyses revealed that Wnt and Neurotrophin pathways play crucial roles in the process of glial precursor cell differentiation into neuronal-like cells. After knocking down cadherin-associated protein beta 1 (Ctnnβ1) in the SNI model, the number of apoptotic cells was significantly reduced, and the expression of Wnt4 and Ntrk3 was significantly increased. The Ctnnβ1 gene may be a crosstalk factor between the Wnt and Neurotrophin pathways that negatively regulates the differentiation of glial precursor cells.

Palmitic acid (PMA) is abundantly present in substantial quantities within palm oil and manifests neurodegenerative propensities. Conversely, the ingestion of Hesperidin (HSD) is correlated with a reduction in inflammatory markers and mediators. This investigation was meticulously devised to scrutinize the protective potential of HSD against the deleterious repercussions of PMA administration on the cerebral cortex. A cohort comprising forty albino Wistar rats was stratified into four groups, each receiving supplements of HSD and PMA. Remarkably, HSD was observed to fortify the histological framework of the cerebral cortex subsequent to PMA exposure, concurrently diminishing the percentage of apoptotic cells. Furthermore, HSD upregulated the levels of antioxidant markers, preserved the levels of neurotransmitter-associated enzymes, and downregulated the expression of inflammation-regulating genes. In conclusion, PMA exerts toxic effects on the cerebral cortex of albino Wistar rats, leading to increased apoptosis and neuroinflammation, thereby reducing brain cholinergic activity. HSD was found to attenuate the cerebral cortex content of MPO, 5-NTD, ROS, MDA, and NF-κB. Additionally, it elevated the cerebral cortex content of antioxidants and anti-inflammatory markers, thereby shielding it from the deleterious effects of PMA.

Leiomyomas (fibroids) are the most common benign tumors of the uterus and are present in greater than half the female population over 50 years old in the United States. Leiomyosarcomas are the malignant variation of leiomyomas and, while far less common, have a high mortality rate. Differential protein expression between both benign and malignant tumors and normal tissue samples forms the basis of many treatment strategies. This study evaluated protein expression of several markers using immunohistochemistry (IHC) methods on 74 leiomyomas, 14 uterine leiomyosarcomas, and 26 normal uterine myometrial samples which had been formalin-fixed and paraffin-embedded. Markers included the Ki-67 proliferation marker, estrogen receptor (ER), progesterone receptor (PR), aldehyde dehydrogenase (ALDH), B-cell lymphoma 2 (BCL-2), and cytokeratin 8/18 (CK 8/18). The Ki-67 positivity was significantly higher in leiomyosarcomas when compared with benign uterine tissues and was also higher in leiomyomas than in normal uterine smooth muscle. ER and PR were highly expressed in benign tissues but exhibited reduced expression in malignant lesions. Both CK 8/18 and ALDH were expressed in a significantly higher proportion of normal myometrial tissues as compared with leiomyomas and leiomyosarcomas. Conversely, BCL-2 expression in normal tissues was lower than in both leiomyomas and leiomyosarcomas with leiomyosarcomas producing the highest expression. The Ki-67 value can reliably differentiate between benign and malignant smooth muscle uterine tissues. Because CK 8/18 and ALDH were more frequently or strongly expressed in normal myometrium vs. leiomyoma or leiomyosarcoma, pathological changes to the cells may be the cause for a reduction in protein production. Future investigations may determine that upregulation of either of these two markers in leiomyomas or leiomyosarcomas could lead to slower tumor growth.

We investigated the effects of ecstasy on the expression of heat shock protein 70 (HSP70) and apoptosis in rat kidney. We used 20 adult male Wister rats divided into four groups of five: control, sham, Ecs 5 and Ecs 10; the latter two groups were administered by intraperitoneal (i.p.) injection 5 and 10 mg/kg ecstasy, respectively. At the end of the experiment, the kidneys were removed, fixed, and prepared for immunohistochemistry and TUNEL staining to evaluate the expression of HSP70 and apoptosis, respectively. HSP70 expression and apoptosis cells were significantly increased in most parts of the kidneys, and kidney weight and volume were decreased in rats administrated 10 mg/kg ecstasy compared to the control group. Administration of 5 mg/kg ecstasy significantly increased HSP70 expression in the distal and collecting tubules and the number of TUNEL-positive cells in the proximal, distal convoluted tubules and renal corpuscles compared to the control group. We found that ecstasy increases HSP70 expression and apoptosis in renal tissue.