European Journal of Histochemistry最新文献

This study aimed to evaluate the therapeutic efficacy of camellia oil on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) in mice, as well as its effect on the expression of skin-barrier-related proteins. A mouse model of AD was created via topical application of DNCB; subsequently, the animals were randomly divided into four groups: the blank control (Control), model (Model), moisturizing cream (Moisturizer), and camellia oil (Camellia) groups. The Camellia group received camellia oil, whereas the Moisturizer group was treated with moisturizing cream, as a positive control. Skin lesions, ear and back tissue morphology, and the serum levels of IgE, IL-4, and IFN-γ were analyzed. Compared with the Control group, AD mice exhibited erythema, papules, dryness, peeling, and significantly higher serum IgE and IL-4 levels. Compared with the Model group, treatment with camellia oil and moisturizing cream considerably reduced skin inflammation, ear thickness, and scratching frequency. A histopathological analysis revealed that camellia oil reduced inflammatory-cell infiltration and edema in the AD-affected skin. Furthermore, camellia oil upregulated filaggrin (FLG), thus aiding in skin-barrier repair. These findings suggest that camellia oil significantly improves AD symptoms, enhances FLG expression, and restores the damaged skin barrier in AD mouse models.

Accurate differentiation of homologous proteins that share high sequence identity remains a significant challenge in biomedical research, as conventional antibodies often lack sufficient specificity, leading to potential misinterpretations. This issue is particularly evident in the study of hexokinases, a family of isoenzymes that catalyze the first step of glycolysis by phosphorylating glucose. Beyond their canonical metabolic roles, hexokinases play critical non-glycolytic functions, especially in cancer biology. However, their unique tissue distributions and context-dependent roles are often obscured by the overlapping specificities of commercially available antibodies, which can produce misleading results. In this study, we rigorously evaluated a panel of antibodies targeting hexokinase isoenzyme 3 (HK3), highlighting the widespread issue of cross-reactivity and insufficient validation. Through this process, we identified and validated a highly specific antibody for HK3, demonstrating its reliability in western blot and immunohistochemistry applications. Using this validated tool, we reveal the distinct localization of HK3 in myeloid cell populations, providing new insights into its potential functional roles in these cells. This work addresses a critical gap in antibody specificity and establishes HK3 as a uniquely expressed gene in myeloid and immune cells and is absent in other cell types under basal conditions. Providing a foundation for future investigations into its context-dependent functions.

Muscle spindles are skeletal muscle sensory receptors composed of intrafusal fibres, partially encapsulated by connective tissue capsule. This capsule encloses the central A and B regions while leaving the distal C region extracapsular. Several past studies in rat have shown that muscle spindles typically contain a single bag1 fibre, a single bag2 fibre, and two smaller chain fibres. Intrafusal fibres co-express multiple myosin heavy chain (MyHC) isoforms: -slow or -1, -slow-tonic, -α, -2a, -2b, -embryonic, and -neonatal. While MyHC-2x was previously thought absent, the recently discovered MyHC-15 isoform has been identified in the C region of rat bag fibres. Using antibodies specific for nine MyHC isoforms and analyzing four different rat skeletal muscles-soleus, extensor digitorum longus, and the lateral and medial heads of gastrocnemius-we aimed to further characterize the co-expression pattern of MyHC-15 with other known isoforms and to determine whether MyHC-2x is expressed in rat intrafusal fibres. While rodents are widely used as animal models in skeletal muscle research, notable species-specific differences in MyHC isoform expression exist. Our findings revealed that MyHC-15 expression in rat intrafusal fibres is less abundant than in human fibres. MyHC-15 was primarily observed in bag fibres but was not detected in the C region, contrary to previous reports in both rat and human. We confirmed the absence of MyHC-2x in rat intrafusal fibres. Similarly, MyHC-embryonic and -neonatal were not detected in the analyzed spindles, suggesting that previously used antibodies may have cross-reacted with MyHC-2a and -2b. While our results partially corroborate previous extensive studies, discrepancies suggest that MyHC expression in intrafusal fibres varies not only along the fibre length but also across muscles.

This corrects the article published in European Journal of Histochemistry 2024;68:4140 doi: 10.4081/ejh.2024.4140 IF: 2.1 Q4 B4 IF: 2.1 Q4 B4.

Carbonic anhydrase (CA) has been localized to many structures involved in ion transport including the acini and ducts of the major (parotid, sublingual and submandibular) salivary glands of humans and rodents. It also has been localized by enzyme histochemistry and by immunohistochemistry for CA isoenzyme VI (CA VI) to the acini and ducts of rat serous lingual glands of von Ebner. The purpose of this study was to explore the intracellular distribution by cell type of three CA isoenzymes in these glands. Immunohistochemistry was undertaken with antibodies to human CAs I, II and VI in paraffin sections of rat tongues that had been fixed in Helly's fluid. The density of the reaction product was scored as 0 (none) to 5 (strongest). Reactions in the acini with CA I and II antibodies were weak luminally to moderate basally and generally moderate, respectively, moderate in the intercalated ducts, and moderate basally to strong luminally in the excretory ducts. Weak to moderate CA VI reactions occurred in the acini and ducts. The stronger luminal reactions to CAs I and II in the excretory ducts suggest that they contribute to pH regulation in the saliva of von Ebner's glands via HCO3- transport.

Intestinal barrier damage causes an imbalance in the intestinal flora and microbial environment, promoting a variety of gastrointestinal diseases. This study aimed to explore the mechanism by which adipose-derived stem cells (ADSCs) repair intestinal barrier damage. The human colon adenocarcinoma cell line Caco-2 and rats were treated with lipopolysaccharide (LPS) to establish in vitro and in vivo models, respectively, of intestinal barrier damage. The expression of inflammatory cytokines (TNF-α, HMGB1, IL-1β and IL-6), antioxidant enzymes (iNOS, SOD and CAT), and oxidative products (MDA and 8-iso-PGF2α) was detected using ELISA kits and related reagent kits. Apoptosis-related proteins (Bcl-2, Bax, Caspase-3 and Caspase-9), tight junction proteins (ZO-1, Occludin, E-cadherin, and Claudin-1) and p38 MAPK pathway-associated protein were detected by Western blotting. In addition, cell viability and apoptosis was determined by a CCK-8 kit and flow cytometry, respectively. Cell permeability was assayed by the transepithelial electrical resistance value and FITC-dextran concentration. The homing effect of ADSCs was detected by fluorescence labeling, and intestinal barrier tissue was observed by HE staining. After ADSC treatment, the level of phosphorylated p38 MAPK protein decreased, the expression of inflammatory factors, oxidative stress and cell apoptosis decreased, the expression of tight junction proteins increased, and cell permeability decreased in Caco-2 cells stimulated with LPS. In rats, ADSCs are directionally recruited to damaged intestinal tissue. ADSCs significantly decreased the levels of D-lactate, diamine oxidase (DAO) and FITC-dextran induced by LPS. ADSCs promoted tight junction proteins and inhibited oxidative stress in intestinal tissue. These effects were reversed after the use of a p38 MAPK activator. ADSCs can be directionally recruited to intestinal tissue, upregulate tight junction proteins, and reduce apoptosis and oxidative stress by inhibiting the p38MAPK signaling pathway. This study provides novel insights into the treatment of intestinal injury.

Colorectal cancer (CRC) is a major public health concern and identifying prognostic molecular biomarkers can help stratify patients based on risk profiles, thus enabling personalized medicine. Epitranscriptomic modifications play a relevant role in controlling gene expression, N6-methyladenosine (m6A) regulators play crucial roles in cancer progression, but their clinical significance in CRC cancer has thus far not been elucidated. Thus, we aimed to examine by immunohistochemical techniques and RT-qPCR, protein levels and RNAs expression of m6A writers (METTL3, WTAP) and eraser (FTO) in a cohort of 10 patients affected by CRC. The patients were followed for 5 years and values of METTL3, WTAP and FTO RNAs in alive vs dead patients were compared. Proteins expression and RNAs expression had a different trend, METTL3, WTAP and FTO proteins' expression showed an increasing trend from non-cancerous adjacent (N) tissue vs carcinoma (CA) tissue G1 stage, and then a decreasing trend from G1 to G2 and G3 stages. The most marked increase was observed in WTAP that, from a 40% of protein expression positivity in N tissue raised to the 81% of positivity in G1 stage K tissue. RNAs expression of METTL3, WTAP and FTO genes in N tissue vs G1 stage CA tissue was significantly different, the analysis and comparison of RNAs values in patient alive after 5 years (0.58±0.04) vs patients dead after 5 years (1.69±0.29) showed that only WTAP values resulted significantly high in dead patients. The fact that WTAP protein expression levels lower while WTAP RNA expression remains high, lets us hypothesize a sort of inhibition of protein expression, but further studies are needed to clarify the mechanism. Although the results suggest a relationship between biological meaning and prognostic utility of WTAP, this prognostic utility must be confirmed by further studies on a larger sample.

Previous ultrasound studies suggest that patients with adenomyosis (AM) exhibit increased uterine cavity stiffness, although direct evidence regarding extracellular matrix (ECM) content and its specific impact on endometrial stiffness remains limited. This study utilized atomic force microscopy to directly measure endometrial stiffness and collagen morphology, enabling a detailed analysis of the endometrium's mechanical properties: through this approach, we established direct evidence of increased endometrial stiffness and fibrosis in patients with AM. Endometrial specimens were also stained with Picrosirius red or Masson's trichrome to quantify fibrosis, and additional analyses assessed α-SMA and Ki-67 expression. Studies indicate that pathological conditions significantly influence the mechanical properties of endometrial tissue. Specifically, adenomyotic endometrial tissue demonstrates increased stiffness, associated with elevated ECM and fibrosis content, whereas normal endometrial samples are softer with lower ECM content. AM appears to alter both the mechanical and histological characteristics of the eutopic endometrium. Higher ECM content may significantly impact endometrial mechanical properties, potentially contributing to AM-associated decidualization defects and fertility challenges.

Retinopathy is a common complication of diabetes mellitus and the leading cause of visual impairment. Danggui Buxue decoction (RRP) has been used as a traditional drug for the treatment of diabetic nephropathy for many years. The aim of this study was to investigate the effects of RRP on hypoxia-induced retinal Müller cell injury. A model of retinal Müller cell damage was created using high glucose levels (25 mmol/L) and/or exposure to low oxygen conditions (1% O2). RRP was given to rats by continuous gavage for 7 days to obtain drug-containing serum. After sterilization, the serum was added to the culture medium at a ratio of 10%. Cell viability, apoptosis, and cell proliferation were assessed using the CCK-8 kit, Annexin V-FITC/propidium iodide apoptosis kit, and EdU kit. The mRNA levels of angiogenesis factors (ANGPTL4, VEGF) and inflammatory factors (IL-1B, ICAM-1) were detected by RT-qPCR. Western blot analysis was employed to assess the levels of proteins related to the ATF4/CHOP pathway. Following hypoxia for 48 h and 72 h, there was a significant decrease in cell viability and proliferation, as well as a notable increase in apoptosis compared to the control group (21% O2). However, high glucose stimulation had no significant effect, and high glucose combined with hypoxia had no further damage to cells. After 48 h of exposure to low oxygen levels, the mRNA expression levels of ANGPTL4, VEGF, IL-1B, and ICAM-1 in retinal Müller cells were significantly higher than in the control group (21% O2). RRP treatment significantly alleviated the increase of cell apoptosis and the upregulation of IL-1B and-1 in retinal Müller cells induced by hypoxia. RRP has the potential to reduce the suppression of the ATF4/CHOP pathway in hypoxia-induced retinal Müller cells, and it significantly alleviates cell apoptosis through regulating inflammatory factors and the ATF4/CHOP pathway.

Proceedings of the 34th National Conference of the Italian Group for the Study of Neuromorphology "Gruppo Italiano per lo Studio della Neuromorfologia" G.I.S.N., Catania, November 22-23, 2024.