Masson's Trichrome Staining Research Articles

Abstract Tu135: FYCO1 ameliorates cardiac remodeling in response to ischemia by amplifying autophagic flux

Background: Dysregulation of autophagy is known to have detrimental effects on cardiac remodeling. FYCO1, a novel regulator of autophagy mediates the transport of autophagosomes, thereby amplifying autophagic flux. Here, we investigate the impact of FYCO1 on myocardial healing via autophagy modulation. Methods: FYCO1-Tg mice crossed with RFP-EGFP-LC3 reporter mice (FYCO1-TGxRGFP) were utilized to analyze autophagic flux via confocal microscopy following permanent ligation of the left anterior descending artery (LAD) to induce myocardial ischemia. Autophagy activity was evaluated via Western blot of p62 and LC3 II. Macrophage infiltration and apoptosis was assessed by immunofluorescence microscopy, while Masson's Trichrome staining detected fibrosis and infarct size. Cardiac function was determined by echocardiography. Results: FYCO1-TGxRGFP mice displayed significantly enhanced autophagic flux post MI, as evidenced by increased p62 (WT LAD: 2,9±0,4; TG LAD:4,0±0,5) and LC3 II (WT LAD: 1,6±0,2; TG LAD:14,0±1,4) protein levels. Confocal microscopy revealed an accumulation of autolysosomes in WT mice 30 days post MI (WT autophagosomes: 1±0,4; WT autolysosomes: 38,6±3,8), contrasting with the sustained ratio of autophagosomes to autolysosomes in FYCO1-TGxRGFP mice (TG autophagosomes:43,3±5,1; TG autolysosomes: 73,5±12,4), implying the preservation of adequate autophagic flux. FYCO1 overexpression reduced macrophage infiltration into the border zone post MI (WT LAD: 132,4 ±14,3; TG LAD: 18,91±3,7 and inhibited apoptosis induction shown by cleaved caspase7 activity (WT LAD: 64,6±10,1; TG LAD: 9,2±2,1). Moreover, FYCO1-TGxRGFP mice exhibited significantly reduced fibrosis (WT:19,6±1,8%; TG:10,6±1,6%) and infarct size (WT:34,5 ±3,9%; TG:5,5±2%) in the left ventricle (LV), accompanied by marked improvements in global heart function post MI (EF WT: 32,7±3,2%; TG: 56,4±3,9%) compared to WT mice. Conclusion: In conclusion, FYCO1 mitigates adverse consequences of ischemic injury via induction of autophagy and a previously unrecognized role in modulating inflammation in response to myocardial infarction.

Abstract Tu063: Acute sleep deprivation induces cardiac remodeling via activation of AT1R/ERK/GSK-3β signaling

Background: Sleep deprivation (SD) has an adverse effect on cardiovascular system. However, whether acute SD contributes to the morphological and functional changes of the heart remains unclear. Approach and Results: The modified multiple platform method (MMPM) was used to establish the SD model in Sprague-Dawley rats. Acute SD significantly enhanced cardiac ejection fraction and fractional shortening in rats as observed by echocardiography. The elevated ratio of heart weight to body weight (HW/BW) and enlarged cross-section area of cardiomyocytes by WGA staining were detected in SD groups (3, 7,10 days). The upregulation of ANP and BNP was also detected by western blotting. Masson-trichrome staining showed significant fibrosis in the SD groups, which was confirmed by western blotting of the fibrosis markers α-SMA and collagen 1. Furthermore, SD increased the level of angiotensin II (Ang II) and angiotensin type 1 receptor (AT1R) in the heart tissue of SD groups accompanied by the activation of the ERK/GSK-3β pathway. Administration of valsartan and benazepril dramatically ameliorated SD-induced cardiac fibrosis and hypertrophy by downregulating Ang II/AT1R and phosphor-ERK/GSK-3β levels. Conclusion: Our results indicated that cardiac remodeling was induced by the acute and short duration of SD, in which the AT1R/ ERK/GSK3β signal pathway was involved. Our study demonstrates a novel complementary phenomenon induced by SD and provides detailed evidence for the risk of SD on the cardiovascular system.

Cardioprotective effect of chelidonic acid against doxorubicin-induced cardiac toxicity in rats

Introduction and objectivesThe current study evaluates the effect of chelidonic acid on doxorubicin-induced cardiac toxicity. Chelidonic acid (CA) is a natural pyran-skeleton heterocyclic compound found in rhizomes of the perennial plant, celandine (Chelidonium majus). MethodsWistar rats were given an intraperitoneal injection of doxorubicin (1.25 mg/kg, cumulative dose of 20 mg/kg) four times per week for a duration of four weeks to induce cardiotoxicity. CA treatment (10, 20, and 40 mg/kg orally for four weeks) was started together with doxorubicin. ResultsCA treatment reduced myocardial damage and improved cardiac dysfunction in doxorubicin-treated rats. It improved blood pressure, restored ST wave height and normalized the QTc interval compared to the rats treated only with doxorubicin. Administration of CA for four weeks reduced left ventricular end-diastolic pressure. Moreover, CA treatment decreased the level of cardiac markers such as creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and cardiac troponin-T. Masson's trichrome, hematoxylin, and eosin staining of heart tissue revealed that CA attenuated the deleterious effects of doxorubicin and prevented further damage and fibrosis in rats. ConclusionThe study findings confirm that CA treatment can protect the myocardium against doxorubicin-induced cardiotoxicity.

Reduced endometrial glycolysis concomitant with increased lesional fibrosis in patients with adenomyosis who complained of heavy menstrual bleeding

RESEARCH QUESTIONWhat role, if any, does the extent of lesional fibrosis play in adenomyosis-associated heavy menstrual bleeding (ADM-HMB)? DESIGNForty-eight patients with ADM-HMB were recruited, among them 25 reported moderate/heavy bleeding (MHB) and the remaining 23, excessive bleeding (EXB). The full-thickness uterine tissue columns were processed for Masson trichrome staining and immunohistochemistry analyses. The levels of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2—proteins critically involved in glycolysis—in endometrial epithelial cells cultured on substrates of different stiffness, and the levels of glycolysis were quantitated. A mouse experiment with induced adenomyosis and simulated menstrual bleeding was conducted to assess the impact of adenomyosis on immunoexpression of proteins involved in glycolysis and inflammation as well as on endometrial repair and bleeding. RESULTSThe endometrial staining of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2 was significantly lower in the EXB group as compared with MHB patients, concomitant with higher extent of fibrosis. The expression of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2 was significantly reduced when endometrial epithelial cells were cultured in stiff substrate, concomitant with reduced glycolysis. Mice with induced adenomyosis exhibited reduced immunoexpression of Hif-1α, as well as those proteins each of which plays a vital, rate-limiting role in different steps of the glycolysis pathway, such as Glut1, Hk2, Pfkfb3 and Pkm2, and elevated fibrosis in endometrium, concomitant with disrupted endometrial repair and more bleeding. CONCLUSIONSLesional fibrosis results in reduced endometrial glycolysis in eutopic endometrium and subsequent imbalance in pro- and anti-inflammatory response, leading to ADM-HMB.

Histogenesis of the cornea in sheep fetuses

In this study, 36 samples of sheep fetuses at different ages were used. After preparation and determination of age, the samples were immediately fixed and subjected to gross anatomy and histology examination. In addition to routine H&E staining, two specific staining methods, Verhoeff and Masson's trichrome, were used in the histological study. The results showed that the corneal forming layer completely loses its contact with the lens and posterior layers and the eyelid ridges start to emerge at around 30 days of gestation. However, the cornea becomes fully differentiated in the second month of gestation and only undergoes growth stages in subsequent ages. From the beginning, the cornea in sheep fetuses is formed as a horizontal ellipse, with its inner curvature larger than its outer curvature. The growth process in terms of dimensions, including longitudinal diameter, transverse diameter, and corneal height, shows a regular ascending trend with increasing age. The upper and lower eyelids fuse in early second month and separate again in late third month. The third eyelid appears at approximately 120 days of gestation in the inner angle of the eye. The outer epithelial covering of the cornea changes from simple cuboidal to stratified squamous epithelium. With increasing age, the matrix is strengthened both in terms of cellular components (fibroblasts) and fibers (collagen). Additionally, an increase in elastic fibers is noticeable in the late fetal stages. The Descemet's membrane appears as an inner limiting membrane beneath the inner epithelial covering in early second month and also undergoes growth with increasing age. Descemet's membrane lacks elastic fibers. Bowman's layer or the outer limiting membrane was not observed in this animal. Masson's trichrome staining confirms the growth of collagen fibers, while Verhoeff staining confirms the growth of elastic fibers with increasing age.

TMAO is involved in kidney-yang deficiency syndrome diarrhea by mediating the “gut-kidney axis”

BackgroundTrimethylamine-N-oxide (TMAO) is a harmful metabolite dependent on the intestinal microbiota and excreted through the kidneys. According to numerous investigations, rich circulation concentrations of TMAO have been linked to kidney and gastrointestinal disorders. Through the “gut-kidney axis” mediated by TMAO, this research attempted to clarify the microbiological causes of kidney-yang deficiency syndrome diarrhea. MethodsAdenine and Folium Sennae were used to create a mouse model of kidney-yang deficiency syndrome diarrhea. 16S rRNA sequencing was used to identify the traits of the intestinal mucosal microbiota. ELISA was used to assess TMAO, transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3). Kidney tissue fibrosis was evaluated using Masson's trichrome staining, and immunohistochemical labeling was used to investigate the protein expression of occludin and Zonula Occludens-1(ZO-1) in small intestine tissue. Microbial activity was determined by using fluorescein diacetate (FDA) hydrolysis spectrophotometry. ResultsTMAO showed a positive correlation with NLRP3, IL-1β and TGF-β1, all of which exhibited substantial increases (P < 0.05). Significant renal fibrosis and decreased ZO-1 and occludin expression in small intestine tissues were detected in the model group. The sequencing results revealed alterations in both α and β diversities of small intestinal mucosal microbiota. Elevated TMAO concentrations were potentially associated with increasing Firmicutes/Bacteroidota (F/B) ratios, Streptococcus, Pseudomonas and unclassified Clostridia UCG 014, but with decreasing Rothia and RB41 abundances. ConclusionThis study establishes a link between intestinal microbiota dysbiosis and elevated TMAO concentrations. TMAO can activate inflammatory responses and cytokines, contributing to kidney-yang deficiency syndrome diarrhea via the “gut-kidney axis”. Moreover, TMAO may coincide with disruptions in the intestinal barrier and renal fibrosis. Dysfunction of the “gut-kidney axis” further elevates TMAO levels, perpetuating a vicious cycle.

Simvastatin suppresses ethanol effects on the kidney of adolescent mice.

Adolescents tend to experiment with ethanol which often results in heavy episodic drinking patterns leading to serious health concerns later in life. Chronic ethanol use damages renal tissue, promotes collagen deposition, and induces renal inflammation, thereby causing renal dysfunction. Therefore, an intervention such as simvastatin (a blood cholesterol-lowering drug) that could suppress the effects of ethanol on the kidney may be beneficial. This study explored the impact of simvastatin against the onset of renal morphological damage, fibrosis, and inflammation caused by ethanol exposure in mice. Ten four-week old C57BL/6J mice (F = 5; M = 5) were assigned to each experimental group: (I) NT; no administration of ethanol or simvastatin; (II) EtOH; 2.5 g/kg/day of 20% ethanol, intraperitoneal injection (i.p.); (III) SIM; 5 mg/kg/day of simvastatin, orally; (IV) EtOH + SIM5; 5 mg/kg/day of simvastatin, orally, followed by 2.5 g/kg/day of 20% ethanol, i.p.; and (V) EtOH + SIM15; 15 mg/kg/day of simvastatin, orally, followed by 2.5 g/kg/day of 20% ethanol, i.p. After the 28-day treatment period, the right kidney was removed and processed for haematoxylin and eosin staining, Masson's trichrome staining, or tumour necrosis factor-alpha (TNF-α) immunohistochemistry. The renal corpuscular area, glomerular area, and urinary space area were measured and the area of collagen or TNF-α expression was quantified using ImageJ software. Ethanol administration significantly increased the renal corpuscular area, the glomerular area, the area of collagen, and the area of tissue with TNF-α immunoreactivity but decreased the area of urinary space. Simvastatin generally suppressed the ethanol effects in both sexes, although to varying degrees. Simvastatin proved to suppress collagen deposition and the TNF-α production induced by ethanol in the kidney of mice thus indicating its effectiveness in the treatment of ethanol-related renal diseases.

A preclinical model of severe NASH-like liver injury by chronic administration of a high-fat and high-sucrose diet in mice

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease, affecting 38% of adults globally. If left untreated, NAFLD may progress to more advanced forms of the disease, including non-alcoholic steatohepatitis (NASH), liver cirrhosis, and fibrosis. Early NAFLD detection is critical to prevent disease progression. Using an obesogenic high-fat and high-sucrose (HF/HS) diet, we characterized the progression of NAFLD in male and female Collaborative Cross CC042 mice after 20-, 40-, and 60-week intervals of chronic HF/HS diet feeding. The incidence and severity of liver steatosis, inflammation, and fibrosis increased in both sexes over time, with male mice progressing to a NASH-like disease state faster than female mice, as indicated by earlier and more pronounced changes in liver steatosis. Histopathological indication of macrovesicular steatosis and gene expression changes of key lipid metabolism genes were found to be elevated in both sexes after 20 weeks of HF/HS diet. Measurement of circulating markers of inflammation (CXCL10 and TNF-α), histopathological analysis of immune cell infiltrates, and gene expression changes in inflammation-related genes indicated significant liver inflammation after 40 and 60 weeks of HF/HS diet exposure in both sexes. Liver fibrosis, as assessed by Picosirius red and Masson's trichrome staining and changes in expression of key fibrosis related genes indicated significant changes after 40 and 60 weeks of HF/HS diet exposure. In conclusion, we present a preclinical animal model of dietary NAFLD progression, which recapitulates human pathophysiological and pathomorphological changes, that could be used to better understand the progression of NAFLD and support development of new therapeutics.

Targeting Angiotensin-converting Enzyme Inhibitor via Enalapril Reduces Postsurgical Adhesion Band Formation

Background: Postoperative adhesions commonly occur after abdominal surgery and can lead to significant complications. There is increasing evidence that targeting the renin-angiotensin system (RAS) can reduce inflammation and fibrosis. This study investigates the therapeutic potential of enalapril, an RAS inhibitor, in a rat model of postsurgical adhesion band formation. Methods: A total of 12 male albino Wistar rats received intraperitoneal administration of enalapril (10 mg/kg). After 9 days, the anti-inflammatory and antifibrotic effects were evaluated using RTPCR and ELISA, alongside hematoxylin and eosin and Masson's trichrome staining. Result: The statistical analysis of findings showed that enalapril significantly reduced the frequency and stability of adhesion bands. It attenuated submucosal edema by suppressing pro-inflammatory cytokines, decreasing pro-inflammatory cell infiltration, and inhibiting oxidative stress at the peritoneal surgery site. Additionally, enalapril inhibited fibrotic adhesion band formation by reducing collagen deposition and suppressing the expression of profibrotic genes in peritoneal adhesion tissues. Conclusion: These findings demonstrate the therapeutic potential of enalapril in preventing postsurgical adhesion band formation by inhibiting key pathological responses of inflammation and fibrosis, supporting its use as a preventive treatment in postoperative adhesion management.

Curcumin analogue EF24 prevents alveolar epithelial cell senescence to ameliorate idiopathic pulmonary fibrosis via activation of PTEN

BackgroundTreating Idiopathic pulmonary fibrosis (IPF) remains challenging owing to its relentless progression, grim prognosis, and the scarcity of effective treatment options. Emerging evidence strongly supports the critical role of accelerated senescence in alveolar epithelial cells (AECs) in driving the progression of IPF. Consequently, targeting senescent AECs emerges as a promising therapeutic strategy for IPF. PurposeCurcumin analogue EF24 is a derivative of curcumin and shows heightened bioactivity encompassing anti-inflammatory, anti-tumor and anti-aging properties. The objective of this study was to elucidate the therapeutic potential and underlying molecular mechanisms of EF24 in the treatment of IPF. MethodsA549 and ATII cells were induced to become senescent using bleomycin. Senescence markers were examined using different methods including senescence-associated β-galactosidase (SA-β-gal) staining, western blotting, and q-PCR. Mice were intratracheally administrated with bleomycin to induce pulmonary fibrosis. This was validated by micro-computed tomography (CT), masson trichrome staining, and transmission electron microscope (TEM). The role and underlying mechanisms of EF24 in IPF were determined in vitro and in vivo by evaluating the expressions of PTEN, AKT/mTOR/NF-κB signaling pathway, and mitophagy using western blotting or flow cytometry. ResultsWe identified that the curcumin analogue EF24 was the most promising candidate among 12 compounds against IPF. EF24 treatment significantly reduced senescence biomarkers in bleomycin-induced senescent AECs, including SA-β-Gal, PAI-1, P21, and the senescence-associated secretory phenotype (SASP). EF24 also effectively inhibited fibroblast activation which was induced by senescent AECs or TGF-β. We revealed that PTEN activation was integral for EF24 to inhibit AECs senescence by suppressing the AKT/mTOR/NF-κB signaling pathway. Additionally, EF24 improved mitochondrial dysfunction through induction of mitophagy. Furthermore, EF24 administration significantly reduced the senescent phenotype induced by bleomycin in the lung tissues of mice. Notably, EF24 mitigates fibrosis and promotes overall health benefits in both the acute and chronic phases of IPF, suggesting its therapeutic potential in IPF treatment. ConclusionThese findings collectively highlight EF24 as a new and effective therapeutic agent against IPF by inhibiting senescence in AECs.

Biological characteristics related to treatment effects of the levonorgestrel-releasing intrauterine system on adenomyosis-associated dysmenorrhoea

Research questionTo investigate the correlations among the biological expression of steroid receptors, fibrosis levels in adenomyotic lesions, and the treatment effectiveness of the levonorgestrel-releasing intrauterine system (LNG-IUS) on alleviation of dysmenorrhea. DesignIn this retrospective cohort study, 125 women with adenomyosis who underwent hysterectomy were included. The tissue samples were collected from 41 patients who had been treated by the LNG-IUS prior to surgery and these patients were further categorized into an effective group (n = 18) and a failure group (n = 23) according to their self-reported relief from dysmenorrhea after 6-month treatment. The expression levels of estrogen receptor-α (ER-α), progesterone receptor (PR), and the extent of fibrosis levels in the adenomyotic lesions were measured by immunohistochemistry and Masson's trichrome staining respectively. ResultsPatients in the failure group demonstrated lower expression levels of ER-α and PR and more pronounced fibrosis in the stroma of adenomyotic lesions compared to those in the effective group. In the glandular epithelium of lesions, ER-α expression was significantly reduced in the failure group, whereas no notable difference in PR expression was observed. Notably, the staining intensity of ER-α in the stroma of the lesions was found to have the strongest positive correlation with the degree of symptom alleviation for dysmenorrhea (r=0.47) with an area under the curve of 0.894 for prediction. ConclusionsThe findings suggest that the reduced expressions of steroid receptors in adenomyotic lesions especially ER-α in the stroma were associated with an increased likelihood of treatment failure of LNG-IUS in alleviating dysmenorrhea.

Carbon monoxide attenuates cellular senescence-mediated pulmonary fibrosis via modulating p53/PAI-1 pathway

PurposeIdiopathic pulmonary fibrosis (IPF) is a fatal progressive condition often requiring lung transplantation. Accelerated senescence of type II alveolar epithelial cells (AECII) plays a crucial role in pulmonary fibrosis progression through the secretion of the senescence-associated secretory phenotype (SASP). Low-dose carbon monoxide (CO) possesses anti-inflammatory, anti-oxidative, and anti-aging properties. This study aims to explore the preventive effects of CO-releasing molecule 2 (CORM2) in a bleomycin-induced pulmonary fibrosis model. MethodsWe established an pulmonary fibrosis model in C57BL/6J mice and evaluated the impact of CORM2 on fibrosis pathology using Masson's trichrome staining, fluorescence staining, and pulmonary function tests. Fibrogenic marker expression and SASP secretion in tissues and AECII cells were analyzed using qRT-PCR, Western blot, and ELISA assays both in vivo and in vitro. Additionally, we investigated DNA damage and cellular senescence through immunofluorescence and SA-β-gal staining. ResultsCORM2 showed a preventive effect on bleomycin-induced lung fibrosis by improving pulmonary function and reducing the expression of fibrosis-related genes, such as TGF-β, α-SMA, Collagen I/III. CORM2 decreased the DNA damage response by inhibiting γ-H2AX, p53, and p21. We identified PAI-1 as a new target gene that was downregulated by CORM2, and which was associated with cellular senescence and fibrosis. CORM2 effectively inhibited cellular senescence and delayed EMT occurrence in AECII cells. ConclusionOur study highlights the potential of CORM2 in preventing DNA damage-induced cellular senescence in bleomycin-induced pulmonary fibrosis through modulation of the p53/PAI-1 signaling pathway. These findings underscore the promising prospects of CORM2 in targeting cellular senescence and the p53/PAI-1 pathway as a potential preventive strategy for IPF.

Approaches to scarless burn wound healing: application of 3D printed skin substitutes with dual properties of anti-infection and balancing wound hydration levels

Severe burn wounds face two primary challenges: dysregulated cellular impairment functions following infection and an unbalanced wound hydration microenvironment leading to excessive inflammation and collagen deposition. These results in hypertrophic scar contraction, causing significant deformity and disability in survivors. A three-dimensional (3D) printed double-layer hydrogel (DLH) was designed and fabricated to address the problem of scar formation after burn injury. DLH was developed using methacrylated silk fibroin (SFMA) and gelatin methacryloyl (GelMA) for the upper layer, and GelMA and hyaluronic acid methacryloyl (HAMA) for the lower layer. To combat infection, copper-epigallocatechin gallate (Cu-EGCG) was incorporated into the lower layer bioink, collectively referred to as DLS. To balance wound hydration levels, HaCaT cells were additionally encapsulated in the upper layer, designed as DLS/c. DLH demonstrated suitable porosity, appropriate mechanical properties, and excellent biocompatibility. DLS exhibited potent antimicrobial properties, exerted anti-inflammatory effects by regulating macrophage polarisation, and may enhance angiogenesis through the HIF-1α/VEGF pathway. In the DLS/c group, animal studies showed significant improvements in epidermal formation, barrier function, and epidermal hydration, accompanied by reduced inflammation. In addition, Masson's trichrome and Sirius red staining revealed that the structure and ratio of dermal collagen in DLS/c resembled that of normal skin, indicating considerable potential for scarless wound healing. This biomimetic matrix shows promise in addressing the challenges of burn wounds and aiming for scarless repair, with benefits such as anti-infection, epidermal hydration, biological induction, and optimised topological properties. Shown in Acknowledgements.

Proanthocyanidin surface preconditioning of dental pulp stem cell spheroids enhances dimensional stability and biomineralization invitro.

Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold-free microtissue-based dental pulp regeneration. Surface collagen cross-linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross-linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)-derived 3D cell spheroids. Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross-linking. Microtissue viability was examined by live/dead staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA-preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA-preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F-actin expressions. PA-preconditioned macrotissues were subjected to histological analysis, including haematoxylin-eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP-1) expressions. PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse-section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP-1 expressions in macrotissues. Additionally, PA preconditioning induced higher F-actin and DSPP expression in microtissues, while inhibition of F-actin activity by cytochalasin B attenuated PA-induced dimensional change and DSPP upregulation. PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC-derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold-free tissue engineering applications in regenerative dentistry.

Pathological characteristics analysis of children with intermittent and persistent hydronephrosis due to uretero-pelvic junction obstruction.

To analyze from a pathological perspective the differences between intermittent and persistent hydronephrosis in children with uretero-pelvic junction obstruction. 23 children who underwent unilateral dismembered pyeloplasty (Anderson-Hynes operation) for intermittent hydronephrosis from September 2017 to March 2024 were included in the observation group. They were compared with a control group consisting of 23 children with persistent hydronephrosis matched for age, gender, and affected side. All children had the narrowed segment surgically excised during the operation, while other obstructive causes (such as polyps, crossing vessels, or tumor compression) were excluded. The specimens were analyzed for muscle and collagen content using Masson's trichrome staining, and the collagen-to-muscle ratio (CMR) was calculated. The number of Cajal-like cells was quantified with c-kit immunohistochemical staining. For all slides, 10 random fields of view were selected under a 400× optical microscope to record pathological data and calculate mean values. Pathological indicators between the two groups were compared using the T-test and the Chi-square test, with P < 0.05 considered statistically significant. The observation group showed a significant difference in the number of fields with low, medium, and high densities of Cajal-like cells compared to the control group [132 (57.4%) vs. 173 (75.2%); 70 (30.4%) vs. 38 (16.5%); 28(12.2%) vs. 19 (8.3%), P < 0.001]. The uretero-pelvic junction in children with intermittent hydronephrosis had lower collagen content, higher muscle content, and a more regular arrangement. The collagen-muscle ratio was significantly lower than that in children with persistent hydronephrosis [(1.59 ± 0.65) vs. (3.98 ± 1.19), P < 0.001]. Compared with persistent hydronephrosis, the narrowed segment at the uretero-pelvic junction in children with intermittent hydronephrosis has a higher density of Cajal-like cells; lower collagen content, and higher muscle content (lower collagen-muscle ratio).

Renal denervation alleviates vascular remodeling in spontaneously hypertensive rats by regulating perivascular adipose tissue.

Vascular remodeling is the main pathological process that causes the damage of the target organ of hypertension. Perivascular adipose tissue (PVAT) surrounds blood vessels and plays a key role in the pathogenesis of various cardiovascular diseases. This study aimed to investigate the effects of renal denervation (RDN) on hypertensive vascular remodeling and to elucidate the role of PVAT in this process. Male spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat were selected. Aortic vascular remodeling was evaluated using hematoxylin and eosin (H&E) staining and Masson's trichrome staining. Morphological changes in the PVAT were observed through H&E and Oil Red O staining. Dihydroethidium was used to measure oxidative stress levels in PVAT, while western blot analysis was used to determine the expression levels of proteins associated with vascular remodeling. The results showed that the aortic medial thickness, media thickness/lumen diameter, collagen volume fraction, and reactive oxygen species (ROS) level in PVAT were significantly higher in the SHR group than in the WKY group. The indexes mentioned above were lower in the SHR-RDN group than in the SHR group. H&E staining revealed that fat droplets in PVAT in the SHR-RDN group became smaller and browning occurred. Moreover, the protein expression of uncoupling protein-1 (UCP-1) and neuregulin 4 (Nrg4) was significantly increased in the SHR-RDN group. In addition, the expression of adiponectin increased and the expression of leptin decreased in the SHR-RDN group compared to the SHR group. In conclusion, RDN can relieve hypertensive vascular remodeling, which may be associated with PVAT.

Trimetazidine improves angiogenesis and tissue perfusion in ischemic rat skeletal muscle.

Introduction: Peripheral artery disease (PAD) is an increasingly common disease, causing significant complications for patients. Trimetazidine (TMZ) not only improves clinical symptoms in PAD patients but also facilitates angiogenesis in ischemic hind limbs. Our aim was to find the function of TMZ in promoting angiogenesis and tissue perfusion in ischemic rat skeletal muscle. Methods: The rats underwent femoral artery ligation (FAL) and then treated with TMZ and saline. Hematoxylin-eosin and Masson's trichrome stain in the ischemic gastrocnemius muscle to analyze muscle morphology and atrophy. To identify angiogenesis and the tissue perfusion, CD31 immunohistochemical staining and laser speckle contrast imaging was conducted. Additionally, hind limb motor ability was measured. Finally, qRT-PCR and Western blotting were used to statistically analyze the expression levels of HIF-1α and VEGF. Results: Our study demonstrated significant enhancement in angiogenesis and tissue perfusion after FAL when treated with TMZ compared to the saline group. Histologically, it mitigates ischemia-induced muscle atrophy and inflammation, as well as reduces fibrosis progression in the TMZ group. Additionally, hind limb motor ability improved in rats treated with TMZ during motor experiments. Discussion: It suggests that TMZ can promote angiogenesis and improve tissue perfusion in ischemic skeletal muscle of rats by activating the HIF-1α/VEGF signaling pathway. Additionally, it leads to significant improvement in ischemia-induced motor limitations in the hind limbs of rats.

Differential effects of clopidogrel and/or aspirin on the healing of tooth extraction wound bone tissue.

A multitude of variables influence the healing of tooth extraction wounds, and delayed or non-healing extraction wounds might complicate later prosthodontic therapy. In this research, we analyzed the effects of systemic clopidogrel and aspirin alone or in combination on the healing of tooth extraction wounds in mice in order to provide experimental evidence for the healing of extraction wounds in patients who are clinically treated with the two medicines. 7-week-old ICR mice were randomly divided into four groups: control group (CON), clopidogrel group (CLOP), aspirin group (ASP), and clopidogrel combined with aspirin group (CLOP + ASP); left upper first molar was extracted, after which mice in 1week of adaptive feeding, CLOP/ASP/CLOP + ASP groups were respectively administered with clopidogrel (10mg/kg/d), aspirin (15mg/kg/d), clopidogrel (10mg/kg/d)+aspirin (15mg/kg/d), and the control group was given an equal amount of 0.9% saline by gavage. Mice in each group were euthanized at 14 and 28days postoperatively, and the maxilla was extracted. The tissues in the extraction sockets were examined using MicroCT and sectioned for HE staining, Masson staining, and TRAP staining, and immunohistochemistry staining (for TRAP, RANKL and osteoprotegerin). MicroCT analysis showed that at day 14, BS/BV was significantly lower in CLOP and CLOP + ASP groups compared to control and ASP groups, while BV/TV, Tb.Th was significantly higher. At day 28, BV/TV was significantly higher in the CLOP + ASP group compared to the CLOP group, with p < 0.05 for all results. HE staining and Masson trichrome staining findings revealed that at day 28, the mesenchyme in the bone was further decreased compared to that at day 14, accompanied with tightly arranged and interconnected bone trabeculae. In the quantitative analysis of Masson, the fraction of newly formed collagen was significantly higher in the CLOP group in comparison with that in the CON group (p < 0.05). At day 14, the ASP group had substantially more TRAP-positive cells than the CLOP and CLOP + ASP groups (p < 0.05). In immunohistochemical staining, RANKL expression was found to be significantly higher in the ASP group than those in the other three groups at day 28 (p < 0.05); OPG expression was significantly higher in the CLOP group and the CLOP + ASP group compared with that at day 14, and was higher than that in the ASP group at day 14 and day 28. OPG/RANKL was significantly higher in the CLOP and the CLOP + ASP groups than in the ASP group (p < 0.05). Clopidogrel alone promotes osteogenesis in the extraction wound, whereas aspirin alone inhibits alveolar bone healing. When the two drugs were combined, the healing effect of the extraction wound was more similar to that of the clopidogrel alone group. These results indicated that clopidogrel could promote the healing of the tooth extraction wound, and neutralize the adverse effect of ASP on osteogenesis when the two drugs were used in combination.

Enhanced Cardiomyocyte NLRP3 Inflammasome-Mediated Pyroptosis Promotes d-Galactose-Induced Cardiac Aging.

Cardiac aging represents an independent risk factor for aging-associated cardiovascular diseases. Although evidence suggests an association between NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome formation and numerous cardiovascular diseases, its role in cardiac aging remains largely unclear. The longevity of mice with wild-type and NLRP3 knockout (NLRP3-/-) genotypes was assessed, with or without d-galactose treatment. Cardiac function was evaluated using echocardiography, and cardiac histopathology was examined through hematoxylin and eosin and Masson's trichrome staining. Senescence-associated β-galactosidase (SA-β-gal) staining was employed to detect cardiac aging. Western blotting was used to assess aging-related proteins (p53, p21) and pyroptosis-related proteins. Additionally, dihydroethidium staining, lactate dehydrogenase release, and interleukin-1β ELISA assays were performed, along with measurements of total superoxide dismutase and malondialdehyde levels. Invitro, H9c2 cells were exposed to d-galactose for 24 hours in the absence or presence of N-acetyl-l-cysteine (reactive oxygen species inhibitor), BAY-117082 (nuclear factor κ-light-chain enhancer of activated B cells inhibitor), MCC950 (NLRP3 inhibitor), and VX-765 (Caspase-1 inhibitor). Immunofluorescence staining was employed to detect p53, gasdermin D, and apoptosis-associated speck-like protein proteins. Intracellular reactive oxygen species levels were assessed using fluorescence microscopy and flow cytometry. Senescence-associated β-galactosidase staining and Western blotting were also employed invitro for the same purpose. The results showed that NLRP3 upregulation was implicated in aging and cardiovascular diseases. Inhibition of NLRP3 extended life span, mitigated the aging phenotype, improved cardiac function and blood pressure, ameliorated lipid metabolism abnormalities, inhibited pyroptosis in cardiomyocytes, and ultimately alleviated cardiac aging. Invitro, the inhibition of reactive oxygen species, nuclear factor κ-light-chain enhancer of activated B cells, NLRP3, or caspase-1 attenuated NLRP3 inflammasome-mediated pyroptosis. The reactive oxygen species/nuclear factor κ-light-chain enhancer of activated B cells/NLRP3 signaling pathway loop contributes to d-galactose-treated cardiomyocyte senescence and cardiac aging.

The particularities of connective fibers from the wall of varicose veins extirpated by cryostripping.

The varicose vein affects more than 30% of the general population. Significantly increased rates were noticed in women and older population. From the histopathological point of view, venous arterialization, smooth muscle cell hypertrophy, and hyperplasia are the main changes noticed in varicose vein disease. Some of the main therapeutic methods used in the management of varicose disease are injection sclerotherapy, conservative, surgical, saphenous vein inversion and removal, high saphenous ligation, ambulatory phlebectomy, transilluminated powered phlebectomy, endovascular management, cryostripping. The aim of this study was to evaluate the morphology of connective fibers from the wall of the varicose veins extirpated by cryostripping. The study included 109 samples taken by cryostripping method. Hematoxylin-Eosin, Masson's trichrome, Silver and Orcein staining were applied. The assessment of fibers was made according to score values between 0 and 3. It was found no major structural differences in terms of alterations of collagen fibers induced by the applied surgical procedure. It was noticed duplications and multiplications of the internal elastic lamina, as diffuse and nodular forms. Depletion of elastic fibers at the media was a lesion identified in most of the specimens. The depletion of reticulin fibers correlates with the accumulation of collagen fibers, which partially or completely replace the network in the media and intima. No correlation was found between changes in the reticulin network and the time between prelevation and buffered formalin fixation, the maximum time investigated being five days. The Orcein staining in the venous vessel evaluation panel may be a useful investigation.