Trichrome Research Articles

Characterization of decellularized porcine oviduct- and uterine-derived scaffolds evaluated by spermatozoa-based biocompatibility and biotoxicity

Extracellular matrix-derived scaffolds (dECM) are widely utilized in regenerative medicine and tissue engineering due to their ability to promote cell growth, proliferation, and differentiation. In reproduction, research is focused on using these scaffolds to treat pathologies causing reproductive dysfunction or to improve assisted reproduction technologies (ARTs). We developed an efficient protocol employing the immersion-agitation technique to decellularize porcine oviductal and uterine sections, comparing the efficacy of fresh versus frozen treatments. Both methods successfully generated acellular matrices with less than 3% residual DNA, effectively preserving structural and protein integrity. Scanning and transmission electron microscopy confirmed the ultrastructural integrity, whereas Masson's Trichrome staining highlighted better collagen preservation in frozen treatments. Proteomic analysis of decellularized scaffolds revealed collagen and key macromolecules such as laminin, filamin, dermatopontin, and fibronectin, which are essential for extracellular matrix structure and cell functions such as adhesion and migration. Innovatively, we assessed the biocompatibility and cytotoxicity of the scaffolds using spermatozoa, demonstrating that thorough washing ensures the scaffold biocompatibility without compromising sperm viability or motility. Our findings not only contribute to the standardization of decellularization protocols for female reproductive organs but also emphasize the importance of evaluating sperm biocompatibility to ensure the safety of dECM scaffolds.

The Therapeutic Potential of Metformin for Attenuating Postsurgical Adhesion Band Formation

Objectives: Intra-abdominal adhesions are the most frequently occurring postoperative complication following abdominopelvic surgery. Peritoneal adhesion formation can lead to infertility, chronic pelvic pain, and intestinal obstruction. Several studies have shown the potential role of metformin in reducing inflammation. Here, we explored the therapeutic potency of Metformin against postsurgical adhesion band formation. Methods: We adopted Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the experimental protocol. Peritoneal adhesions were examined macroscopically .Metformin (100 mg/kg) was administered intraperitoneally in male albino Wistar rats. After 9 days, macroscopic evidence and score of fibrotic bands based on scaling methods were performed and evaluated by the Nair and Leach scoring system. Moreover, the anti-inflammatory and antifibrosis effects were investigated by using hematoxylin and eosin, Masson's trichrome staining, the enzyme-linked immunosorbent assay (ELISA), and reverse transcription polymerase chain reaction (RT-PCR). Results: Metformin inhibited the stabilization of adhesion bands and appeared to elicit antiinflammatory responses by attenuating submucosal edema, suppressing proinflammatory cytokines, decreasing proinflammatory cell infiltration, and inhibiting oxidative stress at the site of peritoneal surgery. We also showed that metformin prevents fibrotic adhesion band formation by reducing excessive collagen deposition and suppression of profibrotic gene expression at the peritoneum adhesion tissues via modulation of Col 1A1/A3. Conclusion: These results supported the potential application of metformin in preventing postsurgical adhesion band formation by inhibiting key pathologic responses of inflammation and fibrosis in patients post-surgery.

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Aldo-Keto reductase 1C3 reduces myocardial cell damage after acute myocardial infarction by activating the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2-antioxidant response element pathway to inhibit ferroptosis.

Acute myocardial infarction (AMI) is a high-risk cardiovascular condition associated with increased cellular damage and oxidative stress. Aldo-Keto Reductase 1C3 (AKR1C3) is a stress-regulating gene. Nevertheless, its specific role and mechanisms regarding AMI remain unclear. We assessed cardiac function through echocardiography; tissue damage was evaluated using Hematoxylin and Eosin (HE) and Masson trichrome staining. AKR1C3 expression levels were measured through Reverse transcription-quantitative polymerase chain reaction and western blot. Assessed cell viability using Cell Counting Kit-8 and lactate dehydrogenase (LDH) assays. The extent of ferroptosis was determined by measuring the levels of Fe2+, boron-dipyrromethane (BODIPY) and malondialdehyde (MDA), the glutathione/glutathione disulfide (GSH/GSSG) ratio, and the expression of Glutathione Peroxidase 4 (GPX4) and Solute carrier 7A11 (SLC7A11). Kelch-like ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2-Antioxidant response element (Keap1-Nrf2-ARE) pathway activation was analyzed through western blotting. Nrf2 was inhibited with ML385 and activated with (R)-Sulforaphane to investigate the Keap1-Nrf2-ARE pathway. The rats in the AMI group displayed reduced heart function, more tissue damage, and lower AKR1C3 expression compared to the Sham group. Similarly, hypoxia-treated H9C2 cells showed reduced viability, and decreased AKR1C3 expression. Overexpressing AKR1C3 in H9C2 cells enhanced viability. Knocking down AKR1C3 exhibited the opposite effect. Of the inhibitors tested, Ferrostatin-1 most effectively restored cell viability in hypoxia-treated H9C2 cells. Moreover, H9C2 cells subjected to hypoxia suggested Keap1-Nrf2-ARE pathway inhibition. Overexpressing AKR1C3 reduced ferroptosis and activated the Keap1-Nrf2-ARE pathway in hypoxia-treated cells, knocking down AKR1C3 exhibited the opposite effect. Further experiments using ML385 in hypoxia-treated H9C2 cells with overexpressed AKR1C3 showed decreased viability and increased ferroptosis compared to the control. Using (R)-Sulforaphane in hypoxia-treated H9C2 cells with knocked-down AKR1C3 exhibited the opposite effect. This study's findings indicate that AKR1C3 plays a role in regulating ferroptosis in myocardial cells, with the Keap1-Nrf2-ARE pathway likely being a key mechanism behind it.

Efficacy of Avocado Oil Cream in Wound Repair: Macroscopic Analysis, Inflammatory Cells Count, and Collagen Density

The wound healing process goes through a series of complex stages that are mutually continuous, namely inflammation, proliferation, and maturation. Following wound formation and the release of proinflammatory cytokines and growth factors, inflammation occurs immediately after the hemostasis phase. Proliferation occurs when products processed by the growth factors are present. The final stage, maturation, is portrayed by the plan of the extracellular network. This study utilized oil from avocado fruit (Persea americana), which contains linoleic and oleic acid content that supports skin tissue repair. The study aimed to explore the effects of using avocado oil cream on the area of wound healing, the number of inflammatory cells, and the collagen density. Twenty-four female mice, aged eight weeks, were used as experimental animals by making excision wounds using a 4 mm biopsy punch on the dorsal skin on the left and right sides. The mice were divided into four groups based on the percentage of avocado oil in the topical cream included Group K (control, topical cream without avocado oil), Group P1 (5% topical avocado oil cream), Group P2 (10% topical avocado oil cream), and Group P3 (15% topical avocado oil cream). Macroscopic examination of the wounds was conducted daily on days 3, 6, and 9 after topical cream treatment using a digital caliper. A total of 48 skin tissue samples were collected from days 3, 6, and 9 after cream application, which were then processed for histopathology evaluations using hematoxylin-eosin staining and Masson’s Trichrome staining. Hematoxylin-eosin staining was used to count the inflammatory cells, and Masson's Trichrome staining was employed to assess collagen density. The results revealed that avocado oil had a great impact on wound closure after 9 days of 15% avocado oil cream treatment, reducing the inflammatory cells after 3-6 days of 10% avocado oil cream therapy, and increasing collagen density after 9 days of 15% avocado oil cream application, as compared to the control, non-avocado oil cream group. Avocado oil can help close wounds, reduced the number of inflammatory cells, and increased collagen density when used in topical pharmaceutical formulations. Avocado oil cream may, therefore, be considered a viable option for wound repair treatment.

Comprehensive Postnatal Anatomical, Histological and Geometric Morphometric Analysis of Thymus Development in Dromedary Camels (Camelus dromedarius).

The thymus, a primary lymphoid organ, plays a critical role in T lymphocyte development and adaptive immunity. This study focuses on the anatomical, histological and geometric morphometric characteristics of the thymus in dromedary camels (Camelus dromedarius) during postnatal development. Thymus samples were collected from camels aged approximately 4, 8, 12 and 16 months. Using photogrammetry and 3D modelling, the samples were analysed to generate landmarks and conduct geometric morphometry with the 3D Slicer and ALPACA algorithm. Principal component analysis (PCA) was then performed to evaluate shape variations. Histologically, the samples underwent Haematoxylin and Eosin and Masson's trichrome staining. Image analysis using QuPath software quantified trabeculae, adipose tissue and Hassall's corpuscles. The results revealed significant anatomical and histological changes in the thymus across the different age groups. Notable variations in tissue composition and structural integrity were observed, with the PCA highlighting distinct morphometric patterns associated with age-related development. These findings provide a deeper understanding of thymus maturation in dromedaries and offer valuable data for comparative anatomy and veterinary medicine. This comprehensive analysis enhances our knowledge of species-specific immune development, with important implications for the health and resilience of these animals in arid environments.

Encapsulation of Mentha aquatica methanol extract in alginate hydrogel promotes wound healing in a murine model of Pseudomonas aeruginosa burn infection

Burn injuries are the fourth most prevalent devastating form of trauma worldwide. Among the most extensively explored materials, composite dressings with alginate loaded with herbal extract can be mentioned. This research aimed to develop a sodium alginate (SA)-based hydrogel encapsulated with Mentha aquatica (MA) methanol extract and investigate its therapeutic efficacy in the infected burn mouse model. SEM, FTIR, in vitro extract release, HPLC, mechanical test, contact angle, swelling, degradability, and temperature response properties were used to analyze the hydrogel scaffold's physicochemical structure. Additionally, the antibacterial activity and MIC level of the extract, cell cytotoxicity, and macroscopic and microscopic analysis of the wound healing process were done using Masson's trichrome and hematoxylin-eosin staining. The physicochemical properties of the SA hydrogel encapsulated with MA extract were verified. The lowest inhibitory dose of the extract was determined to be 12.5 mg/ml. Application of SA/MA hydrogel to localized wounds of deep third-degree burns demonstrated faster tissue regeneration, collagen recovery, and eradication of bacterial infection. This research focused on the design and the preparation of a novel and effective biomaterials-based medical product, which has the potential to rehabilitate infected and injured skin tissue; therefore, it can be a promising candidate for wound dressing applications.

Histological Changes in Skin and Subcutaneous Cellular Tissue in Patients with Massive Weight Loss After Bariatric Surgery.

Morbid obesity is a public health problem with high social and economic impact. Due to failure in its treatment with traditional weight loss strategies, surgical interventions are usually required, which give rise to massive weight loss. Until now, the studies made are inconclusive regarding the histological changes in the skin caused by massive weight loss, which may later generate post-surgical complications. Therefore, the objective of this study is to evaluate the cutaneous histological changes before and after bariatric surgery. Two skin biopsies were taken from nine different patients who underwent bariatric surgery. The first biopsy was taken before the surgical intervention, and the second biopsy was taken a year after the surgery and massive weight loss. Histological analysis was performed using haematoxylin & eosin staining, Weigert's Resorcin-Fuchsin, and Masson's trichrome stain to analyse the percentage of collagen fibres, percentage of fibrosis, percentage of vascularity, vascular layer involvement and adipocyte population. The differences between the biopsies were evaluated with the Student's T test and Mann-Whitney U test, with a p-value of <0.05. Biopsies from post-bariatric patients with MWL showed an increase in fibrosis percentage, and a decrease in collagen fibres, elastic fibres of the dermis, adipocyte population, as well as reduced vascular proliferation. MWL after bariatric surgery gives rise to changes in the skin and subcutaneous cellular tissue. Skin shows an increased fibrosis percentage after massive weight loss. Elastic and collagen fibres become disorganized after massive weight loss. Vascular proliferation is decreased after massive weight loss. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Virtual multi-staining in a single-section view for renal pathology using generative adversarial networks

Sections stained in periodic acid-Schiff (PAS), periodic acid-methenamine silver (PAM), hematoxylin and eosin (H&E), and Masson's trichrome (MT) stain with minimal morphological discordance are helpful for pathological diagnosis in renal biopsy. Here, we propose an artificial intelligence-based re-stainer called PPHM-GAN (PAS, PAM, H&E, and MT-generative adversarial networks) with multi-stain to multi-stain transformation capability. We trained three GAN models on 512 × 512-pixel patches from 26 training cases. The model with the best transformation quality was selected for each pair of stain transformations by human evaluation. Frechet inception distances, peak signal-to-noise ratio, structural similarity index measure, contrast structural similarity, and newly introduced domain shift inception score were calculated as auxiliary quality metrics. We validated the diagnostic utility using 5120 × 5120 patches of ten validation cases for major glomerular and interstitial abnormalities. Transformed stains were sometimes superior to original stains for the recognition of crescent formation, mesangial hypercellularity, glomerular sclerosis, interstitial lesions, or arteriosclerosis. 23 of 24 glomeruli (95.83 %) from 9 additional validation cases transformed to PAM, PAS, or MT facilitated recognition of crescent formation. Stain transformations to PAM (p = 4.0E-11) and transformations from H&E (p = 4.8E-9) most improved crescent formation recognition. PPHM-GAN maximizes information from a given section by providing several stains in a virtual single-section view, and may change the staining and diagnostic strategy.

Understanding the hierarchical structure of collagen fibers of the human periodontal ligament: Implications for biomechanical characteristics

The periodontal ligament (PDL) is a unique fibrous connective tissue that regulates periodontal homeostasis mechanisms. Its biomechanical properties primarily reside in the hierarchical and non-uniform collagenous network. This study aimed to investigate the region-specific structure and composition of collagen fibers in the PDL at various scales and to explore their relationship with mechanical properties in a split-mouth design. Fresh human cadaver transverse PDL specimens of maxillary anterior teeth were categorized into cervical, middle, and apical groups. These specimens were analyzed via Masson’s trichrome staining, scanning electron microscopy, picrosirius red (PSR) staining, three-dimensional (3D) reconstruction, Raman spectroscopy, and uniaxial tensile test. Statistical analyses were performed to compare the structural, compositional, and tensile properties among the groups. Notably, the middle PDL samples exhibited superior tensile strength and higher fiber area fraction than the other two transverse sections. Despite a higher mineral-to-matrix ratio and a different collagen secondary structure, the apical PDL demonstrated a relatively weaker tensile strength, possibly associated with its discovered sparser collagen fiber areal fraction. The cervical region, characterized by a mediocre fiber areal fraction, displayed diminished tensile strength. The 3D reconstructed collagenous network model and PSR staining exposed the fiber interaction and the micropores. Microscale porosity and variations in collagen secondary structure, particularly in the apical region, suggest adaptive mechanisms for accommodating compressive forces and maintaining functional integrity. Variance in the tensile properties of samples in different force directions indicated the significant influence of fiber orientation and root level on tissue mechanics. Statement of significanceThis study provides critical insights into the biomechanical and structural properties of the human periodontal ligament (PDL), particularly focusing on the underexplored anterior teeth. Through advanced techniques like SEM, histological staining, 3D reconstruction, Raman spectroscopy, and tensile testing, we reveal significant regional variations in PDL collagen organization, composition, and biomechanical properties. Our findings address a crucial knowledge gap concerning the material mechanics of the PDL, offering a foundational understanding for future periodontal tissue engineering and biomimetic material development. This multi-scale analysis underscores the importance of both mesoscale structural characteristics and nanoscale molecular structures in maintaining PDL mechanical integrity.

A novel pulmonary fibrosis NOD/SCID murine model with natural aging

BackgroundIdiopathic pulmonary fibrosis (IPF) is an age-related disease severely affecting life quality with its prevalence rising as the population ages, yet there is still no effective treatment available. Cell therapy has emerged as a promising option for IPF, however, the absence of mature and stable animal models for IPF immunodeficiency hampers preclinical evaluations of human cell therapies, primarily due to rapid immune clearance of administered cells. This study aims to establish a reliable pulmonary fibrosis (PF) model in immunodeficient mice that supports autologous cell therapy and to investigate underlying mechanism.MethodsWe utilized thirty 5-week-old male NOD/SCID mice, categorizing them into three age groups: 12weeks, 32 weeks and 43 weeks, with 6 mice euthanized randomly from each cohort for lung tissue analysis. We assessed fibrosis using HE staining, Masson’s trichrome staining, α-SMA immunohistochemistry and hydroxyproline content measurement. Further, β-galactosidase staining and gene expression analysis of MMP9, TGF-β1, TNF-α, IL-1β, IL-6, IL-8, SOD1, SOD2, NRF2, SIRT1, and SIRT3 were performed. ELISA was employed to quantify protein levels of TNF-α, TGF-β1, and IL-8.ResultsWhen comparing lung tissues from 32-week-old and 43-week-old mice to those from 12-week-old mice, we noted a marked increase in inflammatory infiltration, fibrosis severity, and hydroxyproline content, alongside elevated expression levels of α-SMA and MMP9. Notably, the degree of fibrosis intensified with age. Additionally, β-galactosidase staining became more pronounced in older mice. Quantitative PCR analyses revealed age-related, increases in the expression of senescence markers (GLB1, P16, P21), and proinflammatory genes (TGF-β1, TNF-α, IL-1β, IL-6, and IL-8). Conversely, the expression of anti-oxidative stress-related genes (SOD1, SOD2, NRF2, SIRT1, and SIRT3) declined, showing statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). ELISA results corroborated these findings, indicating a progressive rise in the protein levels of TGF-β1, TNF-α, and IL-8 as the mice aged.ConclusionsThe findings suggest that NOD/SCID mice aged 32 weeks and 43 weeks effectively model pulmonary fibrosis in an elderly context, with the disease pathogenesis likely driven by age-associated inflammation and oxidative stress.

Abstract B075: Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant cancer- associated fibroblasts (CAFs) which contribute to a tumor-promoting, chemotherapy-resistant and immunosuppressive tumor microenvironment (TME). However, CAF-ablating therapeutic strategies have thus far failed in clinical trials. One potential reason is that PDAC CAFs are heterogeneous and plastic. It was recently discovered that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) can originate from pancreatic stellate cells (PSCs) via TGF-β and IL-1 signaling, respectively. However, their functional heterogeneity remains to be elucidated. We hypothesize that distinct CAF subtypes exhibit different tumor-promoting roles mediated by targetable signaling mechanisms. To this end, we aimed at characterizing the functions of TGF- β-dependent myCAFs in shaping the TME and influencing PDAC progression. Methods: We generated novel myCAF-depleted PDAC mouse models by orthotopically injecting PDAC organoids derived from KPC (Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre) mice into Fap-Tgfbr2 knockout (Fap-tTA; Rosa26 LSLtdTom/+ ; tet-O-Cre; Tgfbr2 fl/fl ) mice. The pancreatic tumors were analyzed by flow cytometry, histology and single-cell RNA-sequencing (scRNAseq). In a complementary approach, we generated myCAF-enriched PDAC mouse models by orthotopically co-injecting KPC PDAC organoids and novel TGF-β-driven ‘myCAF-locked’ murine PSCs. In addition, to further dissect myCAF-malignant cell signaling crosstalk, KPC PDAC organoids and myCAF-locked PSCs were co-cultured and then flow-sorted for bulk RNA- sequencing. Results: MyCAF-depleted PDAC tumors from Fap-Tgfbr2 knockout mice had a significant decrease in myCAFs, and a significant expansion of iCAFs, compared to control mice. Additionally, Masson’s trichrome staining indicated significant reduction in collagen content in the TME upon myCAF depletion. Moreover, scRNAseq analysis revealed that myCAF depletion shaped the composition of innate immune cells, including macrophages and neutrophils. Notably, although primary tumor growth was not altered, liver and lung metastases decreased, while diaphragm metastases and ascites increased, in myCAF-depleted PDAC mice. Conversely, liver and lung metastases increased, while diaphragm metastases and ascites decreased in myCAF-enriched PDAC mouse models, suggesting that TGF-β-dependent myCAFs play a role in promoting distant metastases. In line with this, transcriptomic analysis of PDAC organoids co-cultured with myCAF-locked PSCs showed enrichment of pathways in epithelial-mesenchymal transition, hypoxia, VEGF, WNT and cell-matrix interactions. Conclusion: Our study demonstrates that TGF-β-dependent CAFs are not only largely responsible for collagen deposition in PDAC, but also possess immune-modulating properties. Moreover, our study supports a role of TGF-β-dependent CAFs in promoting PDAC metastasis to the liver and the lungs, potentially through upregulating pro-metastatic pathways in the malignant cells. Citation Format: Priscilla S.W. Cheng, Muntadher Jihad, Judhell S. Manansala, Weike Luo, Gianluca Mucciolo, Sara Pinto Teles, Giulia Biffi. Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B075.

Matairesinol blunts adverse cardiac remodeling and heart failure induced by pressure overload by regulating Prdx1 and PI3K/AKT/FOXO1 signaling

BackgroundPathological cardiac remodeling is a critical process leading to heart failure, characterized primarily by inflammation and apoptosis. Matairesinol (Mat), a key chemical component of Podocarpus macrophyllus resin, exhibits a wide range of pharmacological activities, including anti-hydatid, antioxidant, antitumor, and anti-inflammatory effects. PurposeThis study aims to investigate whether Matairesinol alleviate cardiac hypertrophy and remodeling caused by pressure overload and to elucidate its mechanism of action. MethodsAn in vitro pressure loading model was established using neonatal rat cardiomyocytes treated with angiotensin Ⅱ, while an in vivo model was created using C57 mice subjected to transverse aortic constriction (TAC). To activate the PI3K/Akt/FoxO1 pathway, Ys-49 was employed. Moreover, small interfering RNA (siRNA) and short hairpin RNA (shRNA) were utilized to silence Prdx1 expression both in vitro and in vivo. Various techniques, including echocardiography, wheat germ agglutinin (WGA) staining, HE staining, PSR staining, and Masson trichrome staining, were used to assess cardiac function, cardiomyocyte cross-sectional area, and fibrosis levels in rats. Apoptosis in myocardial tissue and in vitro was detected by TUNEL assay, while reactive oxygen species (ROS) content in tissues and cells was measured using DHE staining. Furthermore, the affinity of Prdx1 with Mat and PI3K was analyzed using computer-simulated molecular docking. Western blotting and RT-PCR were utilized to evaluate Prdx1 levels and proteins related to apoptosis and oxidative stress, as well as the mRNA levels of cardiac hypertrophy and fibrosis-related indicators. ResultsMat significantly alleviated cardiac hypertrophy and fibrosis induced by TAC, preserved cardiac function, and markedly reduced cardiomyocyte apoptosis and oxidative damage. In vitro, mat attenuated ang Ⅱ - induced hypertrophy of nrvms and activation of neonatal rat fibroblasts. Notably, activation of the PI3K/Akt/FoxO1 pathway and downregulation of Prdx1 expression were observed in TAC mice; however, these effects were reversed by Mat treatment. Furthermore, Prdx1 knockdown activated the PI3K/Akt/FoxO1 pathway, leading to exacerbation of the disease. Molecular docking indicated that Molecular docking indicated that Mat upregulated Prdx1 expression by binding to it, thereby inhibiting the PI3K/Akt/FoxO1 pathway and protecting the heart by restoring Prdx1 expression levels. ConclusionMatairesinol alleviates pressure overload-induced cardiac remodeling both in vivo and in vitro by upregulating Prdx1 expression and inhibiting the PI3K/Akt/FoxO1 pathway. This study highlights the therapeutic potential of Matairesinol in the treatment of cardiac hypertrophy and remodeling, providing a promising avenue for future research and clinical application.

Mapping Staphylococcus aureus at Early and Late Stages of Infection in a Clinically Representative Hip Prosthetic Joint Infection Rat Model.

Prosthetic joint infection (PJI) continues to be a devastating complication following total joint replacement surgeries where Staphylococcus aureus is the main offending organism. To improve our understanding of the disease pathogenesis, a histological analysis of infected peri-implant tissue in a hip PJI rat model was utilized to assess S. aureus spread and tissue reaction at early and late stages of infection. Sprague-Dawley rats were used and received a left cemented hip hemiarthroplasty using a 3D-printed titanium femoral stem. The rats received an intra-articular injection of S. aureus Xen36. These infected rats were sacrificed either at 3 days post-infection (early-stage infection) or at 13-days post-infection (late-stage infection). The femoral and acetabular tissues of all animals were harvested at euthanasia. Histological analysis for the harvested tissue was performed using immunohistochemistry, hematoxylin and eosin, as well as Masson's trichrome stains. Histological examination revealed significant quantitative and qualitative differences in peri-implant tissue response to infection at early and late stages. This hip PJI rat model identified clear histologic differences between early and late stages of S. aureus infection and how quickly bacterial infiltration could occur. These findings can provide insight into why certain surgical strategies like debridement and antibiotics may be associated with high failure rates.

PSIII-27 Blood biochemistry, histology, and metabolomic profiling of contaminated with mycotoxin: A repeated dose oral toxicity study in rats

Abstract Mycotoxins causes diseases and physiological responses in humans and livestock. This study was performed to estimate the effects of binary mixtures mycotoxin {e.g., deoxynivalenol (DON) and fumonisin (FB)] on blood biochemistry, histology, and metabolic profiles in rats fed with different toxin concentrations. Experimental animals were administered by oral gavage with 0.9% saline, DON (1, 5, and 10 mg/kg BW), FB (1, 5, and 10 mg/kg) at 8 wk of age. Histological changes, metabolomic profiling, and blood biochemical analysis were performed Masson’s trichrome stain kit, liquid chromatography mass spectrometry, and VetTest chemistry analyzer, respectively. The final BW of the rats contaminated with mixtures mycotoxin significantly decreased compared with the control group. Biochemistry results and fibrosis were also significantly differentially regulated according to mycotoxin concentrations. Additionally, we observed collagen fibers in the liver and kidney showed blue staining after toxin contamination. For metabolomic profiling, data were analyzed using MetaboAnalyst 4.0. Results were analyzed by multivariate analysis to elucidate the potential compounds. The toxin-treated groups observed discriminating metabolites in the liver and kidney tissue but no differences in ileum. The profiling revealed potential metabolites such as glycine, serine and threonine metabolism. Phosphatidylethanolamine and tryptophan were identified with high sensitivity as candidate biomarkers via variable importance in the projection plot in piglets fed with mycotoxin (FDR &amp;lt; 0.05; P &amp;lt; 0.05). The differently treated groups showed discriminating metabolites in different concentrations. In conclusion, these findings suggest that mycotoxin exposure disturbs histological changes and metabolites. Dietary toxins in rats may better understand the physiological effects consistent with metabolism, which are commonly reported in animals.

Caecal villi? A comparative histological and morphometric study of caecal and jejunal mucosa in adult rabbits

BackgroundRabbits are herbivores with a distinctive digestive strategy that differs significantly from other caecal fermenters (e.g., horses, guinea pigs) and ruminants. In view of this, the current study aimed to highlight distinctive histological and morphometric features of the caecal mucosa in adult rabbits that accentuate its major role in digestion. The caecal and jejunal samples were harvested from five 1-year-old domestic rabbits and processed by regular paraffin-embedding histological technique followed by Goldner’s trichrome staining. A comprehensive morphological and morphometrical analysis of the jejunal mucosa vs. caecal mucosa was performed.ResultsMicroscopically, as in the case of the jejunal mucosa, the caecal mucosa presents long and often branched finger-like villi covered by a simple columnar epithelium mostly made of enterocytes with a prominent microvillous brush border. Besides, the caecal villi include a lacteal along with the villous muscle. Statistically, except for villus length, all the parameters assessed in the caecal mucosa, including villus width, villus count, thickness of the brush border and enterocyte/goblet cells ratio, revealed a high grade of similarities with the jejunal villi.ConclusionsAccording to the obtained results, the caecal mucosa in adult domestic rabbits includes unique features, namely caecal villi, structures infrequently presented in the large intestine of other adult mammals. Those structures once more emphasize the major role of the caecum not only in fermentation but also subliminally in local absorption. To our knowledge, this is the first reliable microanatomical and morphometric report of caecal villi in adult domestic rabbits.

Effect of fibroblasts small- conductance Ca2+ -activated potassium channel subtype 2 (SK2) on myocardial fibrosis in pressure overload mouse

Studies have shown that Small conductance Ca2 + −activated K+ (SK) channel are expressed in fibroblasts. We aimed to determine the expression of SK2 channels in cardiac fibroblasts during myocardial hypertrophy and investigate its relationship with fibrotic remodeling. Myocardial hypertrophy and fibrotic remodeling induced by transverse aortic constriction (TAC) were assessed by echocardiography, Masson's trichrome staining and Western blot. Knockdown and overexpression of the SK2 protein were used to assess relationship between SK2 expression in fibroblasts and myocardial fibrosis. There is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy. The differentiation and secretion of fibroblasts in mice with cardiac hypertrophy are enhanced, and the expression of SK2 channel protein is increased. Manipulating SK2 levels in fibroblasts can either promote or inhibit their differentiation and secretory function. Knocking down SK2 reduces the up-regulation of TGF β1, p-Smad2/3/GAPDH, p-p38/GAPDH, p-ERK1/2/GAPDH, and p-JNK/GAPDH proteins induced by Ang II in cardiac fibroblasts without significantly affecting total protein levels. AAV9-SK2-RNAi injection in mice improves cardiac function. Collectively, our study suggests that the expression of the SK2 channel is significantly increased in fibroblasts of mice with myocardial hypertrophy, potentially impacting myocardial fibrosis remodeling via the TGF-β signaling pathway.

Similarities and differences in morphology, CD31 and CD68 expression of male vs. female sinoatrial node and its surrounding atrial muscle in ageing and obesity

Research purposeThe sinoatrial node (SN) is the main pacemaker site, and it is located in the junctional area of the superior vena cava within the right atrium (RA). The precise micro-anatomy of the SN in males and females in ageing and obesity remains unclear. Basic proceduresHuman SN/RA specimens were dissected from 25 post-mortem hearts (preserved in 4 % formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin. Masson's Trichrome staining and immunohistochemistry for CD31 (a marker of endothelial cells) and CD68 (a marker of macrophages) were performed. Images at different magnifications were taken and analysed. 12-lead ECGs from 24 patients under Polish local ethical rules were obtained. Heart rate and P wave morphologies from lead II, lead III and lead aVF were analysed. Statistical analysis was performed with the unpaired t-test. Principal resultsHeart weight to body weight ratio was significantly higher in aged obese males vs. their female counterparts. In the RA samples, there was an increase in connective tissue and decreased myocyte content from aged obese females compared to aged obese males. Aged non-obese males had significantly increased cellular hypertrophy than the aged non-obese females. Both the aged obese and aged non-obese females showed more CD3 but less CD68 expressing cells than males. In the SN samples, CD31 and CD68 expressing cells were higher in both aged non-obese and aged obese males than their female counterparts. Major conclusionsAgeing and/or obesity are more likely to impact these cardiac tissues through increased inflammation, increased immune response and hypertrophy.

First Fully Endoscopic End-to-End Colonic Anastomoses With a Novel Endoscopic Device: A Feasibility Study in a Porcine Model.

Continuous advancements and breakthroughs in flexible gastrointestinal endoscopy have led to alternatives to colonic anastomosis. This study aimed to evaluate the feasibility and safety of end-to-end colonic anastomosis using a single flexible endoscope with the novel through-the-scope "bow-tie" (TTS-BT) device and conventional metal clips in a porcine model. Animal study. Animal laboratory at China Medical University. Eight healthy pigs were included. Eight animals underwent total colonic severance and anastomoses with through-the-scope "bow-tie" devices and metal clips. The primary outcomes were the success rate of the anastomosis and survival rate during 3-month follow-up. Furthermore, the secondary outcomes were anastomotic site healing, reintervention rate, and rate of anastomotic complications such as bleeding, leakage, stenosis, and obstruction. Six pigs were euthanized, and necropsies were performed 3 months postoperatively, while two pigs were fed for long-term observation. The anastomotic stoma was histologically analyzed using Hematoxylin-eosin and Masson's trichrome staining. End-to-end colonic anastomoses were successfully performed using through-the-scope "bow-tie" devices, and satisfactory healing was achieved in all pigs. The success rate of anastomosis was 100% (8/8). All animals survived postoperatively without anastomotic complications, including bleeding, leakage, or obstruction; however, two cases of stenosis occurred (25%), and one case (12.5%) required reintervention. Large-scale studies should be conducted to verify the feasibility and safety of the through-the-scope "bow-tie" device in other parts of the intestine. Flexible endoscopy with the through-the-scope "bow-tie" device is feasible and safe for intraluminal colonic anastomosis. This study may expand the indications for full-thickness endoscopic resection in the future. See Video abstract.

Evaluation of the novel ALK5 inhibitor EW-7197 on therapeutic efficacy in renal fibrosis using a three-dimensional chip model.

EW-7197, a potent oral ALK5 inhibitor, was assessed for its impact on transforming growth factor beta 1 (TGF-β1)-induced fibrosis in a three-dimensional (3D) renal fibrosis-on-a-chip and a mouse model. The evaluation included tubular epithelial-mesenchymal transition, angiogenesis, and inflammatory cytokine expression. In a 3D renal fibrosis-on-a-chip model, three cell types (kidney fibroblasts, human proximal tubular cell line, and human umbilical vein endothelial cells) were cultured and treated with TGF-β1 and EW-7197. Expression of alpha smooth muscle actin (α-SMA) and keratin 8 (KRT-8) was assessed, angiogenesis was observed via confocal microscopy, and cytokine levels were measured using real-time polymerase chain reaction, immunoassay, and enzyme-linked immunosorbent assay. In a cisplatin-induced renal fibrosis mouse model, blood urea nitrogen levels, TGF-β, and Smad 2/3 were determined, and renal fibrosis was assessed with Masson's trichrome stain. The α-SMA expression was significantly lower in the EW-7197 group than in the TGF-β fibrosis group. TGF-β decreased the expression of the epithelial marker KRT-8, an effect that was reversed by EW-7197 and SB431542. In the TGF-β-induced fibrosis model, the length of the thick vessels was reduced, and the diameter of both thick and thin vessels was decreased, but EW-7197 reversed these effects. EW-7197 significantly reduced the messenger RNA expression of TGF-β and increased the levels of vascular endothelial growth factor receptor 2, interleukin (IL)-10, and IL-6. EW-7197 reduced the levels of secretory cytokines TGF-β1, TGF-β3, IL-1β. In the cisplatin-induced renal fibrosis mouse model, EW-7197 reduced renal fibrosis by down-regulating TGF-β signaling. EW-7197 attenuated the TGF-β1-induced fibrotic cellular response in the 3D chip model and animal model. These findings indicate the potential effect of EW-7197 in attenuating renal fibrosis.