Characterization of a decellularized rat larynx: comparison between microscopy techniques

Chronic intestinal pseudo-obstruction (CIPO) is caused by a plethora of primary and secondary causes, dominantly involving the neuromuscular tissue, interstitial cells of Cajal, or the connective tissue framework. The lack of the connective tissue framework, known as desmosis, is evaluated by Masson's trichrome (MT) or picrosirius red stains, both of which are recommended in the London classification. We evaluated the orcein stain in detecting desmosis in comparison to the MT stain. We performed both orcein and MT stains in six previously published cases of complete or partial desmosis along with six age-matched controls. Our results showed comparable results of the orcein stain as compared to the MT stain. Additional advantages of lower cost and a clearer background in orcein stain were noteworthy, whereas MT stain can be used for the detection of additional pathology. We believe that orcein stain can be used as a cheap alternative in resource-limited settings.

Background: Fibrosis can define as the development of excessive fibrous tissue in an organ or tissue. In the situation of the uterus, leiomyoma or scar tissue may involve fibrosis state. The main feature of fibrosis in myoma is the existence of disordered smooth muscle cells and collagen that can be recognized via histological examinations in addition to extracellular component deposition. Leiomyoma is the most public uterine disorder that is mainly consists of smooth muscle cells, myo-fibroblast, and an obvious content of extracellular matrix. The myofibroblast in myoma lesion produced an elevated content of ECM. This paper displays the fibrotic circumstantial in leiomyoma and the mechanisms related to deposition of ECM components. Methods: this study involved (50) patients of 18-80 years old women diagnosed with benign uterine fibroid. Tumor samples were attained by consent from women undergoing hysterectomy or myomectomy at the Al Karama Teaching Hospitals in Kut, Wasit Province, Iraq between October 2022 to March 2023. samples from normal and fibroid were subjected to routein histological processing and subsequently stained with routein stains for general description, Masson Trichrome stain, Van Gieson's stain to visualize collagen content and histochemical stain (Alcian Blue-Periodic Acid Schiff) to evaluate the profile of mucins secreted via tumor cells.Normal myometrium and myoma tissue expression of TGF β1 growth factor was compared using immunohistochemical staining. Qupath and Image J programs were used to analysis the content of fibrous tissue within normal and myoma tissue. Results: Microscopical results revealed that myoma have a proliferation of overlap fusiform cells organized in bundles alienated by variable amounts of connective tissue fibers, enclosed peripherally by a pseudo capsule. Special stained sections analysis revealed deposition of extracellular martix components specially collagen fibers that vary within each nodular fibroid (intrafascicular, interfascicular) enclosed myocytes that reflect the state of fibrosis that appears as blue – green colors enclosed in Masson's trichrome stain, while in Van Gieson's stain appears as red color. Proliferative tumor cells were revealed via Alcian Blue-PAS stain in dark blue color. results showed distribution of perivascular fibers in myoma as compared with normal adjacent myometrium, which appears in red color in Van Gieson's stain. Immunostaining recorded the significantly higher intensity of TGF-β in the leiomyoma tissue than in the normal myometrium (P < 0.05). The effects of the TGF-β1 inhibitor significantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell survival in the leiomyoma tissue (P < 0.05). Conclusion: highly fibrous tissue contents showed within myoma and perivascular than adjacent myometrium, these fibrous tissues were detected via special colours in both Masson Trichrome and Van Gieson's stains. The fibrous tissue within leiomyoma showed higher intensity to TGF than normal myometrium.

Altered smooth muscle development and innervation in the lower genitourinary and gastrointestinal tract of the male human fetus with myelomeningocele.

1235 STRUCTURAL ALTERATIONS IN THE EXTRACELLULAR MATRIX OF HUMAN FORESKIN CAUSED BY CHRONIC SMOKING MAY EXPLAIN HIGH LEVELS OF URETHRAL RECONSTRUCTION FAILURE

To study pathological changes of fibromuscular system and the role of TGF-β1/Smad pathway in the urethra of a parturition-induced stress urinary incontinence (SUI) rat model. Twenty-eight 8-week-old Sprague-Dawley female rats at gestational day 16 were used and randomized into two groups: sham group and SUI group. After delivery, rats in the SUI group underwent postpartum vaginal balloon dilation and bilateral ovariectomy. 1 month after ovariectomy, urodynamics was assessed. Histological examination (Masson's trichrome stain, picrosirius red stain, Hart's elastin stain, Gordon & Sweet's stain, and immunohistochemical stain) and Western blot were performed on urethral tissues. Both leak point pressure and maximal bladder capacity were significantly decreased in the balloon-injured ovariectomized rats, compared with the sham rats. Muscle was significantly decreased in the urethra of SUI rats compare with sham rats. Collagen I/III and reticular fibers from SUI group were also significantly lower than sham group. Meanwhile, elastic fibers and reticular fibers showed fragmentation and disorganization indicating impairment in the fibromuscular system in SUI rats. TGF-β1, MMP-9, and phosphorylated Smad2 (p-Smad2) were expressed significantly higher in SUI than in sham rats. Simulated birth trauma and menopause induced an upregulation of the TGF-β1/Smad pathway and impairment of the fibromuscular system in the urethra.

Arthritis is characterized by the progressive degeneration of articular cartilage, and the avascular nature of cartilage limits its capacity for self-repair. Stem cells are considered a promising treatment option due to their multipotent differentiation potential. The aim of this work was to investigate the structural changes in the hyaline articular cartilage of the knee joint in a model of arthritis induced by complete Freund's adjuvant, and to assess intra-articular injection of bone marrow mesenchymal stem cells (BM-MSCs) through both histological and immunohistochemical study. Adult male albino rats were divided into four groups: group 0 (donor group), group I (control group), group II (arthritis group) and group III (BM-MSCs treated arthritis group). Samples were collected 2, 6 and 10weeks after the onset of the experiment. Sections were stained with; hematoxylin and eosin, Safranin O fast green stain, Masson's trichrome stain and anti-MMP9 antibody. In Group II (arthritis group), the articular cartilage showed signs of degeneration, including chondrocyte extensive proliferation, fibrillations, fissuring, and denudation, with fibrous tissue covering the exposed surface. There was a significant decrease in cartilage thickness, collagen content, and proteoglycan levels. The integrated density of MMP9 in the cartilage was significantly increased compared to Group I (control group). In contrast, Group III (BM-MSCs-treated arthritis group) exhibited a continuous cartilage surface with no cracks or fissures. There was a significant increase in cartilage thickness, collagen content, and proteoglycan levels, while the integrated density of MMP9 was significantly decreased compared to Group II (arthritis group).

The endovascular technique of mechanochemical ablation (MOCA) has become popular in treating patients with saphenous reflux. We reported the histopathological findings in human ex-vivo incompetent great saphenous veins following treatment with saline, polidocanol, mechanical ablation and MOCA using ClariVein device. Twenty-four vein GSV specimens were obtained via traditional surgery and treated with four methods: Group A: 0.9% normal saline (NS); Group B: 3% polidocanol; Group C: mechanical ablation + 0.9% NS; Group D: mechanical ablation + 3% polidocanol (MOCA). Hematoxylin and eosin (HE), Masson's trichrome and immunohistochemical staining were performed on each specimen and integrated optical densities were measured with vWF and a-SMA stains and statistically evaluated. vWF staining was used to assess endothelial damage and a a-SMA staining was used to assess media injury. HE and Masson's trichrome staining of Groups C and D revealed severe damage to the endothelium and media compared to Groups A and B. The statistical result of vWF staining showed the damage of endothelium was significantly increased by Group D compared to Groups A, B and C. The statistical result of a-SMA staining showed the damage of media was significantly increased by Groups C and D compared to Groups A and B. The mechanism of MOCA was caused by both endothelium damage and media tearing. The damage of endothelium was significantly increased by MOCA when compared with mechanical ablation alone.

The accuracy of Masson's trichrome stain to predict the presence of immune complexes was determined in 63 renal biopsies. When immunofluorescence was defined as the reference method, the histologic method correctly predicted the presence or absence of deposits in 70% of biopsy specimens, while electron microscopy was accurate in 79% of specimens. This difference was not statistically significant. The commonest error in our assessment of the Masson's trichrome-stained specimens was the erroneous interpretation of specimens showing minimal change nephropathy or ischemic glomerulopathy by immunofluorescence and electron microscopy. This resulted in a falsely positive diagnosis of one or another of the glomerulonephritides in 13% of cases. Thus, the routine study of renal biopsies with Masson's trichrome stain is clearly useful and should be applied with caution, but it does not replace electron-microscopic and immunofluorescence studies.

Background and Objectives:Coronary microvascular remodeling is one of the complications associated with diabetic cardiovascular disease. We investigated the effect of long-term treatment with ramipril, an an- giotensin converting enzyme inhibitor, on coronary microvascular remodeling on a type II diabetic model, the Otsuka Long-Evans Tokushima Fatty (OLETF rat. Materials and Methods:Twenty OLETF diabetic, and twenty LETO non-diabetic rats at twenty-four weeks old, were randomized into 4 equal groups for treatment with either an aqueous solution of ramipril (5 mg/kg, n=10 for two groups or saline (n=10 for two groups for 24 weeks on a daily basis. The blood glucose levels, body weights and blood pressures of the rats were checked on a regular basis. Masson's trichrome and picrosirius red stains were used for the morphometric analysis of the thickening of the coronary arterial wall and the degree of perivascular fibrosis. The myocardial collagen content was determined by measuring the levels of myocardial hydroxyproline. Results:Marked thickening of the coronary microvascular wall and prominent perivascular fibrosis were detected in the hearts of OLETF rats to a greater extent than in the LETO rats (p<0.01. Ramipril significantly prevented coronary microvascular remodeling in OLETF rats (p<0.01, but there was no significant difference in the collagen content/dry heart weight ratio between the groups. Conclusion:Long-term treatment with ramipril has an antifibrotic effect, and improves the coronary microvascular remodeling in diabetic OLETF rats. (Korean Circulation J 2002;32(8 :680-688

In unruptured brain arteriovenous malformations (bAVMs), microhemorrhage portends a higher risk of future rupture and may represent a transitional state along the continuum of destabilization. Exploration of the molecular and cellular mechanisms of microhemorrhage will provide a possible target for medical treatment to prevent bAVM bleeding. We performed RNA sequencing analysis on 34 unruptured bAVM surgical samples. Functional pathway analysis was performed to identify potential signals associated with the microhemorrhagic phenotype. Candidate gene was then investigated in bAVM specimens by immunohistochemical staining. Several functional assays were used to investigate the effects of candidate genes on the phenotypic properties of cultured human umbilical vein endothelial cells. Then, Masson trichrome staining and immunofluorescence staining were used to evaluate the phenotypic and molecular changes in bAVM tissue. Via RNA sequencing, we identified differential gene expression between 18 microhemorrhagic bAVMs and 16 nonmicrohemorrhagic bAVMs. TGFβ (transforming growth factor-beta)/BMP (bone morphogenetic protein) signaling was associated with the bAVM microhemorrhage group when SMAD6 (SMAD family member 6) was downregulated. Immunohistochemical staining showed that the vascular endothelium of microhemorrhagic bAVMs exhibited decreased SMAD6 expression. Functional assays revealed that SMAD6 downregulation promoted the formation of endothelial cell tubes with deficient cell-cell junctions and facilitated the acquisition of mesenchymal behavior by endothelial cells. Masson trichrome and immunofluorescence staining demonstrated that mesenchymal phenotype of endothelial cells is promoted in microhemorrhagic bAVMs. TGFβ/BMP signaling mediated by SMAD6 in vascular endothelial cells is associated with microhemorrhagic bAVMs, and mesenchymal behavior of endothelial cells induced by SMAD6 downregulation is related with bAVM microhemorrhage.

Canine endocrinopathies, such as hypothyroidism and hyperadrenocortism,induce typical dermatological alterations. Collagen fibers are significant for the maintenance of structural integrity,as well as in the determination of tissue function. This study aimed at assessing the coloration caused by Picrosirius Red staining under circular polarization and Masson Trichrome staining, as tools to quantify the total collagen in the skin of dogs exhibiting endocrine dermatopathies. Skin samples taken from dogs with hypothyroidism and hyperadrenocorticism were stained using Hematoxylin and Eosin (HE), Masson’s Trichrome (MT) and Picrosirius Red (PSR). The animals with hyperadrenocorticism revealed a higher percentage of collagen area than did the animals with hypothyroidism PSRp (hypothyroidism = 43.76 ± 0.8255 and hyperadrenocorticism = 47.08 ± 0.8584). The percentage of the collagen area using MT between the groups is given in parenthesis (hypothyroidism = 52.06 ± 0.9371, hyperadrenocorticism = 61 ± 0.7529 and control = 56.88 ± 0.64) (p &lt;0.05). It is therefore, evident that the special stains employed are useful in estimating the percentage of collagen area in the skin, as they revealed that dogs with hypothyroidism had lower collagen deposition whereas those animals with hyperadrenocorticism showed higher quantity of collagen in the dermis.

Objective: The present research performed a clinical and histopathological analysis of female genital schistosomiasis (FGS) cases. This was a descriptive and retrospective study, with a quantitative approach based on secondary databases belonging to Memorial Nestor Piva from Universidade Tiradentes. Methodology: Medical records and biopsies from patients affected with genital schistosomiasis (1984-1994) 75 pathology reports were found, biopsies of feminine genitals, with the respective paraffin blocks and slides. In these three search blocks, to contain sufficient quantity of biological material underwent histological new. For pathological analysis, hematoxylin-eosin, picrosirius Red and Masson's trichrome staining were used. A distribution of cases per year was performed to obtain an average and a percentage distribution among the most affected organs. In addition, the chi-square test was used to establish an association between schistosomiasis and its anatomical location and with other pathologies. Results: An average of four cases per year was obtained and the infection was found in all genital system organs, with its highest concentration in the ovaries followed by the uterus. In addition, there was an association between the parasitosis and cervical metaplasia, uterine tumors and salpingitis. In the histopathological analysis, several granulomas containing Schistosoma mansoni eggs, in productive and curing phases, were observed. Conclusion: It was concluded that ovaries were the most affected anatomical sites and that the presence of genital schistosomiasis may favor the occurrence of associated pathologies, thus causing a high morbidity rate to women's health.

To investigate the effects of combination combining Percutaneous Needle Fasciotomy (PNF) with stretching(Str) on early-stage skeletal muscle fibrosis in rats, focusing on the transforming growth factor beta-1 (TGF-β1)/Smad signaling pathway. Twenty-four male Wistar rats were randomly divided into Control, immobilization(imm), Acupuncture(Apu) + Str, and PNF + Str groups (n = 6 each). After establishing a gastrocnemius fibrosis model by 4-week ankle imm in the right hindlimb ankle, intervention groups received either PNF (0.35 × 25mm needle insertion with fascial release) or Apu (following an identical protocol), both combined with daily passive Str (3-N dorsiflexion for 7days). Post-intervention assessments included functional tests (ankle joint range of motion, hindlimb grip strength, andgastrocnemius twitch/tetanic contractile forces), histological evaluation (collagen content via Masson's trichrome staining and immunofluorescence), and molecular analysis of TGF-β1/Smad pathway components. Compared to the imm group, the PNF + Str group exhibited significantly better muscle performance, reduced histopathological damage, and favorable modulation of TGF-β1/Smad signaling markers. Importantly, these improvements were more pronounced than those in the Apu + Str group, wich demonstrates PNF's superior therapeutic effects in enhancing functional recovery, reducing fibrosis, and regulating the TGF-β1/Smad pathway. Our findings from this animal model demonstrate that PNF was superior to Apu in alleviating early-stage skeletal muscle fibrosis over the short term, potentially via modulation of the TGF-β1/Smad signaling pathway, though long-term effects warrant further investigation.

Background and Aims Sepsis is the most frequent cause of acute kidney injury (AKI) in the intensive care unit. Methicillin-resistant Staphylococcus aureus (MRSA) sepsis is particularly nefarious, with the kidney frequently affected leading to AKI or chronic complications. Whereas sepsis-associated AKI is often attributed to hemodynamic changes and toxic insults, small cohort studies show increased infiltration of neutrophils and macrophages in post-mortem kidney tissue from sepsis patients. Neutrophil extracellular traps (NETs) are extracellular DNA strands released from activated neutrophils. Their formation is mediated through the activity of the protein citrullinating enzyme peptidylarginine deiminase 4 (PAD4). NETs have been shown to contribute to antibacterial defense in the kidney, but may also propagate thrombosis and vascular dysfunction in the renal microvasculature. We aimed to gain mechanistic insights into the roles of neutrophils and NET release in MRSA-induced kidney injury. Method Wild-type (WT), LysM-GFP (myelomonocytic reporter mice), MRP8Cre+PAD4fl/fl (NET-defective), or PAD4fl/fl (NET-proficient) mice were injected with MRSA (laboratory strain USA300). Granulocyte colony-stimulating factor (G-CSF) was used to induce neutrophilia and prime for NET release prior to MRSA injection. Blood samples were collected to measure creatinine. Kidneys were harvested to visualize structural abnormalities (hematoxylin and eosin stain), bacterial invasion (Gram stain), collagen content (Masson's Trichrome stain), myelomonocytic cells (LysM-GFP), and Padi4 gene expression (RNAscope). Scanning electron microscopy (SEM) was used to assess glomerular structure. Local RNA levels of KIM-1 (kidney injury molecule 1) and NGAL (neutrophil gelatinase-associated lipocalin) were measured using quantitative reverse transcription PCR analyses. Detailed cellular profiling was performed using isolated nuclei from snap-frozen kidneys to perform single-nucleus RNA sequencing and single-cell suspensions for deep immunophenotyping in both kidneys and spleens using spectral flow cytometry. Results MRSA induced abscess and tubular cast formation in kidneys from WT mice, resulting in profound renal fibrosis. Whereas local levels of KIM-1 and NGAL were elevated in MRSA-infected mice as compared with controls (P &amp;lt; 0.001 for both), creatinine levels were not significantly different (P = 0.073). SEM revealed a normal glomerular structure. Myelomonocytic reporter mice showed predominant infiltration of neutrophils and macrophages inside abscesses. Single-nucleus RNA sequencing revealed a heterogeneous set of neutrophils with only a small subset expressing Padi4. RNAscope showed that these Padi4 expressing cells were located at the outer region of abscesses. In the spleen, MRSA induced extramedullary hematopoiesis. Interestingly, G-CSF administration prior to MRSA infection protected against disease severity and mortality. Compared with vehicle-injected controls, G-CSF-treated WT mice showed increased neutrophil numbers prior to sepsis induction and more pronounced neutrophilia following MRSA infection. G-CSF-treated WT mice showed lower levels of creatinine as compared with vehicle-injected controls (P = 0.017), less infiltration of MRSA through the kidney, better bacterial containment in the renal papilla and lack of abscess formation along with an increase in CCR2+ macrophages in the kidney. Interestingly, G-CSF-treated NET-defective mice showed higher levels of creatinine following MRSA infection than similarly treated NET-proficient controls (P = 0.010). NET-defective neutrophils did not entirely constrain bacteria in the renal papilla, thus impacting the dissemination of MRSA throughout the kidney. Recruitment of CD11b+ myeloid cells was impaired in kidneys from NET-defective mice. Conclusion Administration of G-CSF protects against MRSA-induced AKI in mice, which is partially mediated through NET release impacting bacterial containment and immune cell recruitment to the kidney. These data may inform future clinical applications targeting neutrophils to protect the kidney.

The purpose of this research lied in studying the structure and function of connective tissue of superior oblique (SO) in rhesus and rabbit. It was an experimental study. Two adult monkeys (rhesus) and five adult rabbits were involved in this study. The gross anatomy of an orbit in each animal was observed. Meanwhile, the fellow orbit was processed with paraffin imbedding and coronal serial section. Masson trichrome stain was used to distinguish muscle and collagen, and Weigert stain was performed for identification of elastin. Connective tissue around SO in both mammals thickened anteriorly, reflected in the trochlea along with the SO tendon, and became contiguous with the nasal aspect of the SR connective tissue ring, while the tendon passed inferior to the SR pulley to insert on the sclera. The contraction of SO OL can cause SR shift nasally. This may explain the mechanism of vestibulo-ocular reflex (VOR) partly.