Elastic Van Gieson Research Articles

Histological characterization of rat vocal fold across different postnatal periods.

To evaluate the vocal fold histological characteristics during different postnatal periods in rats, especially older rats. Sprague-Dawley rats aged 4 days, 4 and 12 weeks, and 12 and 24 months were used for the experiment. Five larynges were obtained for each age and cut into 5-μm consecutive sections. The expression of Ki-67 was assessed using immunohistochemistry to examine cell proliferation. Elastic van Gieson staining was used to detect the collagen and elastin concentrations. The cell type was determined using multicolor immunofluorescence. Ki-67 was not expressed in the macula flava (MF) of 12-week-, 12-month-, and 24-month-old adults. Collagen fibers in the lamina propria (LP) increased with age. The elastic fiber concentrations in the LP decreased significantly at 24 months (p < .01) but remained stable in the MF. All posterior MF cells showed strong glial fibrillary acidic protein and vimentin-positive reactions with weaker expressions of CD68 and α-smooth muscle actin (α-SMA). The myofibroblasts (α-SMA-positive) and macrophages (CD68-positive) in the LP of the 24-month-old rats were significantly the highest (p < .01). The extracellular matrix in the LP increases with age, presenting as an increase in collagen with the loss of elastin, which may be due to myofibroblast proliferation. Moreover, the cellular properties or extracellular matrix components of the mature MF in rats are comparable to those in humans.

Quercetin Promotes Abdominal Aortic Aneurysm Regression Through the RAGE/PI3K/AKT/mTOR Axis

Objective: Abdominal aortic aneurysms (AAA) are a common critical cardiovascular disease with high morbidity and mortality rates. Quercetin, a flavonoid and a traditional Chinese medicinal ingredient commonly found in vegetables and fruits, has a favorable inhibitory effect on experimental aneurysms without side effects. This study aimed to model elastase AAA and investigate the mechanism of action of quercetin in alleviating AAA. Methods: Quercetin was administered at a dose of 60 mg/kg once daily starting on the day of AAA induction for 5 weeks. The therapeutic effect of quercetin on AAA was demonstrated using biochemical assays, observation of pathological tissue sections, Elastic Van Gieson staining, immunohistochemistry, molecular docking simulations, and Western blot protein validation. Results: Our study showed that quercetin treatment significantly alleviated abdominal aortic vessel wall dilatation, elastin degradation and adhesion to surrounding tissues caused by elastase. Additionally, quercetin significantly inhibited the mRNA and protein expression of the receptor for advanced glycation end products(RAGE)/ phosphatidylinositol 3-kinase (PI3K)/ phosphatidylinositol 3-kinase (AKT) / mammlian target of rapamycin (mTOR) pathway. Conclusion: Quercetin can alleviate abdominal aortic injury caused by elastase via RAGE/PI3K/AKT/mTOR and provide experience in clinical use.

The effect of preoperative hormonal stimulation on the urethral plate; A histologic and histochemical study

Preoperative hormone stimulation (PHS) is used to increase the glans size and may improve the cosmetic appearance after hypospadias surgery. The exact effect of PHS on different penile tissues remains unclear and controversial. Previous studies showed that PHS increased vessel density in the foreskin. However, the effect of PHS on the urethral has never been studied before. In this study we examine the PHS effects on the urethral plate. Specimens of the urethral plate and the underlying tissue were excised to correct severe chordee in 16 children with proximal and perineal hypospadias with severe chordee in 2 groups. Group A consisted of 8 children received PHS prior to surgery. Group B included 8 children with proximal and perineal hypospadias with severe chordee who did not receive PHS and served as a control group. Specimens were examined blindly using hematoxylin-eosin and elastic van-Gieson stain as well as factor 8 and SMA antibodies. The median age of Group A was 13 (range 10-20) months. The median age of Group B (without prior PHS) was 17 (range 14-29) months (p=0,03). The median vessel-density in group A (82 vessels per mm2) was significantly higher than in Group B (65 per mm2), (p<0.05). The median vessel diameter was also significantly higher in Group A (13μm) than in Group B (11μm), (p<0.05). The median epithelial layer thickness in Group A was 110μm and in Group B 98μm, but showed no statistical significance (p=0,16). There was no significant change in dartos fascia layer thickness or androgen receptor expression. There was no visual change in the organization of elastic fibers. This study is the first study to document the effect of PHS on the urethral plate in hypospadias with severe chordee. PHS significantly increased the median vessel-density as well as median vessel diameter as compared to a matching control group. The findings of the study may justify PHS administration before the first operation to improve the vascularity and may reduce the severity of chordee.

Expression and significance of pin1 in the wound healing.

The aim of this study is to delineate the expression patterns of prolyl cis-trans isomerase NIMA-interacting protein 1 (Pin1), Glial cell-derived neurotrophic factor (GDNF), and Angiotensin II (ANG II) during the process of wound repair, and to ascertain the effects of Pin1, GDNF, and ANG II on the healing of wounds in a rat model. A total of 18 rats were allocated into three groups-sham (control), DMSO (vehicle control), and Pin1 inhibitor (treatment with juglone)-with six animals in each group. An animal model of wound healing was established, followed by the intraperitoneal administration of juglone. Tissue samples from the wounds were subsequently collected for histopathological evaluation. Expression levels of Pin1, GDNF, and Ang II were quantified. In addition, an in vitro model of wound healing was created using human umbilical vein endothelial cells (HUVEC), to assess cell proliferation, migration, and tube formation under conditions of juglone pre-treatment. The expression levels of Pin1, GDNF, and ANG II were notably elevated on 7-, and 10- days post-wound compared to those measured on 3-day. Contrastingly, pre-treatment with juglone significantly inhibited the expression of these molecules. Histological analyses, including HE (Hematoxylin and Eosin), Masson's trichrome, and EVG (Elastic van Gieson) staining, demonstrated that vascular angiogenesis, as well as collagen and elastin deposition, were substantially reduced in the juglone pre-treated group when compared to the normal group. Further, immunohistochemical analysis revealed a considerable decrease in CD31 expression in the juglone pre-treatment group relative to the normal control group. Pin1 serves as a pivotal facilitator of wound repair. The findings indicate that the modulation of Pin1, GDNF, and ANG II expression impacts the wound healing process in rats, suggesting potential targets for therapeutic intervention in human wound repair.

Rhabdomyomatous Mesenchymal Hamartoma: Report of 4 Cases With Histochemical and Immunohistochemical Findings and Emphasis on Potential Pitfalls.

Rhabdomyomatous mesenchymal hamartoma (RMH) typically presents as a congenital midline head and neck cutaneous polyp in infants. Perianal and mucocutaneous lesions have been reported, and recently, acquired adult-onset variants have been proposed. This makes the true prevalence, etiopathogenesis, and clinicopathologic distribution and classification of RMHs in children compared with those in adults uncertain. We performed a retrospective review to highlight the salient histopathologic, histochemical, and immunohistochemical features in RMHs and to emphasize their specific clinicopathologic criteria to avoid diagnostic pitfalls. We found 4 (0.3%) infants [2 female infants and 2 male infants, average age: 4 months] with mental, nasal, lingual, and perianal midline RMHs (average size: 1.0 cm) of 1303 patients with cutaneous polypoid lesions. Three were isolated, and 1 was associated with Goldenhar syndrome. The cutaneous polyps demonstrated intermixed skeletal muscle, adipose, and fibrocollagenous core stroma that extended into the dermis and around the dermal appendages. The lingual lesion demonstrated skeletal muscle and fibrocollagenous stroma with prominent nerve bundles and little adipose tissue. All showed interstitial loose mesenchyme. Masson trichome demarcated the triphasic stromal components. Alcian blue demonstrated the loose myxoid mesenchyme. Elastic van Gieson did not show elastic fibers. Desmin demonstrated the skeletal muscle bundles, S100 highlighted the adipose tissue lobules and the nerve bundles, and CD34 displayed the mesenchymal stroma. Ki67 showed a low proliferation index in the loose mesenchyme. Smooth muscle actin did not reveal smooth muscle bundles, but with CD31, they highlighted the thick blood vessels. CD117 revealed prominent mast cells. From our retrospective review series, 4 cases that originally diagnosed as RMHs were excluded. Likewise, we found some examples of the reported cases in the English literature that might have been mistaken for RMHs. This is because they did not fulfill the diagnostic clinicopathologic criteria. RMH constitutes a rare entity with specific clinicopathologic features. Most lesions are isolated. Some are associated with congenital anomalies and syndromes. Strict clinicopathologic diagnostic criteria should be applied to avoid mislabeling look-alike lesions for RMHs.

Establishment and Validation of an Animal Model of Abdominal Aortic Aneurysm in Beagles Through Vascular Patch Angioplasty and Elastase Infusion

To evaluate the effect of an abdominal aortic aneurysm (AAA) animal model established in beagles by way of vascular patch angioplasty combined with elastase infusion. A total of 60 beagle dogs were included in this study. Among them, 10 beagles were assigned to a control group to obtain normal abdominal aortic wall tissue, while the other 50 underwent vascular patch angioplasty combined with elastase infusion in order to establish the AAA disease model. In order to evaluate the outcome of modeling, abdominal vascular ultrasonography was performed 14 days after the modeling surgery was performed and ultrasound and computed tomographic angiography (CTA) were performed 28 days after the modeling surgery. The criterion for evaluating modeling success is that the maximum diameter of the abdominal aortic aneurysm is 50% greater than the diameter of the normal abdominal aorta below the renal artery. A total of 20 beagles of the modelling group and 5 control beagles were sacrificed 35 days after the modeling surgery and infrarenal abdominal aortic wall tissues were harvested. Then, hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and elastic van Gieson (EVG) staining were conducted to observe the pathology features of abdominal aortic wall tissues. A total of 50 beagles underwent the AAA modeling procedures, with the average operative and anesthesia time being (119.4±18.9) and (137.4±15.8) minutes, respectively, the average blood loss volume being (43.6±7.7) mL, the average abdominal aorta block time being (39.7±5.3) minutes during the modeling surgery, and the average abdominal aorta diameter measured during the surgery being (6.5±0.4) mm. Intraoperative mortality was 0%. Mortality within 30 days after the surgery was 2% (1 out of the 50 beagles). Postoperative ultrasound and CTA results revealed that the success rate of AAA modeling was 100%. Pathology examination suggested that the animal model rather successfully simulated the pathophysiologic changes associated with human AAA in regard to the morphological and pathological changes. Vascular patch angioplasty combined with elastase infusion can be used to successfully establish AAA model in beagles. The AAA modeling method described in our report demonstrates stability and reliability in aneurysm formation effect and the surgical procedures are easy to replicate. The method integrates the advantages of previous animal modeling methods and can be used to study the pathogenesis of AAA.

Abstract 15534: Identification of Narciclasine, a Potential Drug to Treat PAH by Unbiased Pharmaco-Transcriptomic Study

Introduction: Pulmonary arterial hypertension (PAH) is a life-threatening vascular disorder characterized by persistent vasoconstriction and detrimental remodeling of the pulmonary blood vessels. This incurable disease is marked by a complex gene expression reprogramming that triggers, among other, metabolic disorders, heightened by inflammation, and the development of a pro-survival phenotype in pulmonary arterial smooth muscle cells (PASMC).We conducted an unbiased transcriptomic study using lung samples from 16 PAH patients and 12 non-PAH controls and identified 4013 differentially expressed genes. Leveraging in silico pharmaco-transcriptomic analysis (utilizing the Lincs and Sigcom databases), we identified narciclasine as a potential drug. Hypothesis: Consequently, we hypothesized that narciclasine could offer improvements in PAH. Methods and Results: In vivo , we administered narciclasine treatment (1 mg/kg, 2 weeks, gavage) in a murine model of PH (monocrotaline model, 60 mg/kg, sc.) and observed improved PH hemodynamics (RVSP, PAAT, TPR), reduced inflammation (CD68 immunofluorescence (IF)), decreased PASMC survival (PCNA and cleaved caspase 3 IF), and alleviated adverse vascular remodeling (Elastic van Gieson Stain Kit). In vitro , narciclasine exhibited a dose-dependent reduction of cell proliferation (IF: ki67; Western blot Survivin, PCNA and PLK1) and increased apoptosis PAH-PASMC (IF: cleaved caspase 3; Wb: Bax and Bcl2). Transcriptomic study (conducted in treated PASMCs) reveals that the therapeutic effects of narciclasine in PAH may be attributed to decreased glycosylation (Gene Ontology analysis). We further observed increased glycosylation in remodeled arteries from PAH lungs, PH preclinical models, and cultured PASMC (Periodic Acid Schiff Kit). Subsequently, we validated that narciclasine reduced glycosylation and downregulated the expression of glycosylation enzymes (by wb: GFAT, NGT, OGT) both in vitro and in vivo. Statistical tests used: unpaired t (2 groups), ANOVA (&gt; 2 groups). Conclusions: In conclusion, our unbiased pharmaco-transcriptomic study identifies narciclasine as a potential therapeutic drug for PAH. Notably, its efficacy appears to be mediated by the reduction of glycosylation.

CD147 monoclonal antibody attenuates abdominal aortic aneurysm formation in angiotensin II-Infused apoE-/- mice.

Abdominal aortic aneurysm (AAA) is a life threatening vascular disease. Our previous study reported the upregulation of CD147 expression in human aortic aneurysms. In this study, we injected apoE-/- mice intraperitoneally with CD147 monoclonal antibody or IgG control antibody to observe its effect on Angiotensin II (AngII) induced AAA formation. ApoE-/- mice were randomly divided into an AngⅡ+CD147 antibody group (n=20) and an AngⅡ+IgG antibody group (n=20). The Alzet osmotic minipump was implanted subcutaneously into the backs of mice to infuse AngII (1000ng/kg/min) for 28days and subsequently treated with CD147 monoclonal antibody or control IgG mAb (10μg/mouse/day) beginning one day after surgery. Body weight, food intake, drinking volume and blood pressure were measured weekly throughout the study. After 4weeks of injection, routine bloodwork measuring liver function, kidney function and lipid levels were recorded. Hematoxylin and eosin (H&E), Masson's trichrome, and Elastic van Gieson (EVG) staining were used to evaluate the pathological changes in blood vessels. In addition, Immunohistochemical assay was used to detect infiltration of inflammatory cells. Tandem mass tag (TMT)-based proteomic analysis was used to define differentially expressed proteins (DEPs) using a p-value<0.05 and fold change>1.2 or<0.83 as the threshold. Subsequently, we conducted protein-protein interaction (PPI) network and GO enrichment analysis to determine the core biological function altered after CD147 antibody injection. The CD147 monoclonal antibody suppresses Ang II-induced AAA formation in apoE-/- mice and reduced aortic expansion, elastic lamina degradation, and inflammatory cells accumulation. Bioinformatics analysis showed that Ptk6, Itch, Casp3, and Oas1a were the hub DEPs. These DEPs in the two group were mainly involved in collagen fibril organization, extracellular matrix organization, and muscle contraction. These data robustly demonstrated that CD147 monoclonal antibody suppresses Ang II-induced AAA formation through reduction of inflammatory response and regulation of the above defined hub proteins and biological processes. Thus, the CD147 monoclonal antibody might be a promising target in the treatment of abdominal aortic aneurysm.

Aberrant p16, p53 and Ki-67 immunohistochemistry staining patterns can distinguish solitary keratoacanthoma from cutaneous squamous cell carcinoma

Keratoacanthoma (KA) is widely considered a benign, usually self-resolving, neoplasm distinct from cutaneous squamous cell carcinoma (cSCC), while some consider KA to be indistinguishable from cSCC. Published studies indicate utility for p16, p53, Ki-67 immunostaining and elastic van Gieson (EVG) in the assessment of KA and cSCC. We compared clinical features and staining patterns for p16, p53, Ki-67 and EVG in fully excised KA, cSCC with KA-like features (cSCC-KAL) and other cSCC (cSCC-OTHER). Significant differences between KA, cSCC-KAL and cSCC-OTHER were found for head and neck location (20%, 86%, 84%), and duration <5 months (95%, 63%, 36%). KA shows both a mosaic pattern for p16 (>25-90% of neoplasm area) and peripheral graded pattern for p53 (up to 50% moderate and strong nuclear staining) in 92% compared with 0% of cSCC-KAL and 0% of cSCC-OTHER. In contrast, a highly aberrant pattern (usually null) for one or both p16 and p53, was present in 0% of KA, 83.8% of cSCC-KAL and 90.9% of cSCC-OTHER. Abnormal distribution of Ki-67 beyond the peripheral 1-3 cells was uncommon in KA (4.2%) and common in cSCC-KAL (67.6%) and cSCC-OTHER (88.4%). Moderate to striking entrapment of elastic and collagen fibres was present in the majority of KA (84%), cSCC-KAL (81%) and cSCC-OTHER (65%). KA are clinically distinct neoplasms typically of short duration occurring preferentially outside the head and neck and generally lacking aberrations of p16, p53 and Ki-67, compared with cSCC that have high rates of aberrant or highly aberrant p16, p53 and Ki-67, but EVG lacked specificity.

DP13 A case of poikilodermatous plaque-like haemangioma: a newly described entity

Abstract A 56-year-old man was referred with a 1.5 cm × 1.5 cm asymptomatic erythematous and scaly plaque on his right upper calf of 6 months’ duration. He had no similar lesions or prior skin issues. He had no significant comorbidities and was on no regular medications. There was no known family history of relevance. The initial clinical findings were felt to be in keeping with Bowen disease. He was treated once with cryotherapy, followed by two cycles of 5-fluorouracil cream, applied once daily over 4 weeks. Despite this, the plaque persisted and increased in size, measuring 3 cm × 2.5 cm. A 4-mm incisional biopsy was performed. Histopathology findings showed an acanthotic epidermis with compact hyperkeratosis, overlying a superficial dermal vascular lesion that consisted of a distinctive band-like proliferation of small endothelial cell-lined vessels. Background fibrosis and oedema were noted, as well as loss of elastic fibres within the lesional area, demonstrated by elastic van Gieson stain. Positive immunohistochemistry staining included CD31 and erythroblast transformation-specific-related gene stains, helping to confirm this lesion’s vascular origin, with a negative result for D2-40 (podoplanin), suggesting no lymphatic involvement. The histological appearances were thought to fit with a diagnosis of poikilodermatous plaque-like haemangioma (PPLH), a newly described entity of acquired vascular proliferation. Poikilodermatous plaque-like haemangioma, first described in 2019, usually presents as a solitary atrophic erythematous-to-violaceous plaque typically found in the lower limbs (Semkova K, Carr R, Grainger M et al. Poikilodermatous plaque-like hemangioma: case series of a newly defined entity. J Am Acad Dermatol 2019; 81:1257–70). Reassuringly, it has a benign course, but it can be persistent (Semkova et al.). Our patient was keen for treatment as the lesion was continuing to grow, causing him concern. However, there is sparse information available in the literature regarding effective treatments for PPLH. The case was discussed at the departmental clinicopathological meeting. The plaque was felt to be too superficial to merit any radiological investigation of the soft tissues. Surgical excision was discussed but given the size and location would most likely require a large skin graft. Sclerotherapy was considered but not felt to be appropriate in this setting. A trial of laser therapy was regarded as the best option given the superficial nature of this vascular lesion. He has since started a course of pulsed dye laser (PDL) at a wavelength of 585 nm, and we wait with interest to determine if it is effective. We therefore present this case to raise awareness of PPLH as a newly described entity and to consider it in the differential of a solitary atrophic erythematous plaque on a sun-protected site.

Immunohistochemical Findings in Retrocorneal Membranes of Eyes with Corneal Decompensation after Complicated Intraocular Surgery.

Retrocorneal membranes (RCMs) may result from epithelial ingrowth, stromal keratocytic downgrowth, fibrous metaplasia of the corneal endothelium, or a combination of these processes. In an institutional case series, the clinical history, ocular findings, and immunohistochemical staining results of RCMs were analysed in patients with unilateral corneal decompensation after complicated intraocular surgery. Between January 2021 and September 2022, six retrocorneal membranes were excised during Descemet's stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) procedures and classified after screening with haematoxylin and eosin, periodic acid-Schiff, elastic van Gieson staining, and immunohistochemical screening with cytokeratin 7 (CK7), anti-cytokeratin (CAM5.2 and AE1/3), cell surface glycoprotein CD34, smooth muscle actin (α-SMA), and vimentin. On the basis of the immunohistochemical screening, the majority of excised RCMs (5 of 6) could histopathologically be classified as membranes originating from fibrous metaplasia of the corneal endothelium. All these RCMs were positive for CK7, α-SMA, and vimentin and negative for CAM5.2 and CD34. In one patient, an RCM had developed after 18 days of corneal contact to a free-floating dexamethasone implant in the anterior chamber and was classified as originating from stromal keratocyte downgrowth (α-SMA- and vimentin-positive, all others negative). All eyes in this series had a previous history of complicated cataract surgery, partially with subsequent intraocular lens exchange. No eyes after previous penetrating keratoplasty were in this series. In this series of eyes with previous complicated intraocular interventions (in most cases cataract surgery and revisions), the dominating RCM belonged to the type originating from fibrous metaplasia of the corneal endothelium.

Aortic calcification: A postmortem CT validation study in a middle-aged population

BackgroundComputed tomography (CT)-detected aortic calcification is strongly associated with aortic stiffness and is an accurate predictor of cardiovascular and all-cause mortality and cognitive decline. Some previous pathologic studies have shown calcium accumulation in the medial layer of the vessel wall, while others have suggested localisation in the atherosclerotic intimal layer. ObjectivesThe aim of this study was to histologically validate CT findings of aortic calcification for detectability and location in the aortic wall. MethodsWe acquired postmortem CT images and collected 170 aortic tissue samples from five different locations in the thoracic and abdominal aorta of 40 individuals who underwent autopsy. Microscopic slides were stained with haematoxylin and eosin and elastic van Gieson stain. Calcified lesions were characterised and calcifications were manually annotated in the intima and media. The presence and morphology of calcifications were scored on CT images. ResultsThe mean age of the autopsied individuals was 63 years, and 28 % died of cardiovascular disease. Calcifications were present in 74/170 (44 %) samples. Calcification was more common in the abdominal aorta than in the thoracic aorta. In all samples with calcifications, 99 % were located in the intimal layer. Only 16/170 samples had a small amount of medial arterial calcification. The histological results showed an 85 % concordance for the presence or absence of CT calcifications. There was complete inter-method agreement for annularity of calcifications in 68 % of the samples (linear weighted kappa 0.68 (95 %CI 0.60–0.77). ConclusionsAortic calcifications visible on CT are located in the intimal layer of the abdominal aorta wall, at least in aortas that are not aneurysmatic or dissected. The presence and annularity of these calcifications can be reliably determined by CT.

A Plaque Instability Index Calculated by Histological Marker Analysis of the Endarterectomy Carotid Artery

(1) Background: Atherosclerosis is a pandemic condition that causes the development of ischemic syndromes including myocardial infarctions and many strokes, in addition to disabling peripheral artery disease. Progression of atheroma plaques has been associated with an increased risk of mortality. It is a challenge to determine in advance if atherosclerotic plaque will become unstable by calculating an index of instability. We proposed a score of ten parameters for the assessment of high-risk plaques, by quantification of 10 risk factors for acute cardiovascular events, most of them representing histological variables. (2) Methods: Carotid endarterectomy samples were collected from 10 highly symptomatic patients who matched for gender, age, risk factors, and plaque morphology. Samples were stained with hematoxylin-eosin, elastic van Gieson, Perls, and Mallory. Immunohistochemistry was performed using specific antibodies, such as CD31 for endothelial cells, CD68 for macrophages, and CD3 for T cells. (3) Results: For each plaque, the presence and/or number of histological features (fibrous cap thickness, lipid core size, plaque and cap infiltration with macrophages and lymphocytes, neovessels, intraplaque hemorrhage, parietal thrombi, and calcium deposits) were recorded on a simple semi-quantitative one- or two-grade scale. The study identified four stable plaques (SPs), two vulnerable plaques (VPs), and four unstable plaques (USPs). We found significantly more macrophages and neovessels in unstable plaques compared with stable plaques. The score for unstable plaques was higher than that for VP. (4) Conclusions: The study showed that symptomatic carotid disease is associated with an increased index score. The proposed algorithm for carotid plaque assessment may be useful for an imaging application.

Cardiac ion channels associated with unexplained stillbirth- an immunohistochemical study.

Despite the use of post-mortem investigations, approximately 20% of stillbirths remain unexplained. Cardiac ion channelopathies have been identified as a cause of death in Sudden Infant Death Syndrome (SIDS) and could be associated with unexplained stillbirths. This study aimed to understand if the expression or localisation of cardiac ion channels associated with channelopathies were altered in cases of unexplained stillbirths. A case control study was conducted using formalin-fixed cardiac tissue from 20 cases of unexplained stillbirth and a control group of 20 cases of stillbirths from intrapartum hypoxia. 4µm tissue sections were stained using haematoxylin and eosin, Masson's trichrome (MT) and Elastic van Gieson (EVG). Immunohistochemistry (IHC) was performed using antibodies against CACNA1G, KCNJ2, KCNQ1, KCNH2 and KCNE1. The cardiac conduction system in samples stained with MT and EVG could not be identified. Therefore, the levels of immunoperoxidase staining were quantified using QuPath software. The nuclear-cytoplasmic ratio of sections stained with haematoxylin and eosin was higher for the hypoxia group (hypoxia median 0.13 vs. 0.04 unexplained, p<0.001). CACNA1G (unexplained median 0.26 vs. hypoxia 0.30, p=0.009) and KCNJ2 (unexplained median 0.35 vs. hypoxia 0.41, p=0.001) had lower staining intensity in the unexplained stillbirth group. There were no statistically significant differences in the staining intensity of KCNQ1, KCNH2 and KCNE1. Two ion channels associated with channelopathies demonstrated lower levels of expression in cases of unexplained stillbirth. Further genetic studies using human tissue should be performed to understand the association between channelopathies and otherwise unexplained stillbirths.

Changes of Age-related Auricular Cartilage Plasticity and Biomechanical Property in a Rabbit Model.

Ear molding is an emerging technique that can correct auricular deformities. Treatment initiation time is the most important prognostic determinant of ear molding. Here, we aimed to examine why auricular cartilage plasticity appeared to diminish with age. Thus, we characterized age-related changes in the biomechanical, biochemical, and morphological properties of auricular cartilage. New Zealand rabbits were used as the experimental animal. We examined immature [postnatal 0 day (P0), 5 days (P5), 15 days (P15)], young [2 months (2M)], and mature [6 months (6M)] rabbits. Rabbits' ears were splinted and folded using adhesive fixation strips. Folding duration ranged from 1 day to 5 days to 10 days. Photographs were taken to calculate the retained fold angle. Cartilage morphology and extracellular matrix (ECM) content were examined histologically (using hematoxylin-eosin, Safranin O, elastic Van Gieson, and Masson's trichrome). Water content, DNA content, and cell density were also analyzed. Biomechanical properties were measured using a Nano indenter. Immature ears had smaller angles after strip removal, and the angled deformation lasted a longer time. Cartilage matrix compositions, including glycosaminoglycan (GAG), elastin fiber, and collagen, increased over development. The water content, DNA content, and cell density decreased with age. Young's modulus was significantly higher in mature cartilage. Here, we successfully established an animal model of ear molding and demonstrated that immature cartilage was associated with better plasticity. We also found that the cartilage's biomechanical property increased with the accumulation of ECM. The biomechanical change could underlie age-related shape plasticity. NA Laryngoscope, 133:88-94, 2023.

Transient atrial inflammation in a murine model of Coxsackievirus B3-induced myocarditis.

Atrial dysfunction is a relatively common complication of acute myocarditis, although its pathophysiology is unclear. There is limited information on myocarditis‐associated histological changes in the atria and how they develop in time. The aim of this study therefore was to investigate inflammation, fibrosis and viral genome in the atria in time after mild CVB3‐induced viral myocarditis (VM) in mice. C3H mice (n = 68) were infected with 105 PFU of Coxsackievirus B3 (CVB3) and were compared with uninfected mice (n = 10). Atrial tissue was obtained at days 4, 7, 10, 21, 35 or 49 post‐infection. Cellular infiltration of CD45+ lymphocytes, MAC3+ macrophages, Ly6G+ neutrophils and mast cells was quantified by (immuno)histochemical staining. The CVB3 RNA was determined by in situ hybridization, and fibrosis was evaluated by elastic van Gieson (EvG) staining. In the atria of VM mice, the numbers of lymphocytes on days 4 and 7 (p < .05) and days 10 (p < .01); macrophages on days 7 (p < .01) and 10 (p < .05); neutrophils on days 4 (p < .05); and mast cells on days 4 and 7 (p < .05) increased significantly compared with control mice and decreased thereafter to basal levels. No cardiomyocyte death was observed, and the CVB3 genome was detected in only one infected mouse on Day 4 post‐infection. No significant changes in the amount of atrial fibrosis were found between VM and control mice. A temporary increase in inflammation is induced in the atria in the acute phase of CVB3‐induced mild VM, which may facilitate the development of atrial arrhythmia and contractile dysfunction.

Platelet protects angiotensin II-driven abdominal aortic aneurysm formation through inhibition of inflammation.

Inflammation is the primary cause of abdominal aortic aneurysm (AAA) formation and development. It has been reported that platelets protect against septic shock by inhibiting inflammation. However, it is unclear whether platelets protect AAA progress via suppressing inflammation. A mouse model of AAA was established by a daily administration of angiotensin II (Ang II, 1000ng/kg/min) for 28-day. The AAA mice received 1×109 platelets transfusion in normal saline every 3rd day for 1month. Hematoxylin and eosin, Masson's trichrome, and elastic van Gieson staining techniques were used to analyze the morphology of the abdominal aorta. Immunohistochemistry was used to detect any infiltration of inflammatory cells, inflammatory factors, and matrix metalloproteins (MMPs) in the aortic tissue. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to examine the inflammatory factor proteins levels in the aortic wall and peripheral blood, respectively. Platelets infusion significantly suppressed the Ang II-driven elevation of aortic diameter, AAA formation (52.38% decrease, P<0.05), aortic expansion, elastic lamina destruction, and inflammatory response. In addition, MMP-2 and MMP-9 production were also reduced by platelets transfusion. For the first time, our study reported the beneficial effect of platelet transfusion in suppressing the Ang II-driven AAA progress in mice through the inhibition of inflammation.

The urethral plate and the underlying tissue; a histological and histochemical study

To examine the urethral plate and the underlying tissues in children with proximal hypospadias associated with severe chordee. The urethral plate and the underlying tissue specimens were excised to correct severe chordee in 17 children with proximal and perineal hypospadias with severe chordee. The median age was 20 months (range 8-36). Sections samples were marked and examined from proximal to distal. Specimens were examined histologically using hematoxylin-eosin (H/E) and Elastic van Gieson (EvG) stain. Histochemical examination was also performed using smooth muscle actin (SMA) and factor 8 antibodies. For control, samples from four patients with hypoplastic urethra proximal to the meatus including the hypoplastic segments until the normal urethra were taken. In addition, the urethra of an adult patient with penile tumor was used as control. The average size of the 17 tissue samples was 0.5cm×0.5cm x 0.3cm in depth. There was a common pattern that was seen in all the 17 specimens with a variable degree of expression. H/E staining showed that the epithelial lining changed from pseudostratified epithelium at the proximal intact urethra to non-keratinized stratified squamous epithelium at the urethral meatus to keratinized stratified squamous epithelium distally at the urethral plate level. EvG staining showed overall very few elastic fibres that increased slightly in the distal urethral plate. SMA staining showed a circular pattern of smooth muscle cells in the proximal intact urethra that changed to a U-shaped pattern at the level of the meatus, to a triangle shaped pattern just distal to the meatus. The distal urethral plate showed an irregular, disorganized rather flat pattern of the smooth muscles. Factor 8 antibodies staining the blood spaces revealed dysplastic unorganized large blood sinusoids underneath the urethral plate that were different from normal capillaries surrounding the proximal urethra. The urethral plate and the underlying tissues in patients with severe chordee have different structure from normal urethra as compared to available literature and the adult control patient. The lack of elastic fibres may help to explain the rigidity of the ventral penis causing chordee. The disorganized irregular distribution of the smooth muscle fibres is suggestive of the hypoplastic corpus spongiosum. The abnormal large blood sinusoids may explain the poor healing quality of the ventral penis in patients with perineal and proximal patients associated with severe chordee. This may explain persistent/recurrent chordee observed later in those patients with severe chordee when dorsal plication is used. The study also supports the recent trend of 2 stage procedure as a plan of management for patients with proximal and perineal hypospadias with severe chordee and excision of all the dysplastic tissues during the first operation.

Effects of Esterified Hyaluronic Acid, Adipose Tissue, and Blood Glue on Survival of Diced Cartilage Grafts.

Diced cartilage grafts are used for correcting nasal dorsal deformities and irregularities. However, cartilage resorption is among most common problems after rhinoplasty. The purpose of this experimental study was to investigate the effects of esterified hyaluronic acid, adipose tissue, and blood glue on the viability of diced cartilage grafts. A total of 24 Wistar albino rats were used for the study. Cartilage grafts were obtained from 1 side ear and diced. The rats were divided into 4 groups (6 in each group): bare diced cartilage (group 1), diced cartilage wrapped with adipose tissue (group 2), diced cartilage blended with blood glue (group 3), and diced cartilage wrapped with esterified hyaluronic acid (group 4). The grafts were inserted into the subcutaneous pockets of the back of same rat. After 2 months follow-up specimens were harvested for histopathological and dimensional examination. The sections were stained with Hematoxylin and Eosin, Masson-Trichrome, and Elastic Van-Gieson. Chronic inflammation, loss of chondrocyte nucleus, vascularization, foreign body reaction, collagen content of matrix, and extent of elastic fiber were assessed under light microscopy. Foreign body reaction in adipose tissue and blood group was significantly higher than bare cartilage and esterified hyaluronic acid group ( P = 0.001). With respect to loss of chondrocyte nucleus esterified hyaluronic acid group had significant higher rate of nucleus loss than other groups ( P = 0.002). This study suggests that blood glue, esterified hyaluronic acid and autologous adipose tissue have not beneficial effects in improving viability of diced cartilage grafts.

HIGHER CIRCULATING GUT MICROBIOTA-DEPENDENT METABOLITE TRIMETHYLAMINE N-OXIDE PLAYS AN ADVERSE EFFECT ON PULMONARY HYPERTENSION

TOPIC: Pulmonary Vascular Disease TYPE: Original Investigations PURPOSE: The potential role of the gut microbiota-dependent metabolite trimethylamine N-oxide (TMAO) in pulmonary hypertension (PH) has never been elucidated. We aim to explore the association between TMAO and PH. METHODS: PH patients at Fuwai Hospital between October 2018 and February 2020 were enrolled. Patients received follow-up every three to six months through clinic visits. Plasma levels of TMAO were measured and associations with clinical markers including WHO-FC, NT-proBNP, hemodynamic parameters, and exercise capacity were explored. In monocrotaline (MCT)-induced PAH rat models, a normal diet and water supplemented with or without 1% 3,3-dimethyl-1-butanol (DMB), a kind of inhibitor of TMAO, were fed with for 4 weeks. Finally, rats received hemodynamic examinations, and after that blood serum as well as heart and lung tissues were collected. The ratio of right ventricle (RV) over left ventricle (LV) plus the septum was used as an index of RV hypertrophy (RVH). Elastic Van Gieson (EVG) stain was used to explore the changes of pulmonary vascular remodeling. RESULTS: One hundred and three consecutive PH patients were enrolled including patients with idiopathic/heritable pulmonary arterial hypertension (IPAH/HPAH, n=30), chronic thromboembolic PH (CTEPH, n=34), and congenital heart disease-associated PAH (CHD-PAH, n=39). After adjusting for confunders, TMAO levels were positively correlated with clinical disease severity assessed by WHO-FC, and also with NT-proBNP, hemodynamic parameters, and exercise capacity. TMAO responded to guideline-recommended treatment in patients with PH. It was noted that TMAO decreased significantly after treatment in patients with IPAH/HPAH. In linear correlation analysis, change in TMAO was positively associated with change in NT-proBNP (P=0.04) in IPAH/HPAH patients. Animal study showed that the level of TMAO in MCT-induced PH rats (22.2±8.7ng/mL) was higher than the MCT+DMB group (6.6±1.1ng/mL) and the control group (4.3±0.2ng/mL). Compared with MCT group, MCT+DMB group obtained a decreased RVH (30% vs 60%, p<0.001), right arterial pressure (4.4mmHg vs 6.2 mmHg, P=0.035), right ventricular systolic pressure (35.3mmHg vs 63.3mmHg, p<0.001), and mean pulmonary arterial pressure (22.5mmHg vs 39.5mmHg, p<0.001) as well as reduced vascular remodeling of lungs under pathological findings. CONCLUSIONS: TMAO was elevated in accordance with WHO-FC and decreased after guideline-recommended treatment in PH patients indicating higher TMAO might play an adverse effect on PH. The animal findings demonstrated that lower TMAO played a protective effect in MCT-induced PAH, which supported the clinical results. CLINICAL IMPLICATIONS: ①Demonstrated elevated TMAO was a risk factor for PH patients;②TMAO might be regarded as a novel indicator of PH patients. DISCLOSURES: No relevant relationships by Jianing Gao, source=Web Response no disclosure on file for Matthew C Konerman; no disclosure on file for Changming Xiong; No relevant relationships by Yicheng Yang, source=Web Response no disclosure on file for Qixian Zeng; no disclosure on file for Lemin Zheng