Elastin Staining Research Articles

P131. DEFINING THE HISTOLOGICAL SUPPORT STRUCTURES OF THE DIGITAL WEBSPACE: NEW ANATOMY AND CLINICAL APPLICATIONS

PURPOSE: Deformity of the digital web space is commonly encountered and can be congenital or acquired. Its correction requires an understanding of the normal anatomy, which is poorly understood, and understudied. The challenge to re-create a web space normal in appearance and function are equally difficult. The purpose of this study is to introduce previously unreported ligamentous structures of the digital web space. METHODS: Forty web spaces were microsurgically dissected in twenty cadaveric hands. Select H&E, Trichrome and Elastin stains were obtained. Anatomic observations were made and correlated with intraoperative findings as they relate to burn reconstruction and congenital syndactylies. RESULTS: Formed by a relatively large, three-dimensional inclined triangle extending from the distal palm to the dorsal metacarpophalangeal joint, the appearance of the web space and its functional demands are completely interdependent. The dynamically changing planes and contours of the web space are the result of a complex subcutaneous endoskeleton consisting of dense fibrous attachments between the palmar and dorsal skin along the whole length and width of the web spaces extending into the fingers in continuity with Cleland’s and Grayson’s ligaments. These tethers are so strong that one can lift the hand up by holding onto them, yet they are flexible enough to allow for finger abduction and adduction. CONCLUSION: The digital webspace is more than skin and fat. A newly described ligamentous framework defines its shape and function. We will discuss the importance of preserving or re-creating this anatomy, and its significance in the management of congenital deformities.

Extramural venous invasion (EMVI) revisited: a detailed analysis of various characteristics of EMVI and their role as a predictive imaging biomarker in the neoadjuvant treatment response in rectal cancer.

To assess whether size, diameter, and large vein involvement of MR-detected extramural venous invasion (MR-EMVI) have an impact on neoadjuvant therapy response in rectal adenocarcinoma. 57 patients with locally advanced rectal adenocarcinoma scanned with MRI before and after neoadjuvant therapy were included. Two abdominal radiologists evaluated the images with special emphasis on EMVI, on initial staging and after neoadjuvant treatment. The sensitivity and specificity of MRI for detection of rest EMVI were determined. The association of various MR-EMVI characteristics including number, size, and main vein involvement with treatment response was investigated. In subjects with discordance of radiology and pathology, elastin stain was performed, and images and slides were re-evaluated on site with a multidisciplinary approach. At initial evaluation, 17 patients were MR-EMVI negative (29.8%) and 40 were MR-EMVI positive (70.2%). Complete/near-complete responders had less number (mean 1.45) and smaller diameter of MR-EMVI (mean 1.8mm), when compared with partial responders (2.54 and 3.3mm; p < 0.005). The sensitivity of MRI for rest EMVI detection was high, specificity was moderate, and in one patient elastin stain changed the final decision. In five patients with rest MR-EMVI positivity, carcinoma histopathologically had a distinctive serpiginous perivascular spread, growing along the track of vascular bundle, although it did not appear in intravascular spaces. This study demonstrates that not only the presence, but also size and number of EMVI that may be significant clinically and thus these parameters also ought to be incorporated to the MRI evaluation and prognostication of treatment response. From pathology perspective, tumors growing alongside major vessels may also reflect EMVI even if they are not demonstrably "intravascular."

2021 Canadian Surgery Forum: Virtual, online Sept. 21-24, 2021.

# 01. Design and validation of a unique endoscopy simulator using a commercial video game {#article-title-2} Procedural simulation has been shown to enhance early endoscopy training. In this proof of concept study, we aimed to show that a first-person shooter (FPS) video game with a novel in-house

Histologic Analysis of Urethral Stricture in 9 Patients Following Dorsal Vaginal Graft Urethroplasty.

To present the pathologic analysis of female urethral strictures obtained during reconstructive urethroplasty. Nine separate female urethral tissue specimens were obtained during dorsal vaginal graft urethroplasty by a single surgeon (S.P.P.). Samples were serially sectioned and fixed in 10% formalin 6 to 12 hours before routine processing in paraffin blocks. Serial 5-µm sections were subjected to H&E, Masson trichrome, and elastin staining. End point analysis included evaluation for epithelial hyperplasia and cell type, mucosal edema, degree of fibroblast/inflammatory cell infiltrate, and elastin fiber density and distribution. Nine specimens were examined. Six specimens had epithelial linings of stratified squamous epithelium overlying fibrosis (67%), 1 had mixed squamous and urothelial epithelium, and 2 had only urothelial epithelium. Two specimens (29%) showed acute injury with prominent squamous papillary hyperplasia, focal erosion, and patchy mucosal hemorrhage. Areas of urethral stricture were variably thickened, with increased, densely packed collagen fibers and associated mucosal lymphocytic inflammation ranging from mild and patchy to focally dense with lymphoid aggregates. The highest elastin fiber density appeared to be associated with vessels and overlying muscle bundles in the submucosa. Further elucidation of histopathologic characteristics may illuminate more appropriate therapeutic pathways for female urethral stricture disease management.

Superimposed tissue formation in human aortic valve disease: differences between stenotic and regurgitant valves

Abstract Introduction The formation of superimposed tissue (SIT), a layer on top of the original valve leaflet, has been described in patients with mitral regurgitation, as a major contributor of valve thickening and probably secondary to increased valve mechanical stress. However, little is known whether SIT formation also occurs in aortic valve disease. Both in the case of aortic stenosis or aortic regurgitation, the aortic valve (AV) is subjected to increased mechanical stresses, although different in type, extent and location. Purpose To characterize SIT formation in aortic stenosis and regurgitation. Methods Human diseased AV leaflets (n=31) were obtained from patients undergoing aortic valve replacement because of aortic stenosis (n=17) or aortic regurgitation (n=14). Histological analysis was performed and elastin staining was used to distinguish the SIT from the original leaflet. Alpha-smooth muscle actin (SMA) staining was performed to identify myofibroblasts and Masson's Trichrome staining to identify collagen fibres. Results In both regurgitant leaflets (RL) and stenotic leaflets (SL) SIT was found at both the ventricular and aortic side (94% of SL, 93% of RL) and could reach up to 50% of total leaflet thickness (Fig. A-C). Although the average SIT thickness did not differ between SL and RL (0.30 mm, standard error of the mean (SEM): ±0.04 for SL vs 0.38 mm, SEM: ±0.05 for RL; p=0.61), the distribution of SIT differed. The SIT at the free edge of the aortic valve was significantly thicker in the RL (0.39 mm, SEM: ±0.06 for SL vs 0.88 mm, SEM: ± 0.07 for RL; p&amp;lt;0.0001), whereas the SIT at the aortic side of the body part was thicker in the SL (0.099 mm, SEM: ±0.023 for SL vs 0.033 mm, SEM: ± 0.021 for RL; p&amp;lt;0.05). Although the SIT comprised of various compositions of extracellular matrix, the overall collagen content was higher in SIT of the SL (212 a.u., SEM: ±4.37 for SL vs 169 a.u., SEM: ±4.06 for RL; p&amp;lt;0.0001). Myofibroblasts were predominantly observed in the SIT as compared to the original leaflet in SL and RL (Fig. D,E; myofibroblast-positive area: 11.6%, SEM: ±3.1 for SIT vs 1.2%, SEM: ±0.3 for original leaflet; p&amp;lt;0.001). The density of myofibroblast in the SIT of the body part of the aortic leaflet, however, was higher in the SL (myofibroblast-positive area: 15.5%, SEM: ±2.0 for SL vs 4.1%, SEM: ±1.3 for RL; p&amp;lt;0.01), whereas the density of myofibroblast in the SIT of the free edge was higher in the RL (myofibroblast-positive area: 15.3%, SEM: ±3.7 for SL vs 4.0%, SEM: ±1.4 for RL; p&amp;lt;0.001). Conclusions Both in aortic stenosis and aortic regurgitation, the AV is characterized by SIT formation but with difference in distribution and composition. These observations suggest the involvement of hemodynamic and mechanical stresses in the regulation of SIT formation of the AV. Understanding the formation of SIT might provide new insights in pathology of AV disease. Funding Acknowledgement Type of funding sources: None.

Elastin in pulmonary pathology: relevance in tumours with a lepidic or papillary appearance. A comprehensive understanding from a morphological viewpoint.

Elastin and collagen are the main components of the lung connective tissue network, and together provide the lung with elasticity and tensile strength. In pulmonary pathology, elastin staining is used to variable extents in different countries. These uses include evaluation of the pleura in staging, and the distinction of invasion from collapse of alveoli after surgery (iatrogenic collapse). In the latter, elastin staining is used to highlight distorted but pre‐existing alveolar architecture from true invasion. In addition to variable levels of use and experience, the interpretation of elastin staining in some adenocarcinomas leads to interpretative differences between collapsed lepidic patterns and true papillary patterns. This review aims to summarise the existing data on the use of elastin staining in pulmonary pathology, on the basis of literature data and morphological characteristics. The effect of iatrogenic collapse and the interpretation of elastin staining in pulmonary adenocarcinomas is discussed in detail, especially for the distinction between lepidic patterns and papillary carcinoma.

Three-dimensional reconstruction of the orbital retrobulbar vasculature

ABSTRACT Purpose The retrobulbar orbital vasculature is known to be complex and variable between individuals. This study aimed to produce a method to map the retrobulbar vessels, to generate 3D reconstructions, to visualise and to improve our understanding of their complexity. Methods Five human orbits donated under the Human Tissue Act (2004) were fixed in formalin, decalcified in 10% formic acid, and dehydrated in acetone at −20°C. Specimens were impregnated with epoxy resin, cured, and cut into 0.3 mm sections. Sections were stained with Gomori’s trichrome stain, imaged, and reconstructed using 3D reconstruction software (BioVis3D, version 3.1). Results The arterial system was reconstructed in all five specimens. The superior ophthalmic vein (SOV) and the central retinal vein (CRV) were reconstructed in four specimens. E12 sheet plastination showed excellent results for histological analysis at a macroscopic level; however, anatomical topology was not entirely preserved on a microscopic level. Gomori’s trichrome stain gave excellent results in highlighting axial sections of the arterial walls and their tunics, including finer calibre vessels, thus allowing detailed reconstruction of the arterial vasculature. Miller’s stain for elastin showed poor results in differentiating vessels from soft tissue; venous vasculature was poorly identified with both stains. Conclusions This study provided a detailed anatomical model of the retrobulbar orbital vascular system, a method that can be used for further studies to form a database relating to the topography of the arterial system. These models may be employed for teaching, and possible surgery planning, for both trainees and ophthalmic surgeons.

Prognostic Value of Venous Invasion Detected by Elastin Stain May Surpass Lymph Node Status in Colon Cancer.

Venous invasion is a poor prognostic factor in colon cancer but is often underreported with significant variability. We aimed to determine the impact of an elastin stain on venous invasion detection in colon cancer and evaluate the value of venous invasion in predicting disease recurrence in combination with lymph node status and other prognostic factors. This is a retrospective analysis of a prospectively collected database. This study was conducted at a tertiary cancer center. A total of 418 patients who underwent curative resection for stage I to III colon cancer and routinely adopted an elastin stain were evaluated. Venous invasion detection rate after adopting elastin stain, prognostic factors influencing disease recurrences by multivariate Cox regression models, and survival were measured. The zones of lymph node metastasis were defined as LNZ1, LNZ2, and LNZ3, corresponding to metastases in the pericolic, intermediate, and apical nodes. Venous invasion detection rate increased from 11.3% to 35.4% compared with the previous period in which only hematoxylin and eosin stain was performed. Cox regression analysis showed venous invasion (HR, 3.856; 95% CI, 1.249-11.910; p = 0.019) and lymph node metastases (HR, 3.156; 95% CI, 1.094-9.108; p = 0.034) in all stages and LNZ 2, 3 (HR, 2.649; 95% CI, 1.244-5.640; p = 0.012) in stage III to be significantly associated with poor disease-free survival. When stratifying all patients by these 3 factors, patients with stage III [LNZ1/venous invasion (-)] had disease-free survival comparable with stage I, but significantly better disease-free survival than those with stage II [venous invasion (+)] (p = 0.018). Patients with stage II [venous invasion (+)] had better disease-free survival by using adjuvant chemotherapy (p < 0.001). This study was limited by its retrospective design. Elastin stain contributed to a considerable increase in venous invasion detection. Venous invasion can be a powerful predictor of poor disease-free survival beyond lymph node metastases when limited to the pericolic area and is useful for deciding the use of adjuvant chemotherapy in stage II colon cancer. See Video Abstract at http://links.lww.com/DCR/B573. ANTECEDENTES:Invasión venosa (IV) es un factor de mal pronóstico en el cáncer de colon, que frecuentemente no se informa con una variabilidad significativa.OBJETIVOS:Nuestro objetivo fue determinar el impacto de tinción de elastina en la detección de IV en el cáncer de colon y evaluar el valor de IV en la predicción de la recurrencia de la enfermedad en combinación con el estado de los ganglios linfáticos y otros factores pronósticos.DISEÑO:Este es un análisis retrospectivo de una base de datos recopilada prospectivamente.ENTORNO CLINICO:Este estudio se realizó en un centro oncológico de referencia de tercer nivel.PACIENTES:Se valoraron un total de 418 pacientes sometidos a resección curativa por cáncer de colon en estadio I-III utilizando de manera rutinaria una tinción de elastina.PRINCIPALES MEDIDAS DE VALORACION:Se midieron la tasa de detección de IV después de adoptar la tinción de elastina, los factores de pronóstico que influyen en las recurrencias de la enfermedad mediante modelos de regresión de Cox multivariados y la supervivencia. La zona de metástasis ganglionares se definió como, LNZ1, LNZ2 y LNZ3, correspondientes a las metástasis en los ganglios pericólicos, intermedios y apicales, respectivamente.RESULTADOS:La tasa de detección de IV aumentó de 11,3% a 35,4% en comparación con el período anterior en el que solo se realizó tinción con hematoxilina y eosina. El análisis de regresión de Cox mostró VI (razón de riesgo, 3.856; intervalo de confianza [IC] del 95%, 1.249-11.910, p = 0.019) y metástasis en los ganglios linfáticos (razón de riesgo, 3.156; IC del 95%, 1.094-9.108, p = 0.034) en todos los estadios y LNZ 2, 3 (cociente de riesgo, 2.649; IC del 95%, 1.244-5.640, p = 0.012) en el estadio III se asociaron significativamente con una pobre supervivencia libre de enfermedad. Al estratificar a todos los pacientes según estos tres factores, los pacientes con estadio III [LNZ1 / VI (-)] tuvieron una sobrevivencia sin enfermedad (SSE) comparable con el estadio I, pero una supervivencia libre de enfermedad significativamente mejor que aquellos con estadio II [VI (+)] (p = 0,018). Pacientes en estadío II [VI (+)] tuvieron una mejor supervivencia sin enfermedad mediante el uso de quimioterapia adyuvante (p <0,001).LIMITACIONES:Estudio limitado por su diseño retrospectivo.CONCLUSIÓN:La tinción de elastina contribuyó a un aumento considerable en la detección de IV. IV puede ser un poderoso predictor de supervivencia sin enfermedad deficiente más allá de las metástasis de los ganglios linfáticos cuando se limita al área pericólica y es útil para decidir el uso de quimioterapia adyuvante en el cáncer de colon en estadío II. Consulte Video Resumen en http://links.lww.com/DCR/B573. (Traducción-Dr. Adrian Ortega).

Signaling pathways associated with structural changes in varicose veins: a case-control study.

In varicose veins, blood pressure increases in the veins of the lower extremities due to mechanical stimulation and function remodeling. The aim of this study is assessment of Signaling pathways associated with structural changes in varicose veins. This pilot study was performed on patients with varicose veins, which had undergone surgery. The healthy tissues from trauma patients or vascular bypass without underlying diseases were used for control samples. Hematoxylin-eosin, trichrome, and elastin staining were used for histopathological examination. The levels of MDA (malondialdehyde), total thiol, SOD (Superoxide dismutase) and NO (nitric oxide) level were measured using Elisa kits to evaluate the oxidative stress level. Gene expression levels of MMP2, MMP9, FOXO3a, APOE and p53 genes were determined using Real-time PCR. This study showed, the vascular Vein wall changes are visible in vascular collagen staining. Although these changes are observed in the structure of vascular wall collagen fibers, the accumulation of collagen and elastin was increased in the walls of varicose veins compared to the control group. The amount of nitric oxide and thiol were increased in the varicose group (P < 0.0001). The expression of metalloproteinase2 gene associated with extracellular matrix change was increased in varicose veins. However, the amount of metalloproteinase 9 was decreased in this group compared to control group. FOXO3a, APOE Genes were down-regulated in the varicose veins compared to control group, while p53 gene expression was significantly increased in the varicose group (P < 0.0001). This study demonstrated changes in oxidative stress, morphological structure, and aging pathways in varicose when compared to non-varicose veins. The changes in oxidative stress may be associated with the variations in morphological structure and aging pathways which contribute to the pathogenesis of varicose veins.

Smooth muscular layer: A new helpful criterion to reclassify tumor deposits into metastatic lymph nodes in patients with colo-rectal adenocarcinoma

ContextThe origin of tumor deposit in colorectal cancer is still unknown, and currently there is no single morphological feature to distinguish a metastatic lymph node from a tumor deposit. Histologically, the normal lymph node capsule and trabeculae contain a smooth muscular layer, which when present in extramural deposits would strongly suggest their lymph node origin. ObjectiveWe analyze the value of the smooth muscular layer criterion in reclassifying tumor deposit into metastatic lymph node. DesignA total of 458 colo-rectal carcinomas surgical specimens treated or not by neoadjuvant (radio)chemotherapy were retrospectively included. Harvested tumor deposits were analyzed by Hematoxylin and Eosin and elastin staining on 10 consecutive serial sections and by α- smooth muscle actin immunostaining. ResultsA total of 129 tumor deposits were identified. 77 (60%) tumor deposits were reclassified into metastatic lymph node, of which 63 (49%) presented a smooth muscular layer on the initial Hematein Eosin staining and/or after serial tissue sections, confirmed by positive α-smooth muscle actin immunostaining in 43 out of 45 cases (90%). Fourteen (18%) additional tumor deposits were reclassified into metastatic lymph node by the appearance of lymphoid tissue after serial sections. ConclusionsThe presence of a smooth muscular layer in a presumable tumor deposit is helpful in pointing out its lymph node origin in patients with colo-rectal carcinomas. This criterion could improve the inter-observer agreement of tumor deposit identification, allowing accurate nodal staging and better assessment of patient's prognosis.

MO763THE VON WILLEBRAND FACTOR IS A KEY PLAYER IN ARTERIOVENOUS FISTULA MATURATION*

Abstract Background and Aims Low arteriovenous fistula (AVF) maturation rate remains a bottleneck in creating a long lasting lifeline to hemodialysis. AVF maturation is determined by the intricate balance between outward remodeling (OR) and formation of intimal hyperplasia (IH), during which vascular smooth muscle cell (VSMC) proliferation is essential. Recently, the von Willebrand Factor (VWF) has been shown to induce VSMC proliferation in vitro and in vivo. In this study we investigate the role of VWF on VSMC proliferation and AVF functionality to gain a better understanding of the relationship between the two latter processes. Method AVFs were created in wild-type (WT) and VWF (-/-) deficient (B6.129S2-Vwf tm1Wgr) mice using the external jugular vein and the common carotid artery (CCA). Mean velocity and diameter of the CCA was measured at baseline before surgery, t=0 immediately post-surgery, t=7 and t=14 using ultrasonography to determine flow volume (mL/s). The mice were sacrificed at t=14, AVFs were collected and OR and IH were analyzed using Weigert’s elastin and aSMA staining, using the Mann-Whitney test. To study VSMC proliferation in vitro, VSMCs were isolated from vena cava explants. VSMCs from VWF -/- mice were cultured with 10% VWF -/- plasma with or without addition of VWF (Wilfactin). Proliferation was measured after 40 hours. Humane patient-matched samples were obtained during two stage brachio-basalic AVF surgery: the native veins at creation and venous AVF samples at transposition. Maturation was defined as a luminal AVF diameter of &amp;gt; 6 mm. Samples were stained for VWF and aSMA. Positively stained tissue in the medial layer was quantified using Histoquant and analyzed with the Wilcoxon signed-rank test. Results WT mice showed better outcomes in AVF functionality. Whereas blood flow did not increase in VWF deficient mice over two weeks after AVF-creation, WT mice showed a steady increase in flow rate from t=0 to t=7 (1.97 fold) and t=14 (3.0 fold) . At 14 days, VWF -/- mice showed significantly reduced OR compared to WT mice, with a 1.46 fold smaller venous perimeter (p=0.008). Formation of IH was also significantly reduced in VWF -/- mice (9.58 fold change, p = 0,0001), with a non-significant 1.42 fold difference in luminal area. Lastly, an 8.05 fold decrease was observed in aSMA+ cells in the IH of VWF -/-mice (p = 0.0002). In vitro, addition of VWF caused an 1.5 fold increase in proliferation compared to VWF deficient VSMCs cultured without VWF (p=0.016). As for chronic kidney disease (CKD) patients, our study showed significant wall thickening and increase in aSMA+ tissue only in matured AVFs. There was significant upregulation of VWF+ tissue in the media from native veins to venous AVF samples, with 183% in failed AVFs (p= 0.026) and 425% in matured AVFs (p= 0.013). Matured AVFs had a 4.6 fold increase in VWF+ area in the medial layer compared to failed AVFs (p= 0.033). Conclusion Our study demonstrates VWF as an inducer of VSMC proliferation and thereby both OR and IH. Secondly, we show that VWF is essential for AVF maturation and that enhanced expression in the media coincides with AVF maturation in CKD patients.

Visualization of elastin using cardiac magnetic resonance imaging after myocardial infarction as inflammatory response

The aim of this study was to investigate the merits of magnetic resonance imaging (MRI) using an elastin-binding contrast agent after myocardial infarction in mouse models with deletions of monocyte populations. Permanent ligation of the left anterior descending (LAD) artery was conducted in 10 wild-type mice and 10 each of three knockout models: CX3CR−/−, CCR2−/−, and MCP-1−/−. At 7 days and 30 days after permanent ligation, cardiac MRI was performed with a 7 T-Bruker horizontal scanner for in vivo detection of elastin with an elastin/tropoelastin-specific contrast agent (ESMA). Histology was performed with staining for elastin, collagen I and III, and F4/80. Real-time PCR was conducted to quantify the expression of genes for collagen I and III, F4/80, and tumor necrosis factor alpha (TNFα). Histological and ESMA-indicated elastin areas were strongly correlated (r = 0.8). 30 days after permanent ligation, CCR2-deficient mice demonstrated higher elastin levels in the scar relative to MCP-1−/− (p < 0.04) and wild-type mice (p < 0.02). The ejection fraction was lower in CCR2-deficient mice. In vivo MRI in mouse models of MI can detect elastin deposition after myocardial infarction, highlighting the pivotal role of elastin in myocardial remodeling in mouse models with deletions of monocyte populations.

The effect of redox signaling on extracellular matrix changes in diabetic wounds leading to amputation

Introduction& Objectives: Redox signaling is a critical regulator in the process of wound healing. This signaling pathway can be effective in the development or healing of diabetic ulcers through the ECM.In this study, the structure of extracellular matrix investigated in relation to redox signaling in the tissue of patients with diabetic ulcers that lead to organ amputation. Materials and methodsThe case-control design on diabetic patients ulcers as case group and non-diabetic limb ischemia as control were used.Hematoxylin-eosin, trichrome, and elastin staining methods were used for pathological evaluations of ECM. MDA, total thiol, and SOD levels were measured using ELISA kits to assess the oxidative stress level. Also, NO level was measured by using ELISA kits in both groups. Expression levels of genes MMP2, MMP9, and HIF were detected using real-time PCR with SYBR-green assay. ResultsThe pathological results showed an increase in the thickness of collagen and elastin fibers. Lipids atrophy was visible in the tissue isolated from the diabetic wound group. The amount of MAD to evaluate the level of lipid oxidation in patients with diabetic Ulcer was significantly higher than the control group(p < 0.01). Thiol level was significantly lower in the diabetic ulcer group than in the control group(p < 0.0001). The expression of metalloproteinases 2 and 9 genes in the tissues isolated from diabetic ulcers was lower than the control group(p < 0.0001). While the expression of the HIF gene in this group was higher than the control group(p < 0.0001). ConclutionIn the diabetic wound, the HIF secretion due to hypoxic conditions is beneficial for matrix deposition and prevents protease activity, but if the hypoxia persists, it can lead to ECM deposition subsequently increases the tissue pressure, increases of the collagen I-to-collagen III ratio in collagen accumulation that due to more hypoxia , lipidsAtrophy and eventually amputation.

Comparison of texture-based classification and deep learning for plantar soft tissue histology segmentation

Histomorphological measurements can be used to identify microstructural changes related to disease pathomechanics, in particular, plantar soft tissue changes with diabetes. However, these measurements are time-consuming and susceptible to sampling and human measurement error. We investigated two approaches to automate segmentation of plantar soft tissue stained with modified Hart's stain for elastin with the eventual goal of subsequent morphological analysis. The first approach used multiple texture- and color-based features with tile-wise classification. The second approach used a convolutional neural network modified from the U-Net architecture with fewer channel dimensions and additional downsampling steps. A hybrid color and texture feature, Fourier reduced histogram of uniform improved opponent color local binary patterns (f-IOCLBP), yielded the best feature-based segmentation, but still performed 3.6% worse on average than the modified U-Net. The texture-based method was sensitive to changes in illumination and stain intensity, and segmentation errors were often in large regions of single tissues or at tissue boundaries. The U-Net was able to segment small, few-pixel tissue boundaries, and errors were often trivial to clean up with post-processing. A U-Net approach outperforms hand-crafted features for segmentation of plantar soft tissue stained with modified Hart's stain for elastin.

ACE Inhibitor, Lisinopril, Affects Age‐Associated Changes in Cardiovascular and Cognitive Function in Female Dahl Salt‐Sensitive Rats

Background Aged Dahl Salt-Sensitive (DSS) rats develop central arterial stiffness (CAS) and hypertension on a normal salt (NS) diet. These changes have effects on cognition via brain structural changes associated with CAS. Previous results showed that ACE inhibitor lisinopril (Lis) improved cardiovascular and cognitive function in male DSS rats. Here we determined whether these effects of Lis on physiology and behavior could be achieved in age-matched female DSS rats. Methods Female DSS rats (n=23) were kept on a NS diet (0.5% NaCl) for the duration of the study. Baseline (BL) measurements were taken at 3- and 6-mo of age. After 6-mo measurements, rats were given either Lis in drinking water (15mg/kg/day, n=12) or control treatment (n=11). Final measurements were taken after 7-mo of Lis treatment, before tissues were collected for histochemistry using Verhoeff's stain for collagen and elastin. Systolic blood pressure (SBP) was measured using tail-cuff plethysmography. Pulse wave velocity (PWV), a measure of CAS, was measured by echocardiography. Open field test (OFT) was performed to assess anxiety-like behavior, and Morris water maze (MWM) was performed to assess spatial memory function. Rats were tested in an operant chambers attention challenge to assess attention and impulsivity. Statistical analyses were performed using Pearson correlation and 2-way ANOVA mixed effects model. Data are presented as mean ± SEM. Results At 3 and 6-mo of age, the treatment groups did not display differences in SBP or PWV. Following treatment, the Lis treated rats had lower SBP vs. controls (116 ± 3.7 vs. 187 ± 9.5 mmHg; p<0.01). Similarly, PWV was lower at 13-mo in treated vs. control rats (4.4 ± 0.3 vs. 5.7 ± 1.0 m/s; p<0.05) (Fig. 1A-B). Histochemical analysis revealed that the aortic media collagen abundance, a measure of fibrosis, was lower (18.5 ± 0.9% vs. 21.5 ± 0.8%, p<0.05) and the elastin/collagen ratio in the aortic media of treated rats was higher (2.5 ± 0.1 vs. 2.0 ± 0.1, p<0.05) than that of controls (Fig. 1C-D). Performance on the OFT at 13-mo compared to 6-mo BL indicated that the treated group had an increase in activity in the center of the field with age (+25% vs. BL) i.e., lower anxiety levels, while in control rats there was an opposite effect (-32% vs. BL) (Fig. 2A). PWV was negatively correlated with distance traveled in the center of the OFT at 13-mo (R=-0.672, P<0.05). MWM results showed that treated rats traveled a shorter distance to a hidden platform compared to control rats (6.5 ± 0.7 vs. 8.8 ± 0.9 m; p=0.039), indicating better spatial memory in the treated group (Fig. 2B). In the attention test at 13-mo of age, treated rats had more correct and fewer premature responses vs. control rats (Fig. 2C-D). Conclusion Female DSS rats show beneficial effects of Lis on cardiovascular and cognitive function. Lower SBP, reduced aortic fibrosis, and stabilized PWV in Lis treated female DSS rats were associated with improved behavioral test performance. The mechanistic basis of the effects of Lis on cognition via cerebrovascular and brain changes is being further investigated.

Evaluation of the effects of Marfan pathogenesis and losartan treatment on coronary and cerebral arteries in the well‐established Marfan Syndrome mouse model

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 (FBN1) gene. This mutation manifests in a variety of phenotypic changes with a notable cardiovascular effect leading to aortic aneurysm, dissection, and rupture. This results in a significant increase in morbidity and mortality in these patients. The angiotensin II type I receptor (AT1R) signalling pathway has previously been shown to contribute to the cardiovascular effects in the progression of this disease. Prior studies have demonstrated the protective effects of Losartan, an AT1R blocker, on slowing the progression of aortic root aneurysm in the well-established mouse model of MFS (Fbn1 +/- p. Cys1041Gly). In this study, we are further investigating the impact of MFS pathogenesis and Losartan treatment on other vessels such as the posterior cerebral artery and coronary artery. Mice (male + female) were divided into experimental groups: Control, MFS, MFS + 0.6g/L losartan. Drug therapy consisted of 0.6g/L of losartan in drinking water. Water intakes were recorded to ensure the mice were receiving the treatment. At 7 months of age, in vivo ultrasound imaging was performed to measure aortic diameters, aortic pulse wave velocity (PWV), and peak blood flow of the coronary and posterior cerebral arteries. In order to assess elastin fragmentation in the aortic wall, 5μm aortic cross-sections were subjected to Van Geison elastin staining. Blood pressure (BP) measurements were also obtained, using the tail-cuff method. Our results demonstrate that Losartan reduces aortic root diameters at the sinus of Valsalva and reduced the PWV, an index of aortic stiffness, in MFS mice at 7 months of age. Losartan also reduces elastin fragmentation in the aortic wall of MFS mice. The systolic coronary peak flow demonstrates a statistically significant decrease in MFS when compared to CTRL. Losartan treatment has no effect on the systolic coronary peak flow. There is no statistical significance in diastolic coronary peak flow between CTRL vs. MFS and MFS vs. MFS + Losartan. There is no statistical significance in comparison of the posterior cerebral artery peak flow between CTRL to MFS and MFS to MFS + Losartan. There is also no statistical significance in the systolic and diastolic BP between the groups. Our results show that Losartan has beneficial effects in delaying the progression of aortic aneurysm, reduces vessel wall stiffness, and reduces elastin fragmentation in the aortic root. This study also provides an early insight into the disease progression of MFS in the less investigated coronary and cerebral arteries, as well as investigates the preliminary potential effects of Losartan on peak flow in coronary and cerebral vessels in the well-established MFS mouse model.

CADASIL-CAUSING NOTCH3 MUTATION CONTRIBUTES TO THE DEVELOPMENT OF PULMONARY ARTERIAL HYPERTENSION IN MALES

Objective: Notch3 signalling has been associated with changes in the pulmonary vasculature leading to pulmonary arterial hypertension (PAH). Mice with the CADASIL-causing R169C Notch3 mutation (TgNotch3R169C) exhibit gain-of-function Notch3 signalling and develop peripheral vascular dysfunction and injury. Here we investigated whether the R169C mutation contributes to PAH development. Design and method: Briefly, 24-week male and female mice overexpressing either a WT or R169C mutant Notch3 gene were exposed to 2 weeks normoxia (N) or chronic hypobaric hypoxia (10% O2) (H). PAH was assessed by in vivo haemodynamic measurements, Fulton index analysis of right ventricle hypertrophy (RVH), and histological remodelling analysis by elastin staining of pulmonary arteries under 80 microns in diameter. Pulmonary artery wire myography was also performed. Results: RV systolic pressure increased in hypoxic animals vs normoxic (WT H 27.5 ± 1.0 vs WT N 22.8 ± 2.2mmHg); and was exacerbated only in male R169C hypoxic animals (R169C H 32.2 ± 1.1 mmHg vs R169C N 23.6 ± 1.4) (p &lt; 0.05). Male R169C hypoxic animals also exhibited significant RV hypertrophy (H 0.26 ± 0.033 vs N 0.16 ± 0.012), p &lt; 0.05. RVH was not significantly different between female groups. R169C pulmonary arteries from males were significantly hypercontractile to 5HT (Emax: R169C 142 ± 10% vs WT 92 ± 6%); an effect attenuated by ROCK inhibitor Fasudil (Emax: R169C ± Fas. 117 ± 20%). Female R169C arteries were not hypercontractile to 5HT (R169C 113.3 ± 11% vs WT 120.3 ± 8%) but Fasudil reduced contraction (Emax: R169C + Fas. 96.8 ± 10%). Notch3 effector gene Hes5, implicated in PAH, was significantly increased in male R169C lung (8.7 fold change vs WT) suggesting increased Notch3 activation. RhoA/ROCK activating Rho-guanine nucleotide exchange factors LARG and PDZ mRNA were upregulated in R169C lung (R169C 33.5 fold vs WT, R169C 18.7 fold vs WT respectively). These changes were not observed in lungs from female mice. Conclusions: Notch3 R169C mutation may contribute to PAH susceptibility and pulmonary vascular hypercontractility in males. ROCK signaling plays an important role in these processes.

Multifocal extramural venous invasion detected with an elastin stain as a predictor of cancer-specific outcomes in stage I-III resected colorectal cancer (CRC).

114 Background: ExtraMural Venous Invasion (EMVI) is considered an indicator of poor prognosis in patients who have undergone resection of primary CRC, but its use has not been widely adopted in staging systems or nomograms. Staining for elastin may facilitate the accurate detection of EMVI and minimize interobserver variability, as well as enable the assessment of specific features of EMVI including focality and size. We examined the prognostic potential of EMVI detected by elastin staining at a tertiary center that performs a high volume of CRC resections. Methods: This is a single-institution, observational study of consecutive patients who underwent resection of primary CRC between 01/2011 and 12/2016 (n=556). All pathology specimens were re-assessed by expert reviewers who were blinded to patient outcomes. Venous invasion was detected using an elastin trichrome stain and classified as IntraMural or ExtraMural. The number of VI foci, as well as the maximum foci width and length, were also determined. Disease-specific and recurrence-free survival (DSS, RFS) were estimated using the Kaplan-Meier method and group differences were assessed using the log-rank test. Cox proportional-hazard models were used to calculate hazard ratios (HR) and 95% CI. For the present analysis, patients with stage IV (n=86) CRC were excluded. Results: The cohort for analysis included 470 patients (264M, 206F; TNM 8th edition Stage I/II n=291; Stage III n=179) with a median follow-up time of 63 months (0.1-114). EMVI was detected in 33% of all cases (20% in Stage I/II vs. 55% in Stage III; p&lt;0.0001). For the entire cohort, DSS and RFS at 5 years were 86% and 76%, respectively. The presence of EMVI was associated with significantly worse DSS and RFS at 5-years (73% and 54%) compared to patients with no VI (92% and 85%) or IMVI alone (93% and 89%; p&lt;0.0001). The majority of EMVI was multifocal (69%) vs. unifocal (31%). Interestingly, multifocal EMVI was prognostic for worse 5-year DSS (65%), whereas unifocal EMVI was similar to the absence of EMVI (91% and 92%, respectively; p&lt;0.0001). A Cox-proportional hazards model showed worsening DSS with increasing number of detected EMVI foci (1 focus: HR 1.1, 2-4 foci: HR 3.3, &gt;4 foci: HR 7.9; p&lt;0.0001). A similar trend was observed for RFS. Neither the maximum width or length of EMVI foci were prognostic of DSS or RFS. Conclusions: To our knowledge, the prognostic role of EMVI focality in CRC has not been previously explored. In this cohort of Stage I - III CRC patients, multifocal EMVI as assessed by elastin staining was a powerful predictor of cancer-specific death and recurrence-free survival. Elastin staining, which improves the accuracy and objectivity of EMVI detection, may allow validation of EMVI as an independent prognostic variable that should be incorporated into staging systems and nomograms.

Submucosal Elastosis of the Right Colon-A Case Report with Review of the Literature

Submucosal elastosis of the colon is a rarely reported condition that may resemble colonic polyp and amyloid deposition. We report a case of 68-year-old male who underwent screening for colorectal malignant neoplasm. Multiple sessile polyps along with innumerable firm yellow nodules of varying sizes are identified throughout the right colon. Histopathologically, the nodules are characterized by extensive submucosal accumulation of eosinophilic elastic fibers, positive for elastin stain and negative for Congo red stain. The overall findings are consistent with fibroelastoma. The clinical, microscopic, and immunohistochemical characteristics of the current case are reviewed in detail in this report. Other 25 reported cases between 2004-2019 are summarized and their clinical and pathological features are compared with the current case

TRPV5 attenuates abdominal aortic aneurysm in mice by regulating KLF4-dependent phenotype switch of aortic vascular smooth muscle cells

Abdominal aortic aneurysm (AAA) is a fatal vascular disease with insidious symptoms. However, the mechanism behind its development remains unclear. The transient receptor potential vanilloid (TRPV) family has crucial protective effects against cardiovascular diseases, but the role of TRPV5 in AAA has yet to be reported. In this study, ApoE−/− mice were intraperitoneally injected with AAV-GFP or AAV-TRPV5. After 30 days, mice were further administered with angiotensin II (Ang II, 1.44 mg/kg/day) by using osmotic pumps to induce the AAA model or Saline for 28 days, (i.e., Saline + AAV-GFP, Saline + AAV-TRPV5, Ang II + AAV-GFP and Ang II + AAV-TRPV5 groups were established). Compared with the control group, the incidence of AAA and the maximal diameter of the abdominal aorta markedly decreased in Ang II + AAV-TRPV5, which was detected by vascular ultrasound at 28 day. Meanwhile, less collagen and elastin degradation were observed in the Ang II + AAV-TRPV5 group by using Masson and Elastin stains. Moreover, more α-SMA and less MMP2 was observed in the abdominal aortas collected at 28 day by immunohistochemistry. In vitro, primary mouse vascular smooth muscle cells (VSMCs) were treated with Ang II (1 μM) to induce phenotype switch. Sh-TRPV5 and AdTRPV5 were used to transfect VSMCs. PCR and Western blotting were used to access the expression of contractile marker, including α-SMA and SM-22α. The results showed that the mRNA and protein level of α-SMA and SM-22α were decreased under the stimulation of Ang II, but could be attenuated by TRPV5 overexpression. The cell scratch assay demonstrated that the migration ability of VSMCs was increased in Ang II treated group and could be ameliorated by TRPV5 overexpression. Above all, VSMCs transformed from the contractile into secretory phenotype under Ang II stimuli, but could be rescued by TRPV5 overexpression. Furthermore, TRPV5 overexpression suppressed the increased expression of KLF4 induced by Ang II treatment in VSMCs. The data demonstrated that TRPV5 could inhibit AAA formation and play a critical role in the VSMC phenotype switch by downregulating KLF4, suggesting TRPV5 as a new strategy for treating AAA.