Verhoeff's Stain Research Articles

Is Floppy Eyelid Syndrome predictive of Intraoperative Floppy Iris Syndrome? A prospective and translational study

Intraoperative floppy iris syndrome (IFIS) is defined as an excessive billowing of the iris during cataract surgery. Floppy eyelid syndrome is defined as an excessive hyperlaxity of the eyelids. Based in our daily experience, we suspected FES to be associated with IFIS. This association has been investigated. The goal of the study was to explore whether FES was predictive of IFIS. We conducted a prospective observational single-center study at the University Hospital of Nice. IFIS and FES were investigated in patients undergoing cataract surgery from November 2019 to May 2021. In addition, tarsus and iris samples were harvested in 2 fresh cadavers, and Verhoeff staining was used to identify elastin fibers. We included 452 eyes (n=312 patients, 50.9% male) with a mean age of 71.7years (±11.4). IFIS was diagnosed in 88 (19.5%) patients, including 20 (4.4%) grade 1, 34 (7.5%) grade 2, and 33 (7.3%) grade 3. FES was diagnosed in 35 (7.7%) patients. PEX was found in 23 (5.1%) patients. On multivariate analysis, FES (P<0.001), pseudo exfoliation (P=0.017), intracameral dilatation (P<0.004), senior surgeons (P=0.009) and α1-ARA (P<0.001) were associated with IFIS. Elastin fibers were identified in small amounts in the tarsus samples but not in the iris samples. FES is predictive of IFIS. Surgeons should be aware of this association to prevent intraoperative complications.

Histogenesis of the cornea in sheep fetuses

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

Preparation and characterization of PVA/chitosan nanofibers loaded with Dragon's blood or poly helixan as wound dressings

Nanofibers have been investigated in regenerative medicine. Dragon's blood (DB)- and poly helixan PF (PHPF) are natural materials used in cosmetics. Herein, we generated DB- and PHPF-loaded polyvinyl alcohol/chitosan (PVA/CS/DB and PVA/CS/PHPF, respectively) nanofibers. PVA/CS/DB and PVA/CS/PHPF nanofibers had an average diameter of 547.5 ± 17.13 and 521 ± 24.67 nm, respectively as assessed by SEM, and a degradation rate of 43.1 and 47.6 % after 14 days, respectively. PVA/CS/DB and PVA/CS/PHPF nanofibers had a hemolysis rate of 0.10 and 0.39 %, respectively, and a water vapor transmission rate of ∼2200 g.m−2.day−1. These nanofibers exhibited favorable antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis in vitro. PVA/CS/DB and PVA/CS/PHPF nanofibers demonstrated a sustained release of 77.91 and 76.55 % over 72 h. PVA/CS/DB and PVA/CS/PHPF nanofibers had a high rate of cytocompatibility and significantly improved the viability of NIH/3T3 cells as compared with free drugs or unloaded nanofibers. Histological inspection via H&E and Verhoeff's staining demonstrated PVA/CS/DB and PVA/CS/PHPF nanofibers enhanced the wound healing and damaged tissue recovery of unsplinted wound models by promoting epithelial layer formation, collagen deposition, and enhancing the presence of fibroblasts. Conclusively, PVA/CS/DB and PVA/CS/PHPF can be introduced as potential wound dressing candidates with favorable properties.

MP36-14 MICRORNA-29B ATTENUATES FIBROSIS IN A RAT MODEL OF PEYRONIE’S DISEASE

You have accessJournal of UrologyCME1 Apr 2023MP36-14 MICRORNA-29B ATTENUATES FIBROSIS IN A RAT MODEL OF PEYRONIE’S DISEASE Patrícia Candido, Caroline Chiovatto, Ruan Pimenta, Poliana Romão, Vitória Ghazarian, Juliana Camargo, Vanessa Guimarães, Gabriel Santos, Iran Silva, Bruno Nascimento, Jorge Hallak, Miguel Srougi, William Nahas, Nayara Viana, Katia Leite, Sabrina Reis, and Feres Camargo Maluf Patrícia CandidoPatrícia Candido More articles by this author , Caroline ChiovattoCaroline Chiovatto More articles by this author , Ruan PimentaRuan Pimenta More articles by this author , Poliana RomãoPoliana Romão More articles by this author , Vitória GhazarianVitória Ghazarian More articles by this author , Juliana CamargoJuliana Camargo More articles by this author , Vanessa GuimarãesVanessa Guimarães More articles by this author , Gabriel SantosGabriel Santos More articles by this author , Iran SilvaIran Silva More articles by this author , Bruno NascimentoBruno Nascimento More articles by this author , Jorge HallakJorge Hallak More articles by this author , Miguel SrougiMiguel Srougi More articles by this author , William NahasWilliam Nahas More articles by this author , Nayara VianaNayara Viana More articles by this author , Katia LeiteKatia Leite More articles by this author , Sabrina ReisSabrina Reis More articles by this author , and Feres Camargo MalufFeres Camargo Maluf More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003270.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Peyronie's disease (PD) is characterized by the formation of fibrotic plaques on the tunica albuginea (TA) of the penis. Effective treatments are limited, and surgery remains the best option, although it does not regress the disease. The therapy with microRNAs (miRNAs) is an attractive alternative for several pathologies because they act in the regulation of gene expression. The miRNA-29b regulates extracellular matrix genes, such as collagen, and its decreased expression can be found in fibrotic diseases. We aimed to investigate the therapeutic potential of miRNA-29b in a fibrin-induced rat model of PD. METHODS: To generate PD-like plaques, rats received injections into the TA at 100 µL of each of the human fibrin and thrombin solutions on days 0 and 5. After fibrin injections, 18 rats were randomly divided into three groups (n=6/group): PD+miRNA29b group, PD+miRNA negative group (Scramble), and sham group. Treatment with mimic miRNA-29b or its negative control was performed on days 14, 21, and 28. The mid-penile shaft was removed on day 30 for histologic examination by Hematoxylin and eosin (H&E), Masson trichrome, and Verhoeff staining and qPCR. RESULTS: The establishment of the animal model was confirmed by the histopathological analyses with the identification of fibrosis (Figure 1a-f), underexpression of miRNA-29b (Figure 1g) and overexpression of COL1A1 and TGF-β1 (Figure 1h-j). After treatment, the Fibrosis+miRNA-29b group showed a decreased fibrotic process in the TA (Figure 2a-i) and observed overexpression of miRNA-29b (Figure 2j) and underexpression of genes COL1A1, COL3A1, and TGF-β1 in the PD+miRNA29b group (Figure 2k-m). CONCLUSIONS: Here, we demonstrated for the first time that overexpression of miRNA-29b attenuated fibrosis in the rat model of PD. The results of this study suggest that treatment with miRNA-29b may represent a new effective tool for PD. Source of Funding: Not applicable © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e484 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Patrícia Candido More articles by this author Caroline Chiovatto More articles by this author Ruan Pimenta More articles by this author Poliana Romão More articles by this author Vitória Ghazarian More articles by this author Juliana Camargo More articles by this author Vanessa Guimarães More articles by this author Gabriel Santos More articles by this author Iran Silva More articles by this author Bruno Nascimento More articles by this author Jorge Hallak More articles by this author Miguel Srougi More articles by this author William Nahas More articles by this author Nayara Viana More articles by this author Katia Leite More articles by this author Sabrina Reis More articles by this author Feres Camargo Maluf More articles by this author Expand All Advertisement PDF downloadLoading ...

Topography and Composition of the Iliolumbar Veins in a South African Cohort: Implications for Spinal Surgery.

A descriptive cross-sectional study of the anatomical variations, morphometry, and histology of the iliolumbar veins (ILVs). This study aimed to describe the anatomical variations of the ILVs and determine their tissue composition in South African cadavers of European descent. A safe anterior surgical approach to the L4/L5 intervertebral disc space requires understanding the anatomy of the ILVs. Limited understanding of ILVs and their variations may lead to inadvertent avulsion of veins with subsequent hemorrhage and damage to the adjacent nerves intraoperatively. Variations in ILVs are population specific, but such reports are limited in the South African population. Eighty-nine adult cadavers were dissected to reveal ILV patterns. The variations (origin, course, and drainage pattern), morphometries, and topography of the ILVs were studied. A total of 19 (10 proximal, nine distal) ILVs were processed for hematoxylin and eosin, Masson's trichrome, and Verhoeff's histological staining to determine the tissue composition. The ILVs were identified in 100% of the cases, and 45% of the ILVs were anastomosed to each other bilaterally. The rightside ILVs terminated into the posterior surfaces of the iliac vessels (p =0.001), whereas the left-side ILVs terminated into the lateral surfaces (p =0.001). The left-side proximal ILVs had higher elastic fiber composition (p =0.030). The ratio of the ILVs' elastic fibers to collagen fibers was 1:9, and 61% of the cadavers exhibited type 1 ILV pattern. Moreover, 42% of the ILVs were at the S1 vertebral level with 31% lying between L4 and L5 spinal nerve roots. The obturator nerve coursed anteriorly to the ILVs in 96% of cases. The ILV variations described for South Africans present new additional patterns, such as bilateral anastomosis and laterality of the terminal drainage. The ILVs have more collagen fibers than elastic fibers, predisposing them to avulsion during surgical retraction. The identification of all the ILVs is crucial to minimize inadvertent hemorrhage and damage to adjacent structures.

Perirenal adipose tissues as a human elastin source, and optimize the extraction process.

Elastin is very rarely repaired extracellular matrix (ECM) in physiological condition. The commercial human elastin for exogenous medical treatment is very expensive, and has a potential for disease transmission. Animal-origin elastin is relatively low price, but has concerns for xenogeneic immune responses. Considering cost and safety, we focused on the perirenal adipose tissue, donated from healthy young people via donor nephrectomy. Until now, all of the perirenal adipose tissues are discarded as a medical waste after kidney transplantation. In the present study, we applied perirenal adipose tissues as the source of human elastin, and optimized the extraction process to get high purified and quantified elastin. Through pre-processing step, the delipidated and decellularized ECM was prepared. Next, with four different elastin extraction process (acidic solvents, neutral salt, organic solvents or hot alkali method), elastin was extracted, and the concentration of amino acid between each product was compared, and bright-field/electron microscopy, Fourier transform infrared (FT-IR) spectroscopy and cytotoxicity analysis were also performed. As controls, bovine neck ligament-derived and human skin-derived elastin were used. Among the elastin extraction methods, the hot alkali insoluble product showed (1) relatively high positive area of Verhoeff's and low Masson's trichrome stain, (2) 64.24% purity, 159.29mg/g quantity, and ∼6.37% yield in amino acid analysis, (3) β-sheet second structure, and (4) thin fiber composed mesh-like sheet structure in SEM image. These values were higher than those of the commercial human skin elastin. When comparing hydrolyzed forms, α-elastin from hot alkali insoluble product showed enhanced cell proliferation and maintained cell properties compared to the κ-elastin. Therefore, we confirmed that the perirenal adipose tissue is an ideal source of human elastin with safety assurance, and the hot alkali process combined with pre-process seems to be the optimal method for elastin extraction with high purity and quantity.

Advanced Maternal Age Impairs Uterine Artery Remodelling in the Pregnant Rat

Advanced maternal age (AMA, ≥35 years) pregnancies are increasingly common, and are at increased risk for pregnancy complications. We previously showed that ex vivo uterine artery constriction due to increasing intraluminal pressures (myogenic tone) was greater in arteries of AMA vs. young pregnant rats. Aging impairs vascular reactivity and increases vascular stiffness via structural changes (e.g. increased collagen), however, the impact of AMA on adaptations to pregnancy in the uterine artery is unknown. We hypothesized that functional and structural uterine artery adaptations to pregnancy are impaired in aged compared with young rats.Pregnant young (4 months) and aged (9 months; ~35 years in humans) rats were studied on gestational day 20 (term=22 days) with age‐matched non‐pregnant rats. Uterine arteries were isolated and mounted in a pressure myograph. Active tone (calcium present in buffer, n=6‐10) and mechanical properties (circumferential stress and strain; calcium absent, n=10‐24) were assessed. Myogenic tone, circumferential stress and strain were calculated from active and passive curves. Collagen and elastin in the tunica media and intima (combined) were visualized with Masson’s trichrome (collagen) or Verhoeff’s (elastin) stains (n=4‐6). Data are mean area under the curve (AUC, myography) or % positive area (histology) ± SEM; analyzed by two‐way ANOVA with Sidak post‐hoc tests (significance: p&lt;0.05).Between 4‐100 mmHg, myogenic tone did not change with age or pregnancy. However, arteries from young pregnant rats displayed forced vasodilation at higher pressures (100‐160 mmHg) that did not occur in arteries from other groups (p&lt;0.0001). Circumferential stress and strain (Figure 1) increased with pregnancy (p&lt;0.0001) but was significantly lower in arteries from pregnant AMA compared to young pregnant dams (p&lt;0.05). Collagen and elastin (% positive areas) did not change with age or pregnancy overall, however, arteries from young non‐pregnant rats had greater collagen:elastin ratios than those from aged non‐pregnant rats (p=0.021). Arteries from aged non‐pregnant, young pregnant and AMA rats had similar ratios.This study illustrates that aging impacts both structural and functional adaptations of uterine arteries during pregnancy. The lower compliancy of arteries from AMA rats may be due to increased crosslinking of the existing collagen within the artery, as it was not explained by collagen:elastin ratio. These structural differences may contribute to the functional differences, potentially reducing uteroplacental blood supply. Thus, impaired uterine artery adaptations may increase the risk of pregnancy complications in women of advanced age.

Histopathological Study of Vascular Changes of Placenta in Preeclampsia Using Histochemistry

Context: Preeclampsia is a threat that affects globally, and more so in developing countries. It occurs in 5%–8% of pregnancies worldwide and is the second-most common cause of maternal and fetal death. Aims: To compare vascular changes in vessels of placental stem villi and terminal villi in the placenta in preeclampsia and compare that with placenta from normal pregnancy using special stain such as Masson's trichrome and Verhoeff's stain. Settings and Design: A total of 120 placentas were included in the study. Sixty cases were from preeclampsia patients of which 20 were from severe preeclampsia and 40 from mild preeclampsia and 60 were gestational age matched controls. Subjects and Methods: Placental tissue was examined for gross and microscopic changes. Representative sections were also screened after staining with special stains such as Masson's trichrome and Verhoeff's stain. Statistical Analysis Used: SPSS 22, USA, was used for descriptive and analytical data. Chi-square was the test of significance. P &lt; 0.05 was considered significant. Results: Placenta from preeclampsia showed significantly low placental weight, less diameter, and thickness as compared to control. The stem villi showed thrombosis and medial hypertrophy. All the parameters were statistically significant when compared between the two groups, and these changes were related to the severity of preeclampsia. Conclusions: Vascular changes and products released may be the reason for the pathogenesis, clinical sequelae onset of disseminated intravascular coagulation, maternal inflammatory syndrome, and poor outcome in preeclampsia, which needed further study.

Automated Tissue Strain Calculations Using Harris Corner Detection.

The elastic modulus, or slope of the stress-strain curve, is an important metric for evaluating tissue functionality, particularly for load-bearing tissues such as tendon. The applied force can be tracked directly from a mechanical testing system and converted to stress using the tissue cross-sectional area; however, strain can only be calculated in post-processing by tracking tissue displacement from video collected during mechanical testing. Manual tracking of Verhoeff stain lines pre-marked on the tissue is time-consuming and highly dependent upon the user. This paper details the development and testing of an automated processing method for strain calculations using Harris corner detection. The automated and manual methods were compared in a dataset consisting of 97 rat tendons (48 Achilles tendons, 49 supraspinatus tendons), divided into ten subgroups for evaluating the effects of different therapies on tendon mechanical properties. The comparison showed that average percent differences between the approaches were 0.89% and -2.10% for Achilles and supraspinatus tendons, respectively. The automated approach reduced processing time by 83% and produced similar results to the manual method when comparing the different subgroups. This automated approach to track tissue displacements and calculate elastic modulus improves post-processing time while simultaneously minimizing user dependency.

Anatomical Parameters of Eye and Associated Histological Features are Potential Risk Factors for Development of Pterygium

&lt;p&gt;&lt;strong&gt;Background and Objectives:&lt;/strong&gt; Pterygium occurs throughout the world but the exact pathogenesis is still not clear. The reports on the association between eye anatomical parameters and presence of pterygium is controversial, similarly how the histological features of the pterygium may differ due to these parameters is not known. Thus, the focus of this study was to explore this gap by assessing and comparing the anatomical parameters of eye in pterygium patients and in healthy controls. In addition, histological features of pterygium were assessed and correlated with the eye anatomical parameters of the patients.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Forty-one pterygium patients of age range 25-70 years undergoing surgery were included in the study after taking written informed consent. Forty-two age and sex matched healthy controls were also recruited for assessing anatomical eye parameters. Relevant history with demographic details of every subject was obtained. All participants underwent a thorough ophthalmic examination. Assessment of orbital protrusion and interpalpebral distance was measured by millimeter scale. Tear film breakup time (TFBUT) and Schirmer test was used for tear film assessment. Presence of any meibomian gland dysfunction (MGD) was examined by slit lamp. Post-surgical tissue samples from patients were assessed for histological features with H&amp;amp; E, PAS &amp;amp; Verhoeff stains. Data were processed and analyzed by using SPSS version 21.0.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;Eyeball protrusion and MGD was found higher in pterygium patients as compared to controls. Basement membrane fragmentation on histology significantly correlated with the eyeball protrusion (P&amp;lt;0.04) and TFBUT (P&amp;lt;0.020), inflammation significantly correlated with the MGD (P&amp;lt;0.05) while elastosis showed significant correlation with TFBUT (P&amp;lt;0.001).&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; It can be concluded that eye anatomical parameters might be the risk factors in the development of pterygium. The correlation between certain histological features &amp;amp; eye anatomical parameters indicate that anatomical eye parameters can be taken as risk factor for recurrence in these patients.&amp;nbsp;&lt;/p&gt;

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.

Electronic Blood Vessel

Summary Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface.

Unique wreath-like smooth muscle proliferation of the pulmonary vasculature in pulmonary veno-occlusive disease versus pulmonary arterial hypertension

Pulmonary veno-occlusive disease (PVOD) is a rare but fatal cause of pulmonary hypertension reported to be linked to mutations of eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4), also known as general control nonderepressible 2 kinase (GCN2). PVOD is difficult to diagnose and often initially misdiagnosed as other types of idiopathic pulmonary arterial hypertension (IPAH). To rapidly and correctly identify PVOD patients and explore the possible pathogenesis, we thoroughly investigated histopathological features and GCN2 protein levels in non-PAH, PVOD and PAH patients. Lung specimens were examined for histopathological changes, including those of pulmonary arteries and veins, by Masson's trichrome, modified Verhoeff's and α-SMA staining in the PVOD, IPAH, and non-PAH groups. GCN2 and α-SMA expression in lung tissue was examined by immunohistochemistry and western blotting. PVOD and IPAH patients showed significant intimal and medial thickening of muscular pulmonary arteries compared with non-PAH patients. PVOD patients had more prominent intimal and medial thickening of muscular pulmonary veins than the other two groups. Interestingly, specialized muscle bundles surrounding the tunica adventitia of the pulmonary artery and vein were observed in PVOD patients. A significant decrease in GCN2 expression in the PVOD group was confirmed by immunohistochemistry and western blotting. Our study is the first to show remarkable histological structures, including the wreath-like arrangement of a hyperplastic muscle bundle in the adventitia of pulmonary arteries, in PVOD patients as a diagnostic clue and to disclose the biological difference between PAH and PVOD in a Taiwanese population.

To Explore the Pathogenesis of Vascular Lesion of Type 2 Diabetes Mellitus Based on the PI3K/Akt Signaling Pathway.

Background Type 2 diabetes mellitus (T2DM) has become a chronic disease, serious harm to human health. Complications of the blood pipe are the main cause of disability and death in diabetic patients, including vascular lesions that directly affects the prognosis of patients with diabetes and survival. This study was to determine the influence of high glucose and related mechanism of vascular lesion of type 2 diabetes mellitus pathogenesis. Methods In vivo aorta abdominalis of GK rats was observed with blood pressure, heart rate, hematoxylin and eosin (H&E), Masson, and Verhoeff staining. In vitro cells were cultured with 30 mM glucose for 24 h. RT-QPCR was used to detect the mRNA expression of endothelial markers PTEN, PI3K, Akt, and VEGF. Immunofluorescence staining was used to detect the expression of PTEN, PI3K, Akt, and VEGF. PI3K and Akt phosphorylation levels were detected by Western blot analysis. Results Heart rate, systolic blood pressure, diastolic blood pressure, and mean blood pressure in the GK control group were higher compared with the Wistar control group and no difference compared with the GK experimental model group. Fluorescence intensity of VEGF, Akt, and PI3K in the high-sugar stimulus group was stronger than the control group; PTEN in the high-sugar stimulus group was weakening than the control group. VEGF, Akt, and PI3K mRNA in the high-sugar stimulus group were higher than the control group; protein expressions of VEGF, Akt, and PI3K in the high-sugar stimulus group were higher than the control group. PTEN mRNA in the high-sugar stimulus group was lower than the control group. Protein expression of PTEN in the high-sugar stimulus group was lower than the control group. Conclusions Angiogenesis is an important pathogenesis of T2DM vascular disease, and PTEN plays a negative regulatory role in the development of new blood vessels and can inhibit the PI3K/Akt signaling pathway.

Histomorphometry of umbilical cord and its vessels in natural and assisted reproduction

Introduction and Aim: Assisted reproductive technique (ART) is an invaluable gift for infertile couple who failed in various infertility treatments. However, there are many aftereffects after assisted reproduction like preterm birth, small for gestational age, congenital anomalies etc. Umbilical cord is of fetal origin, which connects the fetus to placenta. Therefore, changes in the histomorphometry of umbilical cord and its vessels may affect the fetus and its growth. Our present study aimed to analyze the histo-morphometric parameters of umbilical cord and its vessels in natural conception and in assisted reproduction. Materials and Methods: 30 placentas (with umbilical cord) of natural conception and 30 placentas (with umbilical cord) of assisted reproduction (ICSI) were collected immediately after delivery in and around Chidambaram. Umbilical cord bits were fixed in buffered formalin and underwent routine tissue processing procedure. 5 to 10 microns of tissue sections were taken and stained in H & E stain, Verhoeff stain and MTS stain. Histo- morphometric parameters of umbilical cord and its vessels were measured in Olympus microscope using ocular micrometer. Results: There was a reduction in the areas of umbilical cord and its vessels in assisted reproduction when compared to natural conception. Conclusion: These results would be useful for neonatologists who handle the newborns of ART mothers. Keywords: Umbilical cord; umbilical artery; umbilical vein; histomorphometry; connective tissue.

Cutaneous melanoma: a study on the updated pathologic stage, solar elastosis, and tumor-infiltrating lymphocytes

Background Melanoma ranks as the third most common cutaneous malignancy, but it is the most lethal if early surgical treatment is neglected. Recently, updates were made on the American Joint Committee on Cancer staging system, as well as guidelines for surgical excision. Previously, grading systems were made to measure cumulative damage (solar elastosis) and to evaluate the immune reaction against the tumor [tumor-infiltrating lymphocytes (TIL)]. Aim To apply the fore mentioned advances to a series of 146 Egyptian patients primarily treated by surgery. The histochemical stains used for solar elastosis included Verhoeff stain and Alcian blue stain, pH 2.5. For phenotyping of lymphocytes and natural killer cells, the following immune markers were used: CD3, CD8, CD4, and CD56. Results Most tumors (64.4%) presented with an advanced size (>4 mm), lymph node metastases (66.7%), and inadequate excision margin (66.4%). In association analysis, only age more than 50 years, head and neck location, and the superficial spreading histologic type were significantly associated with solar elastosis. Moreover, only old age (>50 years) and large tumor thickness (>4 mm) are associated with low level of TIL, denoting poor immune reaction. Conclusion The presentation of Egyptian patients with advanced stages of melanoma calls for the implementation of public education program including self-examination of skin to detect melanoma at an early stage. Moreover, prospective follow-up studies are needed to develop guidelines of surgical excision suitable for our advanced cases. The negativity of both solar elastosis and TIL in melanoma of lower extremity supports the trauma of combined immunodeficiency and trauma in oncogenesis at this location.

Porcine Mesothelium-Wrapped Diced Cartilage Grafts for Nasal Reconstruction.

Fascia-wrapped diced cartilage grafts have become a useful tool in modern rhinoplasty surgery. Unfortunately, fascial harvest is associated with donor site morbidity; therefore, a nonautologous alternative to fascia would be ideal. Decellularized porcine mesothelium (PM), Meso BioMatrix™, is an acellular scaffold that could potentially fill this need. To determine if PM could serve as an acceptable alternative, we histologically compared diced cartilage grafts wrapped in fascia versus PM. Human rib cartilage and temporoparietal fascia were obtained under an IRB-approved protocol. Cartilage was diced into 0.5 mm pieces and implanted in subcutaneous pockets in nude rats. Implanted materials included cartilage alone, cartilage wrapped in fascia, cartilage wrapped in PM, fascia alone, or PM alone. Specimens were harvested at 8 weeks and stained with hematoxylin and eosin, Masson's trichrome, Safranin-O, and Verhoeff's stain to assess cartilage viability, architecture, and regenerative potential. Unwrapped diced cartilage showed the highest cartilage viability, but was associated with loss of contour and dispersion of the cartilage pieces. Meso BioMatrix-wrapped grafts maintained contour and cartilage pieces had not dispersed; however, there was a significantly lower number of nucleated lacunae and a greater amount of basophilia than both fascia-wrapped cartilage and unwrapped cartilage. There was no significant difference in cartilage resorption between fascia-wrapped cartilage and Meso BioMatrix-wrapped cartilage or in the proteoglycan or collagen content between all groups. Off-the-shelf decellularized PM was associated with lower cartilage viability than unprocessed fascial allograft. No cartilage piece dispersion, fibrosis, resorption, or a foreign body reaction to Meso BioMatrix was observed. PM, although not equivalent to autologous tissue, may be utilized to achieve acceptable clinical results and be a viable alternative that limits donor side morbidity. This experimental study supports further clinical investigation of this material in rhinoplasty procedures.

Age-related histologic and biochemical changes in auricular and septal cartilage.

To characterize the histologic and biochemical properties of auricular and septal cartilage and analyze age-related changes in middle-aged to older adults. Cross-sectional study of auricular and septal cartilage from 33 fresh cadavers. Auricular and septal cartilage specimens were stained using Safranin O for glycosaminoglycans, Verhoeff's stain for elastin, and Masson's trichrome for collagen. Percentage of tissue stained, cell density and size were quantified. Relationships between donor characteristics and histologic properties were evaluated using mixed model analyses. The average donor age was 75 years (standard deviation = 11 years; range, 55-93 years). In auricular cartilage, each 1-year increase in age was associated with a 0.97% decrease in glycosaminoglycans (P < .001) and a 0.98% decrease in elastin (P < .001). In septal cartilage, glycosaminoglycans decreased 2.4% per year (P < .001). Age did not affect collagen content significantly in auricular (P = .417) or septal cartilage (P = .284). Cell density and cell size declined with age in auricular (both P < .001) and septal cartilage (P = .044, P = .032, respectively). Compared to septal cartilage in patients of all ages, auricular cartilage had more glycosaminoglycans, less collagen, higher cell density, and smaller cells. In auricular and septal cartilage, glycosaminoglycans, elastin, cell density, and cell size decrease significantly with age in patients over 55 years of age. Glycosaminoglycan content declines faster with age in septal cartilage than auricular cartilage. These age-related changes may affect biomechanical properties and tissue viability, and thereby have implications for graft choice in functional, aesthetic, and reconstructive nasal surgery. NA. Laryngoscope, 127:E399-E407, 2017.

Changes in CD4+CD25+ Tregs in the pathogenesis of atherosclerosis in ApoE-/- mice.

The goal of this study was to observe the pathological characteristics of atherosclerotic plaques in the aortic walls of ApoE-/- and C57BL/6J mice and the changes of CD4+CD25+ regulatory T cells (Tregs) in atherosclerotic mice. Twenty ApoE-/- mice were split into high-fat diet (AH) and normal diet (AN) groups and 10 C57BL/6J male mice were designated as the control group (BN). The serum concentrations of IL-10 and TGF-β1 were detected by enzyme-linked immunosorbent assay; paraffin sections of the aorta were stained with hematoxylin & eosin, and morphometric parameters were measured using the Image Pro Plus 6.0 system. Verhoeff stain was used to observe the distribution of elastic fibers, and immunohistochemical staining was performed to verify the phenotype of the forkhead box protein 3 (Foxp3+) CD25+ cells in the atherosclerotic tissue. The proportion of CD4+CD25+ Tregs in the spleen was calculated by flow cytometry. The thickness of the intima, the intima/media ratio, the plaque area, and the plaque/lumen ratio of mice in AN group were significantly larger than those of mice in BN group. The thickness of the intima, the plaque area, and the plaque/lumen ratio of the mice in AH group were significantly increased compared with those of the AN group mice. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4+CD25+ Tregs in AN group mice were significantly decreased compared with the control group. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4+CD25+ Tregs in the mice in AH group were significantly decreased compared with those in AN group. The proportions of Foxp3+ and CD25+ cells within the total lymphocyte population were significantly decreased in AH group mice compared with those in AN group mice. Atherosclerosis in an experimental mouse model was correlated with Treg depletion in the lymphoid tissues and plaques, indicating the important antiatherosclerotic role of CD4+CD25+ Tregs. Impact statement In this article, we conclude that Tregs decreased with atherosclerosis (AS) as determined in ApoE knockout mice fed a high fat diet. It is an important matter for understanding the AS pathology.

Histological analysis of retraction pocket pars tensa of tympanic membrane in children

AimsHistological and histochemical analysis of retraction pocket of pars tensa of tympanic membrane in children. Identification of morphological abnormalities in comparison with a healthy tympanic membrane as it is described in standard textbook. Identification of signs typical for cholesteatoma and support for a retraction theory of cholesteatoma formation. Study designA prospective study analysing 31 samples of retraction pockets taken during surgery. DepartmentsUniversity Hospital, Children's Medical Centre MethodsSamples of retraction pockets were processed by a standard process for light microscopy, stained by haematoxylin-eosin. Van Gieson's stain was used for differential staining of collagen, Verhoeff's stain for elastic fibre tissues, Alcian blue for acidic polysaccharides and PAS (Periodic Acid Schiff) method for basement membrane polysaccharides. ResultsThe following findings were observed in the samples of retraction pockets: hyperkeratosis (100%), hypervascularisations (100%), subepithelial fragmented elastic fibres (96%), myxoid changes (87%), subepithelial inflammatory infiltration (84%), rete pegs (71%), papilomatosis (71%), intraepithelial inflammatory cellularizations, (48%), intraepithelial spongiosis (16%) and parakeratosis (3%). No basement membrane continuity interruptions were observed. Thickness of retraction pocket, thickness of epidermis, occurrence of rete pegs and frequency of fragmented elastic fibres was higher in a Grade III stage RP than Grade II stage RP (according to Charachon). ConclusionMorphological abnormalities in the structure of retraction pockets in comparison with a healthy tympanic membrane were described. The changes are typical for a structure of cholesteatoma (these changes are common in matrix and perimatrix), supporting retraction theory of its origin. Our observations show that it is inflammation that probably plays a key role in the pathogenesis of retraction pocket. The frequency of some of the changes increases with the stage of retraction pocket (II–III according to Charachon). Basement membrane continuity interruptions are not typical for retraction pockets.