Verhoeff-Van Gieson Staining Research Articles

The Labiomandibular Fold Anatomy for Comprehensive Lower Facial Rejuvenation: A Micro-Computed Tomography Investigation.

Perioral rejuvenation is challenging due to the lack of spatial anatomical understanding of the labiomandibular fold (LMF). The LMF's formation mechanism remains underexplored due to intricate relationships between musculature and subcutaneous structures. This study aimed to clarify the three-dimensional structures of the LMF using micro-computed tomography and histology. Twenty-two rectangular specimens, following the LMF as the diagonal line, were retrieved bilaterally from eleven fresh frozen cadavers (mean age, 69.3 years). Eleven specimens were imaged with phosphotungstic acid contrast staining for micro-computed tomography, and the rest were histologically examined using Masson and Verhoeff-van Gieson staining. The LMF was divided into three equal-width regions for analysis. The LMF serves as a border between the lateral fat-abundant zone and the medial fat-scant zone statically. Dynamically, the LMF is accentuated with age by the downward pull of depressor muscles via a compact slanted collagen-elastin-muscle (CEM) network composed of the depressor anguli oris, platysma, and buccinator muscle fibers. The LMF formation is governed by subcutaneous composition differences between the lower lip and cheek region and vertical dermal insertions of mimetic muscles. Perioral muscle contractions transmit directly at the skin via the CEM network, governing regional wrinkle severity and appearance. The micro-CT findings provide crucial three-dimensional insights into the layered architecture and intricate musculature correlation of the LMF region, providing regional-specific evidence for holistic rejuvenation practices. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Abstract 4125938: Novel Surgical Model of Thoracic Aortic Aneurysm Mediated by Vasa Vasorum Inhibition

Objective: The adventitial layer of the aorta contains the network of microvessels known as vasa vasorum. Previous work by our team revealed deficient vasa vasorum associated with medial hypoxia and upregulated adventitial thrombospondin-1 (TSP-1), an anti-angiogenic molecule, in human aortic aneurysm. We hypothesize that heightened TSP-1 in the ascending thoracic aorta induces aneurysmal degeneration through its anti-angiogenic effects on the vasa vasorum. We tested this hypothesis in a rabbit model via peri-adventitial delivery of ABT-510, a thrombospondin peptide mimetic. Methods: The ascending aorta was exposed in New Zealand White rabbits (n=16) and treated with ABT-510 proximally and a vehicle control distally. Echocardiography was undertaken immediately prior to the procedure and 14 days post-operatively. Adventitial vasa vasorum were quantified from H&E-stained sections. Elastin and collagen organization were assessed with Verhoeff-Van Gieson (VVG) and pentachrome staining as well as multiphoton microscopy. Immunodetection of Hypoxyprobe™ and alpha smooth muscle actin (αSMA) were also undertaken to identify hypoxia and smooth muscle cell content, respectively. Results: Echocardiography revealed an increase in aortic diameter over 14 days with ABT-510 treatment compared with control (42.27% ± 5.54 vs 20.99% ± 4.10, p=0.004). Histological analysis showed decreased vasa vasorum count (44.7 ± 9.19 vs 85.8 ± 12.89, p=0.048) and decreased vasa vasorum indexed cumulative area (0.013 ± 0.0037 vs 0.021 ± 0.0050 µm 2 / µm 2 adventitial area, p=0.019). Immunodetection of Hypoxyprobe™ showed evidence of increased hypoxia. VVG, pentachrome, and immunodetection of αSMA showed decreased elastin content, increased deposition of ground substance, and areas of smooth muscle cell loss . Multiphoton microscopy demonstrated fragmented elastin fiber microarchitecture and decreased collagen content (Fig 1). Conclusions: Our study findings demonstrate a novel, clinically relevant model for thoracic aortic aneurysm through vasa vasorum inhibition that recapitulates features of cystic medial degeneration. This translational model could enable development of targeted, pro-regenerative therapies that restore perfusion to the aortic wall.

Identification of a Regenerative Protocol for Recellularizing Human Auricular Cartilage Scaffolds.

Utilizing biological scaffolds for cartilage tissue engineering is a promising tool for improving auricular reconstruction. Decellularized auricular scaffolds provide a means of regenerating cartilage for in vivo implantation, but identifying the ideal regenerative mix remains challenging. Human cadaver auricular cartilage was decellularized and recellularized with either auricular chondrocytes alone, auricular chondrocytes with adipose-derived stem cells, or both cells with platelet-rich plasma. Confirmation of decellularization and recellularization was done by hematoxylin and eosin staining. Extracellular matrix preservation and production were determined by Masson's trichrome, Alcian blue, and Verhoeff-van Gieson staining. Collagen II assessments were made using immunohistochemistry. Decellularization of cadaver auricular cartilage was confirmed by the absence of cells, reduction in glycosaminoglycans, and the preservation of collagen and elastin. Recellularization was more efficient when chondrocytes were seeded with adipose-derived stem cells, which was enhanced by adding platelet-rich plasma. Coculture with platelet-rich plasma yielded better total collagen (56% increase) and glycosaminoglycan (47% increase) induction. Moreover, when platelet-rich plasma was added, collagen II induction was significantly increased (42%; P < 0.05). We identified a regenerative protocol that included auricular chondrocytes, adipose-derived stem cells, and platelet-rich plasma, which stimulated chondrogenesis on decellularized auricular cartilage. This finding provides a model to explore cartilage formation and the potential for improving auricular and cartilage-based reconstruction.

Cross-sectional study to evaluate the utility of elastic tissue staining in primary cicatricial alopecia

Background and aimsDiagnosing end-stage primary cicatricial alopecia (PCA) on routine histology is challenging since the major diagnostic feature (inflammatory infiltrate) may be minimal or absent. This study aimed to assess...

Abstract P453: Dissecting tunica responsibility in arterial stress relaxation: Smooth muscle need not apply

Perivascular adipose tissue (PVAT) is being recognized as an important arterial tunica. Stress relaxation, a gradual reduction in stress over time while under a constant strain, is considered a property of smooth muscle (ex. intestine, uterus, bladder). While aortic PVAT (aPVAT) contains a microvasculature, it does not contain organized smooth muscle and isolated PVAT stress relaxes to a greater extent than the vessel itself. This raised the idea that other tunicas lacking smooth muscle may also stress relax. Here we hypothesize that appropriately oriented and functional smooth muscle was not necessary for stress relaxation to occur. To test this, we used histochemical staining and isometric contractility of thoracic aorta from the male Dahl salt-sensitive (SS) rat. Some rings of aorta were separated into adventitia (no muscle), media (muscle), and PVAT (no muscle), and responses of these isolates compared to the intact aortic ring (muscle + integration of tunicas). Masson Trichrome (MT) and Verhoeff Van Gieson (VVG) staining validated the dissection of the different tunicas with minimal contamination of cells from other tunicas. Stress relaxation was measured after increasing, cumulative amounts of passive tension (6 grams total) was applied in the presence or absence of Ca 2+ , essential for smooth muscle contraction. Each step was followed by challenge with a maximum concentration of the a 1 adrenergic agonist phenylephrine (PE, 10 -5 M). Stress relaxation occurred in all tunicas at each passive tension, whether or not Ca 2+ was present (figures). In fact, loss of Ca 2+ improved stress relaxation in adventitia and PVAT. Importantly, in the presence of Ca 2+ , PE contraction was observed at each passive tension in tissues containing smooth muscle: media (min, 250 mg PT: 26.67 +/- 8.51 mg; max, 6000 mg PT: 589.59 +/- 114.53 mg) and aorta + PVAT (min, 250 mg PT: 562.61 +/- 238.45 mg; max, 6000 mg PT: 1322.91 +/- 352.73mg). Little to no measurable contraction was recorded in the adventitia and aPVAT. There was little to no measurable contraction in tunicas in the absence of Ca 2+ . Taken together, these data suggest that all tunicas of the rat thoracic aorta stress relax and can do so without the presence of organized smooth muscle. Of all tunicas, PVAT demonstrated the greatest ability to stress relax. Collectively, these findings force reconsideration of individual responsibilities of arterial tunicas, all of which participate in the (dys)function of an artery.

Nesfatin-1 inhibits cerebral aneurysms by activating Nrf2 and inhibiting NF-κB signaling.

Cerebral aneurysm (CA) has been considered one of the most common cerebrovascular diseases, affecting millions of people worldwide. A therapeutic agent is currently missing for the treatment of CA. Nesfatin-1 (Nes-1) is an 82-amino acid adipokine which possesses a wide range of biological functions. However, the physiological function of Nes-1 in CA is still unknown. Here, we aimed to assess the preventive effects of Nes-1 in the pathological development of CA and elucidate the mechanisms behind this. We used an elastase-induced CA model, accompanied by a high-salt diet to induce hypertension. Additionally, diverse experimental techniques, including Verhoeff-Van Gieson staining, real time PCR, enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence staining, were employed to assess CA formation, gene and protein expression, as well as the macrophage infiltration. Our results indicate that administration of Nes-1 significantly decreased the aneurysm size. Additionally, Nes-1 prevented inflammatory response by inhibiting the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) at both the mRNA and protein levels in the Circle of Willis (COW) region. Also, the increased levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in the COW region were reduced by Nes-1. We found that Nes-1 administration suppressed the invasion of macrophages. Mechanistically, Nes-1 activated Nrf-2 by promoting its nuclear translocation but prevented the activation of the IκBα/NF-κB signaling pathway. These findings suggest that Nes-1 might be used as a promising agent for the prevention of CA.

Tunnel technique and subepithelial connective tissue graft, with or without cross-linked hyaluronic acid, in the treatment of multiple gingival recessions: 12-month outcomes of a randomized clinical trial.

This study evaluated the influence of the adjunctive application of a cross-linked hyaluronic acid (HA) in the treatment of multiple gingival recessions, using a modified coronally advanced tunnel (MCAT) technique and subepithelial connective tissue graft (SCTG) (MCAT+SCTG±HA). A randomized, split-mouth, double-masked comparison of the effects of MCAT+HA+SCTG (test) versus MCAT+SCTG (control) in the treatment of multiple, contralateral gingival recessions with clinical, esthetic, and histological evaluations was carried out. All samples were stained with hematoxylin and eosin, Masson's trichrome, Verhoeff-Van Gieson, and Alcian blue stain for semiquantitative evaluation. The primary outcome variable was 12-month mean root coverage (MRC). Twenty-four patients with 266 gingival recessions received both control and test treatments (133 recessions per group). 12-month MRC of the MCAT+HA+SCTG group was not significantly different from the MCAT+SCTG group with 84.32%±34.46% and 85.71%±36.43%, respectively (p=0.991). Both treatment modes produced favorable esthetic outcomes (root coverage esthetic score [RES] 9.51±1.01 tests vs. 9.26±1.10 controls, p=0.7292). However, the application of HA improved soft tissue texture (p=0.0091). The remaining end point measures did not differ significantly between groups. Histological evaluation showed a significantly greater number of elastic fibers and a moderate increase in collagen fiber density in biopsy samples taken from the test sides when compared to the control sides (p=0.0419 and p=0.300, respectively). MCAT+SCTG is an effective procedure in the treatment of multiple recession Type 1 (RT1) and RT2 recessions. There were no statistically significant differences in evaluated clinical treatment outcomes in the MCAT+HA+SCTG group compared to the MCAT+SCTG group within a period of 12 months. The application of HA increased collagen and elastic fiber density.

Comparison of three techniques for decellularization of porcine bone-tendon-bone grafts

ABSTRACT Anterior cruciate ligament injuries are frequent afflictions related to sports or physical trauma. Autograft reconstruction strategies cause secondary injury to the patient. One alternative, supported by clinical evidence, is porcine xenografts. For clinical use, xenografts must be conditioned to avoid immune rejection. The most widely accepted procedure is tissue decellularization. We analyzed three decellularization strategies: the application of the anionic detergent sodium dodecyl sulfate (SDS), sonication, and freezing and thawing cycles. The treated tissues were evaluated histologically using H&E, Masson’s trichrome, Verhoeff-van Gieson staining, and DAPI for fluorescent staining of nuclei. Finally, collagen fiber preservation was evaluated by quantifying this protein by colorimetry. The most efficient decellularization techniques were sonication and SDS. Collagen fibers were preserved in all experimental conditions.

USF1 transcriptionally activates USP14 to drive atherosclerosis by promoting EndMT through NLRC5/Smad2/3 axis

BackgroundEndothelial-to-Mesenchymal Transformation (EndMT) plays key roles in endothelial dysfunction during the pathological progression of atherosclerosis; however, its detailed mechanism remains unclear. Herein, we explored the biological function and mechanisms of upstream stimulating factor 1 (USF1) in EndMT during atherosclerosis.MethodsThe in vivo and in vitro atherosclerotic models were established in high fat diet-fed ApoE−/− mice and ox-LDL-exposed human umbilical vein endothelial cells (HUVECs). The plaque formation, collagen and lipid deposition, and morphological changes in the aortic tissues were evaluated by hematoxylin and eosin (HE), Masson, Oil red O and Verhoeff-Van Gieson (EVG) staining, respectively. EndMT was determined by expression levels of EndMT-related proteins. Target molecule expression was detected by RT-qPCR and Western blotting. The release of pro-inflammatory cytokines was measured by ELISA. Migration of HUVECs was detected by transwell and scratch assays. Molecular mechanism was investigated by dual-luciferase reporter assay, ChIP, and Co-IP assays.ResultsUSF1 was up-regulated in atherosclerosis patients. USF1 knockdown inhibited EndMT by up-regulating CD31 and VE-Cadherin, while down-regulating α-SMA and vimentin, thereby repressing inflammation, and migration in ox-LDL-exposed HUVECs. In addition, USF1 transcriptionally activated ubiquitin-specific protease 14 (USP14), which promoted de-ubiquitination and up-regulation of NLR Family CARD Domain Containing 5 (NLRC5) and subsequent Smad2/3 pathway activation. The inhibitory effect of sh-USF1 or sh-USP14 on EndMT was partly reversed by USP14 or NLRC5 overexpression. Finally, USF1 knockdown delayed atherosclerosis progression via inhibiting EndMT in mice.ConclusionOur findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

Special Staining and Protein Expression of VEGF/EGFR and P53/NF-κB in Cryptorchid Tissue of Erhualian Pigs.

Erhualian pigs exhibit one of the highest reproductive rates globally, and cryptorchidism is a crucial factor affecting reproductive abilities of boars. This investigation focused on cryptorchid tissues from Erhualian pigs, where the histological structure of cryptorchidism was observed using specialized staining. In addition, protein expression of P53/NF-κB in cryptorchid tissues was assessed using Western blot and immunohistochemistry. In comparison to normal Erhualian testes, Masson's trichrome staining indicated a reduction in collagen fibers in the connective tissue and around the basal membrane of the seminiferous tubules in cryptorchid testes. Moreover, collagen fiber distribution was observed to be disordered. Verhoeff Van Gieson (EVG) and argyrophilic staining demonstrated brownish-black granular nucleoli organized regions in mesenchymal cells and germ cells. When compared to normal testicles, the convoluted seminiferous tubules of cryptorchids exhibited a significantly reduced number and diameter (p < 0.01). Notably, VEGF/EGFR and P53/NF-κB expression in cryptorchidism significantly differed from that in normal testes. In particular, the expression of VEGF and P53 in cryptorchid tissues was significantly higher than that in normal testes tissues, whereas the expression of EGFR in cryptorchid tissues was significantly lower than that in normal testes tissues (all p < 0.01). NF-κB expressed no difference in both conditions. The expressions of VEGF and NF-κB were observed in the cytoplasm of testicular Leydig cells and spermatogenic cells, but they were weak in the nucleus. EGFR and P53 were more positively expressed in the cytoplasm of these cells, with no positive expression in the nucleus. Conclusion: There were changes in the tissue morphology and structure of the cryptorchid testis, coupled with abnormally high expression of VEGF and P53 proteins in Erhualian pigs. We speculate that this may be an important limiting factor to fecundity during cryptorchidism.

A comprehensive evaluation of spontaneous pelvic organ prolapse in rhesus macaques as an ideal model for the study of human pelvic organ prolapse

Pelvic organ prolapse (POP) seriously affects a woman’s quality of life, and the treatment complications are severe. Although new surgical treatments are being developed, the host tissue responses and safety need to be evaluated in preclinical trials. However, there is a lack of suitable animal models, as most quadrupeds exhibit different structural and pathological changes. In this study, 72 elderly rhesus macaques (Macaca mulatta) were physically examined, and the incidence of spontaneous POP was similar to that in humans. The vaginal wall from five control monkeys and four monkeys with POP were selected for further analysis. Verhoeff-van Gieson staining showed that elastin content decreased significantly in monkeys with POP compared with control samples. Immunohistological staining revealed that the smooth muscle bundles in monkey POP appeared disorganized, and the number of large muscle bundles decreased significantly. The collagen I/III ratio in monkey POP also significantly decreased, as revealed by Sirius Red staining. These histological and biochemical changes in monkeys with POP were similar to those in humans with POP. Moreover, we generated a single-cell transcriptomic atlas of the prolapsed monkey vagina. Cross-species analysis between humans and monkeys revealed a comparable cellular composition. Notably, a differential gene expression analysis determined that dysregulation of the extracellular matrix and an immune disorder were the conserved molecular mechanisms. The interplay between fibroblasts and macrophages contributed to human and monkey POP. Overall, this study represents a comprehensive evaluation of spontaneous POP in rhesus macaques and demonstrates that monkeys are a suitable animal model for POP research.

Aerobic Exercise versus Electronic Cigarette in Vascular Aging Process: First Histological Insight.

Smoking is related to vascular aging. However, the hazardous effect of e-cigarette is often debatable, with limited studies available. In contrast, moderate-intensity aerobic exercise is well known to decrease aortic stiffness. We provide novel research to determine the effect of e-cigarette and aerobic moderate-intensity exercise on the aortic structure of Wistar rats. A total of 26 male Wistar rats (Rattus norvegicus) 8 weeks aged, 200-250 g b.w., were randomly divided into 4 groups, namely, K0 (normal rats), K1 (rats were given moderate-intensity aerobic exercise by animal treadmill 20 m/30 min), K2 (rats were given e-cigarette with 6 mg nicotine, 40% propylene glycol, and 60% vegetable glycerine 30 min for 5 days/week), and K3 (rats were given e-cigarette and moderate-intensity aerobic exercise). After exposure for 6 weeks, all animals were sacrificed to isolate the aorta for histopathological analysis with hematoxylin-eosin stain to evaluate the elastic fiber layer and intimal-medial thickness. The Verhoeff-Van Gieson staining was done for quantification elastic lamina fragmentation. Our study found that the e-cigarette group had the highest elastic lamina fragmentation among groups (8.14 ± 2.85). The exercise only group showed the lowest elastic lamina fragmentation (2.50 ± 1.87). Fragmentation in the e-cigarette and exercise group was higher than in the exercise only group (5.83 ± 0.753 vs. 2.50 ± 1.87, p = 0.002). There is a significant difference of NO serum between four groups. The result of post hoc analysis using LSD showed that there is a significant difference of NO serum between K0 and K2, K0 and K3, K1 and K2, and K1 and K3. Therefore, our research demonstrated that the most injury of aorta elastic lamina was in the group that was exposed to e-cigarette that leads to vascular aging while exercise is not yet proven to reverse this effect.

Treatment with imatinib was useful to delay the neointimal hyperplasia of aortocaval fistula in adenine-induced renal failure rats

PurposeThe study was conducted to investigate the effect of the treatment with imatinib, a c-kit specific inhibitor, on the neointimal hyperplasia (NIH) of aortocaval fistula (ACF) in adenine-induced renal failure rats. Materials and methodsAll rats were randomly assigned to 4 groups: rats were fed on a normal diet (normal group); rats were fed on a 0.75% adenine-rich diet (renal failure group). The remaining rats underwent ACF after receiving a 0.75% adenine-rich diet and received daily saline gavage (model group) or imatinib gavage (imatinib group) for 7 days after surgery. Immunohistochemical method was used to detect c-kit expression, and Elastomeric Verhoeff-Van Gieson (EVG) staining was used to observe morphological changes of the ACF. The Pearson correlation analysis was used to evaluate the correlations of c-kit expression with intimal thickness and the percentage of stenosis, respectively. ResultsThe renal failure group showed positive c-kit expression on the intima of the inferior vena cava (IVC), whereas the normal group did not. Compared to the model group, intimal thickness (P = 0.001), the percentage of stenosis (P = 0.006) and c-kit expression (P = 0.04) were decreased in the imatinib group at 8 weeks postoperatively. C-kit expression was positively correlated with both intimal thickness and percentage of stenosis (intimal thickness: R = 0.650, P = 0.003; the percentage of stenosis: R = 0.581, P = 0.011) in both the model and imatinib groups. ConclusionTreatment with imatinib, a c-kit specific inhibitor, was useful to delay the NIH of ACF in adenine-induced renal failure rats.

Configuration of elastin fibers in the intra- and extra-capsule ligaments of the elderly cricoarytenoid joint

PurposeTo define the localization and configuration of the elastic fibers of the cricoarytenoid ligament (CAL) and their relationship with the cricoarytenoid joint (CAJ) capsule.MethodsTwenty-four CAJs from twelve cadavers were analyzed using Verhoeff-Van Gieson staining, and immunohistochemistry methods. This is a prospective study.ResultsThe CAL was classified into two parts: an extra-capsular anterior-CAL and an intra-capsular posterior-CAL. The both parts contained rich elastic fibers. The elastic fibers of the anterior-CAL were orientated in both anterior–posterior and superior–inferior directions and under a relaxation status, whereas the elastic fibers of the posterior-CAL were arranged in a lateral–medial direction and under a taut status.ConclusionsThis study defined the fine configuration of the CAL, particularly its elastic fibers, which may help us to better understand the biomechanics of the CAJ motions, and differential diagnosis of CAJ disorders. The results of the study re-confirm that the P-CAL is the key posterior–lateral passive force to limit the mobility of the muscular process of the arytenoid cartilage and stabilize the CAJ, whereas the A-CAL may protect the CAJ from an over superior–lateral–posterior motion.Level of evidence: H/A.

Multiple coronally advanced flap with a selective use of connective tissue graft: A 3-year prospective clinical and histological study.

The purpose of the present study was to prospectively evaluate the 3-year changes in the gingival dimensions following multiple coronally advanced flap (MCAF) with selective use of connective tissue graft (CTG). In addition, the secondary aim was to histologically identify the factors related to phenotype changes. Twenty patients treated with MCAF and site-specific application of a CTG were available for the 3-year follow-up. Outcome measures included complete root coverage (CRC), recession reduction, keratinized tissue width (KTW), marginal tissue thickness changes, and primary flap position. Biopsies were harvested at one of the sites treated with the adjunct of CTG. All sections were stained with hematoxylin and eosin, Masson trichrome, Verhoeff-van Gieson, tenascin, and alcian blue stain for semiquantitative evaluation. At 3 years, CRC was detected in 86% of sites treated with MCAF alone and 81% of sites treated with MCAF+CTG. The 47% of sites treated with MCAF+CTG presented an apical shift of primary flap from its original position. Linear regression showed a significant association between KTW change and the initial KTW in MCAF-treated sites, while both initial KTW and position of primary flap were statistically significantly associated factors with KTW changes in the MCAF+CTG group. In all the biopsies examined, there is always a marked and clear separation between the connective tissue of the gingival flap and the palatal connective tissue of the graft. The selective use of CTG is an effective treatment for multiple gingival recessions. Only a limited increase in KTW can be expected in a bilaminar technique if, during the healing phases, the connective tissue is maintained completely covered.

Verhoeff van Gieson staining in laser-irradiated gingival tissues

ABSTRACT There have been several studies on the use of the Verhoeff van Gieson staining method to demonstrate thermal effects on tissues. However, this method has rarely been used for the analysis of periodontal tissues. This study was undertaken to compare the quality and effectiveness of the Verhoeff van Gieson (VVG) staining method with conventional hematoxylin & eosin (H&E) in measuring the thermal effects in gingival tissues. Periodontal tissues around bovine mandibular teeth were treated using different surgical lasers (wavelengths of 10,600 nm, 970 nm, and 445 nm) at 2 W power setting. Measurements of the depth of the coagulation zone were recorded for all treatment groups in sample tissues stained with H&E as well as the VVG-staining method. Measures were interpreted by a trained pathologist. A statistical analysis was performed using the Wilcoxon signed-rank test to determine if there was a statistically significant difference between values recorded for the light penetration depth on tissues stained with each of the two staining methods. It was determined that there was no significant difference in the recorded values (P = 0.23). We have concluded that the VVG-stained tissues were better able to visualize the depth of thermal damage and thus may make it easier for someone not well trained to interpret the depth of light penetration in these tissues.

Trehalose attenuates abdominal aortic aneurysm formation by inducing autophagy in smooth muscle cells.

Trehalose is a naturally occurring disaccharide, which has been identified as an autophagy inducer and exhibits protective effect in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of trehalose in abdominal aortic aneurysm (AAA) remains undefined. To study the effect of trehalose in AAA, trehalose (1 g/kg per day) were given for 14 continuous days in a mouse model of elastase-induced abdominal aortic aneurysm. On day 14, ultrasound was performed to measure aortic diameter before the abdominal aortas were harvested and processed for further analysis. Verhoeff-Van Gieson staining and TUNEL staining were performed on paraffin sections to evaluate vascular histology and apoptosis, immunofluorescence staining and Western-blot were performed to evaluate expression of autophagy markers. Echocardiography and in situ pictures demonstrated that trehalose attenuated infrarenal aorta dilation. Verhoeff-Van Gieson staining showed elastin degradation was improved in trehalose-treated group. Compared with vehicle-treated mice, trehalose treatment restored smooth muscle cell contractile phenotype with increased α-SMA, Calponin and Myh11 expression. Furthermore, trehalose also attenuated cell apoptosis and leukocytes infiltration. Importantly, trehalose induced autophagy with decrease SQSTM1/p62 accumulation, increased lamp2 expression and LC3B conversion. Trehalose attenuated AAA progression with decreased inflammation and restored SMC contractile phenotype by inducing autophagy. These results demonstrated the therapeutic potential of trehalose in AAA.

Tumor Microenvironment in Oral Squamous Cell Carcinoma: Special Stains and Scanning Electron Microscopic Study.

Effect of the oral cancerous epithelial cells on the extracellular matrix (ECM) and vice versa is also responsible for the growth and progression of cancer. The effect of inflammation on the stroma and its association with progression has also been stated in literature. The control group consisted of 10 patients with apparently normal mucosa with no history of habits, the study group consisted of 30 patients with oral squamous cell carcinoma (OSCC) confirmed by examination and incisional biopsy. H and E staining, Masson's Trichrome Staining, and Verhoeff's Van Gieson staining were used to study the inflammation, collagen fibers, and elastic fiber, respectively. A few selected specimens were studied under the scanning electron microscope. The grade of inflammation was statistically correlated with collagen and elastic fibers. Grade of inflammation was closely associated with lymphadenopathy and the density of collagenous and elastic components of the ECM. The scanning electron microscopic evaluation of elastic fibers revealed irregular fragmented elastic fibers. The stroma is the key to complete removal and treatment of OSCC; however, more studies are needed to define the role of each component, therefore improving the prognosis of a patient.

Long-term intake of aspartame-induced cardiovascular toxicity is reflected in altered histochemical parameters, evokes oxidative stress, and trigger P53-dependent apoptosis in a mouse model.

Aspartame (ASP) is probably the best known artificial sugar substitute that is used widely in food. Many experimental studies have reported the toxicity of long-term administration of ASP in various organ tissues. However, there is little evidence available about the nature and mechanisms of the adverse effects of long-term consumption of ASP on the cardiovascular system. This study was conducted to evaluate the possible effects of ASP on heart tissue. For this study 36 mature male mice were divided into one control group and three groups which received respectively 40 mg/kg, 80 mg/kg and 160 mg/kg ASP orally, for 90 days. ASP at the doses of 80 and 160 mg/kg increased the serum content of malondialdehyde (MDA), but decreased serum nitric oxide (NO), creatine kinase (CK) and CK-MB, as well as blood superoxide dismutase (SOD) levels. Serum level of total anti-oxidant capacity (TAC) in blood was also reduced in serum at the dose of 80 mg/kg. Histochemical staining, including Periodic acid-Schiff, Masson's trichrome and Verhoeff-van Gieson staining, indicated that ASP at doses of 80 and 160 mg/kg reduced glycogen deposition and decreased the number of collagen and elastic fibres in the cardiac tissue. The cardiac expression of pro-apoptotic genes, including P53, Bax, Bcl-2 and Caspase-3, was modulated at the dose of 160 mg/kg. Moreover, transcription of Caspase-3 was up-regulated at the dose of 80 mg/kg. In conclusion, long-term consumption of ASP any higher than the acceptable daily intake (40 mg/kg) appears to act by promoting oxidative stress, has the potential to alter both histopathological and biochemical parameters, and induces P53-dependent apoptosis in cardiac tissue.

Anatomical study of the adductor canal: three-dimensional micro-computed tomography, histological, and immunofluorescence findings relevant to neural blockade.

A precise anatomical understanding of the adductor canal (AC) and its neural components is essential for discerning the action mechanism of the AC block. We therefore aimed to clarify the detailed anatomy of the AC using micro-computed tomography (micro-CT), histological evaluation, and immunofluorescence (IF) assays. Gross dissections of 39 thighs provided morphometric data relevant to injection landmarks. Serial sectional images of the AC were defined using micro-CT and ultrasonography. The fascial and neural structures of the AC proper were histologically evaluated using Masson's trichrome and Verhoeff-Van Gieson staining, and double IF staining using choline acetyltransferase (ChAT) and neurofilament 200 antibodies. The posteromedial branch insertion of the nerve to vastus medialis (NVM) into the lateral border of the AC proper was lower (14.5 ± 2.4 cm [mean ± SD] above the base of the patella) than the origin of the proximal AC. The AC consists of a thin subsartorial fascia in the proximal region and a thick aponeurosis-like vastoadductor membrane in the distal region. In the proximal AC, the posteromedial branch of the NVM (pmNVM) consistently contained both sensory and motor fibers, and more ChAT-positive fibers were observed than in the saphenous nerve (27.5 ± 11.2 / 104 vs. 4.2 ± 2.6 / 104 [counts/µm2], P < 0.001). Anatomical differences in fascial structures between the proximal and distal AC and a mixed neural component of the neighboring pmNVM have been visualized using micro-CT images, histological evaluation, and IF assays.