Muscle Bundles Research Articles

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.

Duplicated superficial branch of the radial nerve and brachioradialis muscle belly: prevalence and significance.

The superficial branch of the radial nerve (SBRN) is a sensory nerve innervating the dorsoradial part of the hand. It originates in the cubital fossa, runs under the belly of the brachioradialis muscle (BM), emerges from underneath in the distal third of the forearm and continues in the subcutaneous tissue towards the hand. There exist several anatomical variations of its branching and course, including a rare variation of its duplication combined with a duplication of the brachioradialis muscle belly. The aim of this study was to find out the prevalence of this variation on a sample of cadaveric human bodies which has not been reported yet. We have carefully dissected 208 cadaveric upper limbs (Central European population). All cases of limbs containing the variation of a double SBRN and/or a double BM belly were measured and documented. We have identified 2 cases of a double SBRN combined with a double BM belly (0.96%). Both were present in the right forearm of a male donor and in both cases the nerve was impinged by muscle bundles connecting the 2 muscle bellies together. Moreover, we have encountered 1 case of a double SBRN without a double BM belly (0.48%), i.e. the total prevalence of a double SBRN was 1.44%. The duplicated SBRN with the duplicated BM is a relatively rare anatomical variation that might cause complications while performing various surgical procedures in the forearm, moreover it might be a rare cause of Wartenberg's syndrome.

Bioprinting 3D muscle tissue supplemented with endothelial-spheroids for neuromuscular junction model

The neuromuscular junction (NMJ) plays a critical role in muscle contraction, and its dysfunction can result in various neuromuscular disorders. In vitro models for studying NMJ are essential for understanding their functions and pathology. However, the engineering of muscle tissue presents challenges for the organization of myofiber-like oriented muscle bundles as well as the induction of vessel formation and innervation. To address these challenges, we fabricated a hybrid muscle construct comprising uniaxially aligned muscle struts and endothelial cell spheroids using a combination of in situ electric field-assisted bioprinting (E-printing) and microdroplet-based spheroid-forming bioprinting (MDS-printing) techniques. This resulted in self-aggregation of human umbilical vein endothelial cells (HUVECs) into spheroids without attachment to the structures. We tested various fabrication parameters, such as electric field and cross-linking conditions, for E-printing and the deposited cell density of MDS-printing, to stabilize the alignment of the human muscle progenitor cells (hMPCs) and HUVEC spheroids, respectively. The stimulated hMPCs efficiently formed fully aligned myofibers, and the incorporation of HUVEC spheroids induced highly upregulated crosstalk between different cell types compared to a simple E-printed hMPC/HUVEC mixture-loaded construct. This improved myogenesis and vessel formation in vitro. In addition, when co-cultured with a motor neuron-like cell (NSC-34) spheroid separated by a channel, we observed considerably improved neuromuscular junction formation compared to those formed with the normally mixed cell-bearing structures. Our findings suggest that this hybrid muscle construct has the potential to enhance muscle tissue engineering by improving biological activities through the incorporation of HUVEC-spheroids and facilitating neuromuscular junction formation through co-culture with NSC-34 spheroids.

Molecular detection of multidrug-resistant Pseudomonas aeruginosa of different avian sources with pathogenicity testing and in vitro evaluation of antibacterial efficacy of silver nanoparticles against multidrug-resistant P. aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a serious zoonotic pathogen threaten the poultry industry causing severe economic losses therefor, this study aimed to isolation, phenotypic, molecular identification of P. aeruginosa from different avian sources (chickens, turkey, pigeons, table eggs, and dead in shell chicken embryos), from different Egyptian governorates (Giza, Qalubia, Beheira, El-Minya, and Al-Sharqia) with applying of antibiotic sensitivity test on all P. aeruginosa isolates. Highly resistant isolates (n=49) were subjected to molecular identification of P. aeruginosa with detection of resistant genes including carbapenemase-encoding genes blaKPC, blaOXA-48, and blaNDM. On the base of molecular results, a highly resistant P. aeruginosa strain was tested for its pathogenicity on day old specific pathogen free (SPF) chicks. Also, in vitro experiment was adopted to evaluate the efficacy of silver nanoparticles (Ag-NPs) against highly antibiotic-resistant P. aeruginosa strains. The overall isolation percentage was from all examined samples were 36.2% (571/1,576) representing 45.2% (532/1,176) from different birds' tissues and 39/400 (9.7%) from total egg samples. Some of isolated strains showed multidrug resistance (MDR) against kanamycin, amoxicillin, amoxicillin-clavulanic acid, neomycin, chloramphenicol, vancomycin, cefotaxime clavulanic acid, lincomycin-spectinomycin, co-trimoxazole, cefoxitin, gentamycin, and doxycycline. These MDR strains were also molecularly positive for ESBL and carbapenemase-encoding genes. MDR strain showed high pathogenicity with histopathological alterations in different organs in challenged birds. Main histopathological lesions were necrosis of hepatocytes, renal tubular epithelium, and heart muscle bundles. The MDR strain showed in vitro sensitivity to Ag-NPs. In conclusion, MDR P. aeruginosa is a serious pathogen causing high morbidity, mortality, and pathological tissue alterations. Ag NPs revealed a promising in vitro antimicrobial sensitivity against MDR P. aeruginosa and further in vivo studies were recommended.

Omnidirectional compliance on cross-linked actuator coordination enables simultaneous multi-functions of soft modular robots

Earthworms have entirely soft bodies mainly composed of circular and longitudinal muscle bundles but can handle the complexity of unstructured environments with exceptional multifunctionality. Soft robots are naturally appropriate for mimicking soft animal structures thanks to their inherent compliance. Here, we explore the new possibility of using this compliance to coordinate the actuation movements of single-type soft actuators for not only high adaptability but the simultaneous multifunctionality of soft robots. A cross-linked actuator coordination mechanism is proposed and explained with a novel conceptual design of a cross-linked network, characterization of modular coordinated kinematics, and a modular control strategy for multiple functions. We model and analyze the motion patterns for these functions, including grabbing, manipulation, and locomotion. This further enables the combination of simultaneous multi-functions with this very simple actuator network structure. In this way, a soft modular robot is developed with demonstrations of a novel continuous-transportation mode, for which multiple objects could be simultaneously transported in unstructured environments with either mobile manipulation or pick-and-place operation. A comprehensive workflow is presented to elaborate the cross-linked actuator coordination concept, analytical modeling, modular control strategy, experimental validation, and multi-functional applications. Our understanding of actuator coordination inspires new soft robotic designs for wider robotic applications.

Fibro-Adipose Vascular Anomaly (FAVA): Not Another Vascular Anagram.

Background. Fibro-adipose vascular anomaly (FAVA) is a rare benign mesenchymal lesion. Characterized primarily by intramuscular vascular malformation with secondary overgrowth of other mesenchymal elements, particularly fibro-adipose tissue, the condition is sometimes complicated by nonspecific clinical and imaging features, causing diagnostic dilemma. Herein, we attempted to outline and correlate the clinical characteristics, imaging findings, and histopathological features of this unusual entity. Method. The study design was retrospective in nature. Computerized database of our institute was searched for tumors, and archived slides were reviewed. Pertinent clinical data including imaging findings and treatment details were also recovered for correlation. Result. Among total of 24 patients identified, mean age was approximately 16 years, with the presence of nearly equal gender distribution. Pain along with swelling was most common symptoms with the presence of movement limitation, in few. Most lesions were long-standing and anatomically confined to lower limb with no side predilection. Using imaging, the majority of the lesions were identified as vascular anomaly or venous malformation, with FAVA being a differential diagnosis in few lesions. However, in a couple of patients, likelihood of mesenchymal tumors was also suggested, radiologically. On histology, the lesions showed the presence of clustered back to back, abnormal thin-walled, variably dilated, blood-filled sac-like vessels amid skeletal muscle bundles, along with extensive fibro-adipose tissue and variably atrophic skeletal muscle bundles, at the periphery, diagnostic of FAVA. Conclusion. Owing to the presence of overlapping clinical and imaging features, FAVA is often misdiagnosed, causing dilemma in clinical management. Clinical, radiological, and histopathological correlation is thereby warranted for clinching the correct diagnosis.

Divergent effects of myostatin inhibition on cardiac and skeletal muscles in a mouse model of pressure overload.

The transforming growth factor-β (TGF-β) superfamily member, myostatin, is a negative regulator of muscle growth and may contribute to adverse cardiac remodeling. Whether suppressing myostatin could benefit pressure-overloaded heart remains unclear. We investigated the effects of pharmacological inhibition of myostatin on cardiac fibrosis and hypertrophy in a mouse model of pressure overload induced by transverse aortic constriction (TAC). Two weeks after the surgery, TAC and sham mice were randomly divided into groups receiving mRK35, a monoclonal anti-myostatin antibody, or vehicle (PBS) for 8 wk. Significant progressive cardiac hypertrophy was observed in TAC mice, as reflected by the increased wall thickness, ventricular weight, and cross-sectional area of cardiomyocytes. In the groups treated with mRK35, compared with sham mice, cardiac fibrosis was increased in TAC mice, accompanied with elevated mRNA expression of fibrotic genes. However, among the TAC mice, mRK35 did not reduce cardiac hypertrophy or fibrosis. Body weight, lean mass, and wet weights of tibialis anterior and gastrocnemius muscle bundle were increased by mRK35. When compared with the TAC-PBS group, the TAC mice treated with mRK35 demonstrated greater forelimb grip strength and a larger mean size of gastrocnemius fibers. Our data suggest that mRK35 does not attenuate cardiac hypertrophy and fibrosis in a TAC mouse model but has positive effects on muscle mass and muscle strength. Anti-myostatin treatment may have therapeutic value against muscle wasting in cardiac vascular disease.NEW & NOTEWORTHY Recent research has highlighted the importance of inhibiting TGF-β signaling in mitigating cardiac dysfunction and remodeling. As myostatin belongs to the TGF-β family, we evaluated the impact of myostatin inhibition using mRK35 in TAC-operated mice. Our data demonstrate that mRK35 significantly increased body weight, muscle mass, and muscle strength but did not attenuate cardiac hypertrophy or fibrosis. Pharmacological inhibition of myostatin may provide therapeutic benefits for the management of muscle wasting in cardiovascular diseases.

Anatomical and histological investigation of the area anterior to the anorectum passing through the levator hiatus

Objective: To document the anatomical structure of the area anterior to the anorectum passing through the levator hiatus between the levator ani slings bilaterally. Methods: Three male hemipelvises were examined at the Laboratory of Clinical Applied Anatomy, Fujian Medical University. (1) The anatomical assessment was performed in three ways; namely, by abdominal followed by perineal dissection, by examining serial cross-sections, and by examining median sagittal sections. (2) The series was stained with hematoxylin and eosin to enable identification of nerves, vessels, and smooth and striated muscles. Results: (1) It was found that the rectourethralis muscle is closest to the deep transverse perineal muscle where the longitudinal muscle of the rectum extends into the posteroinferior area of the membranous urethra. The communicating branches of the neurovascular bundle (NVB) were identified at the posterior edge of the rectourethralis muscle on both sides. The rectum was found to be fixed to the membranous urethra through the rectourethral muscle, contributing to the anorectal angle of the anterior rectal wall. (2) Serial cross-sections from the anal to the oral side were examined. At the level of the external anal sphincter, the longitudinal muscle of the rectum was found to extend caudally and divide into two muscle bundles on the oral side of the external anal sphincter. One of these muscle bundles angled dorsally and caudally, forming the conjoined longitudinal muscle, which was found to insert into the intersphincteric space (between the internal and external anal sphincters). The other muscle bundle angled ventrally and caudally, filling the gap between the external anal sphincter and the bulbocavernosus muscle, forming the perineal body. At the level of the superficial transverse perineal muscle, this small muscle bundle headed laterally and intertwined with the longitudinal muscle in the region of the perineal body. At the level of the rectourethralis and deep transverse perineal muscle, the external urethral sphincter was found to occupy an almost completely circular space along the membranous part of the urethra. The dorsal part of the external urethral sphincter was found to be thin at the point of attachment of the rectourethralis muscle, the ventral part of the longitudinal muscle of the rectum. We identified a venous plexus from the NVB located close to the oral and ventral side of the deep transverse perineal muscle. Many vascular branches from the NVB were found to be penetrating the longitudinal muscle and the ventral part of rectourethralis muscle at the level of the apex of the prostate. The rectourethral muscle was wrapped ventrally around the membranous urethra and apex of the prostate. The boundary between the longitudinal muscle and prostate gradually became more distinct, being located at the anterior end of the transabdominal dissection plane. (3) Histological examination showed that the dorsal part of the external urethral sphincter (striated muscle) is thin adjacent to the striated muscle fibers from the deep transverse perineal muscle and the NVB dorsally and close by. The rectourethral muscle was found to fill the space created by the internal anal sphincter, deep transverse perineal muscle, and both levator ani muscles. Many tortuous vessels and tiny nerve fibers from the NVB were identified penetrating the muscle fibers of the deep transverse perineal and rectourethral muscles. The structure of the superficial transverse perineal muscle was typical of striated muscle. These findings were reconstructed three-dimensionally. Conclusions: In intersphincteric resection or abdominoperineal resection for very low rectal cancer, the anterior dissection plane behind Denonvilliers' fascia disappears at the level of the apex of the prostate. The prostate and both NVBs should be used as landmarks during transanal dissection of the non-surgical plane. The rectourethralis muscle should be divided near the rectum side unless tumor involvement is suspected. The superficial and deep transverse perineal muscles, as well as their supplied vessels and nerve fibers from the NVB. In addition, the cutting direction should be adjusted according to the anorectal angle to minimize urethral injury.

Combination of ultrasound treatment and starter culture for improving the quality of beef jerky

The effects of treatment by ultrasound (US), inoculation of Pediococcus acidilactici BP2 strain (BP), and their combination (US-BP) on the quality characteristics of beef jerky were investigated during fermentation for 6 days. Moisture contents and water activity were highest after the US and US-BP treatments (P < 0.05). These effects were attributed to the decreased moisture mobility in beef jerky during ultrasonication. Meanwhile, samples treated with US and US-BP displayed more broken muscle fibers and larger gaps and cavities between the adjacent muscle bundles, resulting in lower shear force values compared to the other samples (P < 0.05), which indicated that the US and US-BP treatments improved the tenderness of beef jerky. Moreover, treatment with BP promoted the flavor development of beef jerky. The US-BP treatment improved the sensory attributes of beef jerky. In conclusion, US-BP is a promising strategy to improve the quality of beef jerky.

ISUOG Practice Guidelines (updated): fetal cardiac screening.

ISUOG Practice Guidelines (updated): fetal cardiac screening.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Incremental value of maximum voltage and activation-guided ablation for cavotricuspid isthmus-dependent atrial flutter

Abstract Funding Acknowledgements Type of funding sources: None. Radiofrequency (RF) catheter ablation is an effective method for treatment of typical cavo-tricuspid isthmus (CTI) dependent atrial flutter. However, despite the widespread use of contact force irrigated catheter, 11% of patients don’t achieve a first-pass CTI block, requiring additional RF applications or additional line during the same or a second procedure. From an anatomical point of view, CTI is composed of discrete bundles of muscle with intervening connective tissue, suggesting that its conduction proprieties are dependent of the anatomic architecture of the bundles. The maximum voltage-guided (MGV) ablation technique targets high-voltage electrograms along CTI to ablate the functionally active anatomic muscle bundle, without drawing a complete anatomic line. [1;2] Purpose The aim of this study was to evaluate the efficacy of maximum voltage and activation-guided ablation (MVG/LAT) for CTI atrial flutter in real-world setting. Methods This was a prospective observational study in which all patients undergoing CTI atrial flutter ablation at the Electrophysiology Laboratory of our Department from 01/2021 to 10/2022 were recruited. Patients who were undergoing the ablation procedure after having already made an unsuccessful attempt of the same were excluded from the study. Substrate and activation mapping of CTI, both during flutter and during pacing from the proximal coronary sinus (CSp), was performed in all patients, identifying the earliest activated high-voltage channels, without the use of fluoroscopy, using the Smart-Touch SF catheter (CARTO 3, V7, Biosense Webster). High-voltage channels were validate also during lateral RA pacing. CTI ablation was performed only in channels with high voltage and early activation (W 45, AI 500). Bidirectional CTI block were validate by differential atrial pacing maneuvers and activation mapping during pacing from CSp. Results Procedural data of 39 patients were evaluated (mean age 68±13 years; men 92.6%; mean right atrium volume 149 mL). The mean number of points taken per map per patient was 1151. Bipolar thresholds were customized (0.5-2.5 mV) with the aim of better visualizing high-voltage channels. In 20/39(51.2%) patients ablation was performed during atrial flutter, in 19/39(48.8%) during pacing from proximal CS. In 19/39(48.8%) patients high-voltage channels were not detected at the central isthmus line (6 o'clock, LAO). The median number of RF applications to achieve bidirectional block of the CTI were 6 (1-20) in all patients (p=NS) (Figure 1,2). During a mean follow-up of 260 days (90-531) no recurrences were documented in any patient. Conclusions MVG/LAT-guided ablation, targeting selectively conductive muscle fibers, decreases the number of RF applications and improves efficacy, mostly in patients with challenging anatomies.

TOPOGRAPHIC AND ANATOMICAL RELATIONSHIPS OF INTRAMUSCULAR ARTERIES AND NERVES IN THE SHOULDER REGION OF HUMAN FETUSES

In modern surgical practice, there is a growing need to improve techniques of operative interventions. Despite advancements in diagnosis and treatment, combined shoulder injuries remain a pressing problem. In the past, surgical intervention for combined trauma primarily focused on saving the patient's life. However, today it is essential not only to preserve life but also to restore the function of the injured structures. Humerus fractures are observed in 27.9% of polytrauma cases, significantly complicating urgent diagnostic and treatment procedures and worsening the patient's condition. A comprehensive study of the topographical and anatomical relationships of intramuscular arteries and nerves at the shoulder level can facilitate the selection of optimal surgical approaches, prevent iatrogenic damage, and contribute to the patient's rapid rehabilitation.&#x0D; The objective of this study was to identify the distribution patterns of arteries and nerves at different levels of the shoulder in human foetuses.&#x0D; Materials and Methods. The study involved the examination of upper limb preparations from 48 human foetuses, ranging from 81.0 to 370.0 mm in parietal-caudal length without external signs of anatomical deviations or anomalies in the development of the skeletal and muscular structures of the upper limbs. Macromicroscopic dissection, vessel injection, and morphometry techniques were employed.&#x0D; Results and Discussion. In human foetuses, the upper and middle thirds of the shoulder were found to have the highest concentration of intramuscular arteries and nerves. The entry points of arteries and nerves, their topography and branching patterns within the shoulder muscles did not always align. Arterial branches generally entered the muscles at slightly more distal locations than the nerve trunks, following the shortest path to reach the muscles.&#x0D; The levels at which arteries and nerves start branching to supply and innervate the muscles of the anterior and posterior shoulder groups, vary in human foetuses. This variation depends on the individual anatomical variability of the major vascular and nerve trunks.&#x0D; Conclusion: The levels of arterial and nerve abduction are related to the topographic and anatomical features of a particular shoulder muscle. Considering the topography of intramuscular arteries and nerves, it is advisable to perform incisions along the direction of muscle bundles, particularly near the lower border of the neurovascular unit of the brachial biceps. This approach can offer higher clinical and surgical effectiveness.

Integrated bioinformatics identifies key mediators in cytokine storm and tissue remodeling during Vibrio mimicus infection in yellow catfish (Pelteobagrus fulvidraco).

The pathogenesis of Vibrio mimicus infection in yellow catfish (Pelteobagrus fulvidraco) remains poorly understood, particularly regarding the impact of infection with the pathogen on primary target organs such as the skin and muscle. In this study, we aim to analyze the pathological intricacies of the skin and muscle of yellow catfish after being infected with V. mimicus using a 1/10 LC50 seven-day post-infection model. Furthermore, we have utilized integrated bioinformatics to comprehensively elucidate the regulatory mechanisms and identify the key regulatory genes implicated in this phenomenon. Our histopathological examination revealed significant pathological changes in the skin and muscle, characterized by necrosis and inflammation. Moreover, tissue remodeling occurred, with perimysium degeneration and lesion invasion into the muscle along the endomysium, accompanied by a transformation of type I collagen into a mixture of type I and type III collagens in the perimysium and muscle bundles. Our eukaryotic transcriptomic and 4D label-free analyses demonstrated a predominantly immune pathway response in both the skin and muscle, with downregulation observed in several cell signaling pathways that focused on focal adhesion-dominated cell signaling pathways. The upregulated genes included interleukins (IL)-1 and -6, chemokines, and matrix metallopeptidases (mmp)-9 and -13, while several genes were significantly downregulated, including col1a and col1a1a. Further analysis revealed that these pathways were differentially regulated, with mmp-9 and mmp-13 acting as the potential core regulators of cytokine and tissue remodeling pathways. Upregulation of NF-κB1 and FOSL-1 induced by IL-17C and Nox 1/2-based NADPH oxidase may have held matrix metallopeptidase and cytokine-related genes. Also, we confirmed these relevant regulatory pathways by qPCR and ELISA in expanded samples. Our findings unequivocally illustrate the occurrence of a cytokine storm and tissue remodeling, mediated by interleukins, chemokines, and MMPs, in the surface of yellow catfish infected with V. mimicus. Additionally, we unveil the potential bidirectional regulatory role of MMP-9 and MMP-13. These results provide novel perspectives on the intricate immune response to V. mimicus infection in yellow catfish and highlight potential targets for developing therapies.

(082) The Effect of Sex Steroids on RhoA/ROCK Pathway in Rat Distal Vagina

Abstract Introduction The RhoA/ROCK calcium-sensitizing pathway plays a role in clitoral contractility, and therefore we deemed interesting to investigate its involvement in the vagina. Objective The aim of this study was to investigate the sex steroid regulation of the vagina contractility through the RhoA/ROCK pathway, using a validated animal model. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (E2), testosterone (T), with or without letrozole (T+L), and compared with intact animals. Contractility studies were performed on vaginal strips to test the effect of ROCK inhibitor Y-27632 and NO synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated, mRNA expression was analyzed by semi-quantitative RT-PCR, and RhoA membrane translocation was evaluated by Western blot (Wb) analysis. Finally, rat smooth muscle cells (rvSMCs) were isolated from distal vagina of intact animals and the quantification of RhoA inhibitory protein RhoGDI was performed by Wb after stimulation with NO-donor SNP, with or without the soluble guanylate cyclase inhibitor ODQ or the PKGR1 inhibitor KT5823 administration. Results The results show that ROCK1 was immunolocalized in the smooth muscle bundles and in the blood vessel wall of vagina, while a weak positivity was detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline pre-contracted vaginal strips, decreased by OVX and completely restored by E2, while T and T+L further decreased it even below OVX level increased it. Accordingly, in Wb analysis, compared to controls, OVX significantly induced RhoA activation, as reveled by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing the NO formation via L-NAME increased Y-27632 responsiveness in the OVX+T group; L-NAME had only a partial effect in controls whilst it did not modulate Y-27632 responsiveness in OVX. Finally, stimulation of rvSMCs with SNP significantly increased the RhoGDI protein expression, an effect counteracted by ODQ and, partially, by the protein kinase inhibitor KT5823 incubation. Conclusions In conclusion, in vagina androgen administration in OVX functionally decreases RhoA/ROCK activity by hampering RhoA membrane translocation and plays a critical role in the inhibitory mechanism of the smooth muscle distal vagina contractility. Accordingly, androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring the sexual intercourse. Disclosure No

Scleraxis-lineage cells are required for correct muscle patterning.

Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scx-lineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue-tissue interaction in musculoskeletal morphogenesis.

Small intestinal submucosa promotes angiogenesis via the Hippo pathway to improve vaginal repair.

Vaginal reconstruction has incorporated the use of gastrointestinal segments for decades, but the technique is inevitably associated with complications. Tissue-engineering techniques, however, have brought great hope for vaginal reconstruction. This study aimed to evaluate the utility of small intestinal submucosa (SIS) in reconstructing clinically significant large vaginal defects in a porcine model and to investigate the role of the Hippo pathway in the vascular remodeling process. The composition and mechanical properties of SIS were characterized. Full-thickness vaginal defects were established in 10 minipig donors, with 4 cm lengths removed and replaced by an equal sized SIS scaffolds. The neovaginas were subjected to macroscopic, histological, immunohistochemical and molecular evaluations at 4 and 12 weeks after the surgery. Four weeks after the operation, extracellular matrix reorganization and complete coverage the surface of the luminal matrix by vaginal epithelium were observed, accompanied by the formation of a microvascular network and the regeneration of smooth muscles, albeit disorderly arranged. Twelve weeks after implantation, enhacements were seen in the formation of the multi-layered squamous epithelium, angiogenesis, and large muscle bundle formation in the vagina. Aditionally, the expression levels of angiogenesis-related proteins, proliferation-related proteins and Hippo pathway-related proteins in the neovagina were significantly increased. These results indicate that SIS could be used to reconstruct large vaginal defects and that the vascular remodeling process is potentially regulated by the Hippo pathway.

Angioleiomyoma of parotid gland: a rare case report

Angioleiomyoma or vascular leiomyoma first described by Virchow is a very uncommon benign soft tissue tumor. Angioleiomyoma has been defined as frequently painful, benign subcutaneous or deep dermal tumor composed of mature smooth muscle bundles which are surrounded and interlaced by vascular channels. Only 8.5% cases arise in the head and neck. A 39 years old male presented with swelling right side face and upper neck past five years. The swelling was non tender, immobile and non-compressible. There was lateral pharyngeal wall bulge which was pushing the tonsil medially. The patient underwent total conservative parotidectomy and histopathology (HPE) came out to be angioleiomyoma. Angioleiomyoma is difficult to diagnose correctly from clinical manifestations and imaging alone, and a biopsy is mandatory to make the diagnosis.

Amount and spatial arrangement of muscle fibers in the human laryngealMusculus ventricularis.

Textbooks and atlases of human macroscopic and microscopic anatomy of the larynx generally provide, if at all, only sparse information on the laryngeal Musculus ventricularis. However, several studies indicate that this muscle takes over the function of vestibular (ventricular) fold phonation after denervation of the Musculus vocalis. In the present study, 29 laryngeal specimens were coronally dissected at different levels, i.e. the anterior (L1), middle (L2), and posterior third of the vestibular fold (L3), and they underwent histological analysis. In all specimens the vestibular folds of both hemi-larynxes contained striated muscle bundles in variable amounts, representing a ventricularis muscle. These muscle bundles obviously originated from the lateral (external) and thyroepiglottic part of the thyroarytenoid muscle and the aryepiglottic part of the oblique arytenoid muscle, as has been described by other authors. The areas of vestibular folds and their amounts of ventricularis muscle bundles were measured using image analysis software (imageJ) by manual tracing. The mean area of the vestibular folds of both hemi-larynxes was 27.9 mm2 (SD [standard deviation] ± 9.17), and the area occupied by fibers of the ventricularis muscle was 1.5 mm2 (SD ± 1.78). Statistical analysis comparing the areas of both hemi-larynxes and levels resulted in no significant differences, except for the levels 2 and 3. In level 2, significantly more muscle fibers (2.0 mm2 ; SD ± 2.21) were detectable within the vestibular fold than in level 3 (0.9 mm2 ; SD ± 1.43). Level 1 also contained more muscle fibers (1.1 mm2 ; SD ± 1.06) than level 3, however, without significance. In conclusion, the laryngeal ventricularis muscle is present in the majority of reported cases. Since the muscle is of clinical relevance, it should be included in anatomical textbooks by default.

Effect of strain rates on the mechanical response of whole muscle bundle.

Muscle injuries frequently happen during sports activities and exercise, which could have serious consequences if not diagnosed and treated promptly. This research aims to investigate the quasi-static and dynamic responses of over 30 fresh frog semitendinosus muscles utilizing Split Hopkinson Pressure Bars (SHPB) and a material testing system under strain rates between 0.001 ~ 200s-1. To accommodate the special shape of muscle-tendon-bone samples, PLA clampers were produced by the 3D printer to properly hold and prevent slipping during the testing process. The mechanical characteristics of the whole muscle bundle, including Young's modulus and stress-strain curve, are illustrated at various strain rates. The findings showed that the muscle properties were sensitive to strain rate when under passive deformation. Both maximum stress and Young's modulus increased with the rise of strain rate, and modulus at 200s-1 can be as high as 10 times compared with quasi-static conditions.