Exosomes secreted by urine-derived stem cells improve stress urinary incontinence by promoting repair of pubococcygeus muscle injury in rats.

BACKGROUND: Previous studies revealed that urine-derived stem cells (USCs) could promote myogenesis after the impairment of the sphincter muscles. However, the effects of exosomes secreted by USCs (USCs-Exo) were not elucidated. Exosomes are nanosized membrane vesicles secreted by the cells. They have been proved to be effective in protecting against tissue injury and therapeutic in tissue repair. USCs are ideal sources of exosomes because of the noninvasive obtaining method and self-renewal abilitiy. This study aimed to show the therapeutic effects of USCs-Exo on improving stress urinary incontinence (SUI). METHODS: Rat SUI models were established in this study using vaginal balloon inflation, and urodynamic and histological examination were carried out after exosome application. The proliferation and differentiation of muscle satellite cells (SCs) were evaluated using EdU, Cell Counting Kit 8, immunofluorescence staining, and Western blot analysis. mRNAs and proteins related to the activation of SCs were detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. RESULTS: After exosome injection, the urodynamic parameters significantly improved and the injured muscle tissue recovered well. The activation, proliferation, and differentiation of SCs were promoted. The phosphorylation of extracellular-regulated protein kinases (ERK) was enhanced. When ERK was inhibited, the promoting effect of USCs-Exo treatment disappeared. CONCLUSION: The findings of this study elucidated the functional roles of USCs-Exo in satellite cell ERK phosphorylation and identified a novel agent for skeletal muscle regeneration, providing a basis for further exploring a cell-free correction for SUI.

[Decellularized cadaveric human artery for substitution of connective tissue and smooth muscle defects on the example of tunica albuginea of penis (experimental work)]. / Ispol'zovanie detselliuliarizirovannoi trupnoi arterii cheloveka dlia zameshcheniia defektov soedinitel'notkannykh i gladkomyshechnykh struktur na primere belochnoi obolochki polovogo chlena (éksperimental'naia rabota).

AIM: To analyze experimentally the outcomes of xenotransplantation (cadaveric artery, decellularized cadaveric artery) for tunica albuginea defects repair. MATERIAL AND METHODS: The study included 60 chinchilla rabbits. Rabbits underwent local replacement of tunica albuginea. Animals were divided into 2 groups (group 1 - human cadaveric artery, group 2 - decellularized cadaveric artery). The result was considered after 7, 14 and 30 days postoperatively. RESULTS: Tendency to stasis, sludge-phenomenon and ultimately development of fibrosis of transplantation area were histologically observed in group 1 after 30 days. In group 2 luminal surface of decellularized artery was smoothed, fibrin and blood elements were absent. In group 1 mean level of C-reactive protein was 3.86±0.43, 1.17±0.2 and 0.73±0.16 mg/L after 7, 14 and 30 days respectively. In group 2 the same values were 0.33±0.03, 0.23±0.03 and 0.11±0.02 mg/L. Differences were significant (t =-46.28, t = -25.95, t = -14.84; p<0.0001).

Dynamic biomechanical characterization of colon tissue according to anatomical factors.

INTRODUCTION: The aim of this study was to determine the mechanical response of colonic specimens retrieved from the entire human colon and placed under dynamic solicitation until the tissue ruptured. MATERIAL AND METHODS: Specimens were taken from 20 refrigerated cadavers from different locations of the colonic frame (ascending, transverse, descending and sigmoid colon) in two different directions (longitudinal and circumferential), with or without muscle strips (taenia coli). A total of 120 specimens were subjected to tensile tests, after preconditioning, at the speed of 1m/s. RESULTS: High-speed video analysis showed a bilayer injury process with an initial rupture of the serosa / external muscular layer followed by a second rupture of the inner layer consisting of the internal muscle / submucosa / mucosa. The mechanical response was biphasic, with a first point of initial damage followed by a complete rupture. The levels of stress and strain at the failure site were statistically greater in terms of circumferential stress (respectively 69±22% and 1.02±0.50MPa) than for longitudinal stress (respectively 55±32% and 0.70±0.34MPa). The difference between longitudinal and circumferential stress was not statistically significant (3.17±2.05MPa for longitudinal stress and 3.15±1.73MPa for circumferential stress). The location on colic frame significantly modified the mechanical response both longitudinally and circumferentially, whereas longitudinal taenia coli showed no mechanical influence. CONCLUSION: The mechanical response of the colon specimen under dynamic uniaxial solicitation showed a bilayer and biphasic injury process depending on the direction of solicitation and colic localization. Furthermore these results could be integrated into a numeric model reproducing abdominal trauma to better understand and prevent intestinal injuries.

Myoarchitectural and functional alterations in rabbit external anal sphincter muscle following experimental surgical trauma.

Obstetrical trauma to external anal sphincter (EAS) is extremely common; however, its role in the development of anal incontinence is not clear. We examined the regenerative process and functional impact of experimental surgical trauma to EAS muscle in an animal model. Surgical myotomy, a craniocaudal incision extending along the entire length and thickness of the EAS, was performed in rabbits. Animals were allowed to recover, and anal pressures were recorded at weekly intervals for 12 wk using a custom-designed probe system to determine the length-tension property of EAS muscle. Animals were killed at predetermined time intervals, and the anal canal was harvested for histochemical studies (for determination of muscle/connective tissue/collagen) and sarcomere length measurement. In addition, magnetic resonance diffusion tensor imaging (MR-DTI) and fiber tracking was performed to determine myoarchitectural changes in the EAS. Myotomy of the EAS muscle resulted in significant impairment of its length-tension property that showed only partial recovery during the 12-wk study period. Histology revealed marked increase in the fibrosis (connective tissue = 69% following myotomy vs. 28% in controls) at 3 wk, which persisted at 12 wk. Immunostaining studies confirmed deposition of collagen in the fibrotic tissue. There was no change in the sarcomere length following myotomy. MR-DTI studies revealed disorganized muscle fiber orientation in the regenerating muscle. We conclude that, following experimental injury, the EAS muscle heals with an increase in the collagen content and loss of normal myoarchitecture, which we suspect is the cause of impaired EAS function.

Anatomical disruption and length-tension dysfunction of anal sphincter complex muscles in women with fecal incontinence.

BACKGROUND: Anal sphincter complex muscles, the internal anal sphincter, external anal sphincter, and puborectalis muscles, play an important role in the anal continence mechanism. Patients with symptoms of fecal incontinence have weak anal sphincter complex muscles; however, their length-tension properties and relationship to anatomical disruption have never been studied. OBJECTIVE: This study aimed to assess the anatomy of the anal sphincter complex muscles with the use of a 3-dimensional ultrasound imaging system and to determine the relationship between the anatomical defects and the length-tension property of external anal sphincter and puborectalis muscles in women with incontinence symptoms and in control subjects. DESIGN: Severity of anal sphincter muscle damage was determined by static and dynamic 3-dimensional ultrasound imaging. The length-tension property was determined by anal and vaginal pressure with the use of custom-designed probes. PATIENTS: Forty-four asymptomatic controls and 24 incontinent patients participated in this study. MAIN OUTCOME MEASURES: The anatomical defects and length-tension dysfunction of anal sphincter complex muscles in patients with fecal incontinence were evaluated. RESULTS: The prevalence of injury to sphincter muscles is significantly greater in the incontinent patients than in the controls. Eighty-five percent of patients but only 9% controls reveal damage to ≥2 of the 3 muscles of the anal sphincter complex. Anal and vaginal squeeze pressures increased with the increase in the probe size (length-tension curve) in the majority of controls. In patients, the increase in anal and vaginal squeeze pressures was either significantly smaller than in controls or it decreased with the increasing probe size (abnormal length-tension). LIMITATIONS: We studied patients with severe symptoms. Whether our findings are applicable to patients with mild to moderate symptoms remains to be determined. CONCLUSIONS: The length-tension property of the external anal sphincter and puborectalis muscles is significantly impaired in incontinent patients. Our findings have therapeutic implications for the treatment of anal incontinence.

Treatment of experimental esophagogastric myotomy with bone marrow mesenchymal stem cells in a rat model.

BACKGROUND: Over the last 15 years, many studies demonstrated the myogenic regenerative potential of bone marrow mesenchymal stem cells (BM-MSC), making them an attractive tool for the regeneration of damaged tissues. In this study, we have developed an animal model of esophagogastric myotomy (MY) aimed at determining the role of autologous MSC in the regeneration of the lower esophageal sphincter (LES) after surgery. METHODS: Syngeneic BM-MSC were locally injected at the site of MY. Histological and functional analysis were performed to evaluate muscle regeneration, contractive capacity, and the presence of green fluorescent protein-positive BM-MSC (BM-MSC-GFP(+) ) in the damaged area at different time points from implantation. KEY RESULTS: Treatment with syngeneic BM-MSC improved muscle regeneration and increased contractile function of damaged LES. Transplanted BM-MSC-GFP(+) remained on site up to 30 days post injection. Immunohistochemical analysis demonstrated that MSC maintain their phenotype and no differentiation toward smooth or striated muscle was shown at any time point. CONCLUSIONS & INFERENCES: Our data support the use of autologous BM-MSC to both improve sphincter regeneration of LES and to control the gastro-esophageal reflux after MY.

Endoscopic suture repair of full-thickness esophagotomy during per-oral esophageal myotomy for achalasia.

BACKGROUND: Per-oral endoscopic myotomy (POEM) requires advanced flexible endoscopic skills, especially in the management of complications. METHODS: We present a full-thickness esophagotomy while performing POEM and repair using an endoscopic suturing device. STANDARD OPERATIVE TECHNIQUE: An anterior esophageal 2 cm mucosectomy is created 7-10 cm proximal to the gastroesophageal junction after a submucosal wheal is raised. A submucosal tunnel is created and extended to 2 cm on the gastric cardia. A selective circular myotomy is performed. The mucosectomy is closed using endoscopic clips. CASE PRESENTATION: An inadvertent full-thickness esophagotomy was created while performing the mucosotomy on an inadequate submucosal wheal. We were able to resume the POEM technique at the initial esophagotomy site. There was a discussion to convert to laparoscopy. However, as we succeeded in creating the tunnel, we continued with the POEM technique. After the selective myotomy was completed, we used an endoluminal suturing device (Overstitch, Apollo Endosurgery, Austin TX) to close the full-thickness esophagotomy in two layers (muscular, mucosal). A covered stent was not an option because the esophagus was dilated, which precluded adequate apposition. The patient had an uneventful postoperative course. At 9-month follow-up, had excellent palliation of dysphagia without reflux. CONCLUSIONS: This case demonstrates the importance of identifying extramucosal intrathoracic anatomy, thus emphasizing the need for an experienced surgeon to perform these procedures, or at a minimum to be highly involved. Raising an adequate wheal is crucial before mucosectomy. Inadequacy of the wheal may reflect local esophageal fibrosis. If this fails at multiple locations in the esophagus, it may be prudent to convert to laparoscopy. This case also demonstrates the need for advanced flexible endoscopic therapeutic tools and a multidisciplinary approach to manage potential complications.

Correlating signs and symptoms with pubovisceral muscle avulsions on magnetic resonance imaging.

OBJECTIVE: We sought to correlate signs and symptoms of pelvic organ prolapse (POP) with pubovisceral muscle avulsions on magnetic resonance imaging (MRI). STUDY DESIGN: In this retrospective cohort study of 189 women with recurrent POP or unexplained symptoms of pelvic floor dysfunction, we reviewed T2-weighted pelvic floor MRI and categorized defects as minor or major avulsion, or as no defect present. Outcomes were correlated to quality-of-life questionnaire scores and data on obstetric and surgical history, together with POP-Quantification (POP-Q) measurements. Multivariable ordinal logistic regression analysis with manual backward elimination was applied to calculate odds ratios (ORs). RESULTS: Major pubovisceral avulsions were diagnosed in 83 (44%) women, minor avulsions in 49 (26%) women, while no defects were seen in 57 (30%) women. Women with a history of episiotomy or anterior vaginal wall reconstructive surgery had a higher OR for more severe pubovisceral muscle avulsions (adjusted OR, 3.77 and 3.29, respectively), as did women with symptoms of POP (OR, 1.01, per unit increase) or higher stage POP of the central vaginal compartment based on POP-Q measurement "C" (OR, 1.18). Women with symptoms of obstructive defecation were more likely to have no defect of the pubovisceral muscle on MRI (OR, 0.97, per unit increase). CONCLUSION: The variables episiotomy, previous anterior vaginal wall reconstructive surgery, POP-Q measurement "C," and symptoms scored with the Urogenital Distress Inventory "genital prolapse" and Defecatory Distress Inventory "obstructive defecation" subscales are correlated with pubovisceral muscle avulsions on pelvic floor MRI.

Effect of human muscle-derived stem cells on cryoinjured mouse bladder contractility.

OBJECTIVE: To investigate the effect of human muscle-derived stem cells (hMDSCs) on ameliorating impaired detrusor contractility in a cryoinjured bladder murine model. METHODS: The hMDSCs were isolated and cultured by modified preplate technique, and only CD34-positive hMDSCs were extracted by Mini-MACS kits. Isolated hMDSCs were prelabeled with PKH26 and injected into the cryoinjured bladder to observe the pattern and characteristics. The nude mice were subdivided into three groups: normal group (N), cryoinjury bladder group with saline injection (C), and hMDSCs injection group after cryoinjury (M). At 2 weeks after injecting hMDSCs, we compared the contractility of bladder muscle strip stimulated by electrical field stimulation (EFS), acetylcholine (Ach.), and adenosine triphosphate (ATP), and the bladder smooth muscle tissue was examined by immunohistochemistry. RESULTS: The contractile powers of bladder muscle strip in the C group were more decreased than the N group after EFS, Ach, and ATP treatment (P < .05). The bladder contractility of the M group was more increased than in the C group (P < .05), but was lower than the N group after EFS and Ach treatment. However, there was no significant difference of contractile power between the C and M groups after ATP stimulation. In immunohistochemical staining, the thickness of the bladder smooth muscle layer in the M group was significantly increased compared with the C group, and PKH26-labeled implanted cells were positive for smooth muscle cell differentiation marker (α-SMA) in the injected region. CONCLUSION: hMDSCs injection increased cholinergic bladder contractile power but not the purinergic component of bladder contraction after cryoinjury.

Diagnosing pubovisceral avulsions: a systematic review of the clinical relevance of a prevalent anatomical defect.

The aims of this systematic literature review were to assess whether the detection of pubovisceral avulsions using magnetic resonance (MR) imaging or perineal ultrasonography was clinically relevant in women with pelvic floor dysfunction and to evaluate the relation with anatomy, symptoms, and recurrence after surgery. We performed a systematic literature review using three bibliographical databases (PubMed, Embase, and CINAHL) as data sources. Clinical studies were included in which pubovisceral avulsions were studied in relation to pelvic organ prolapse (POP) stage, pelvic floor symptoms, and/or recurrence of POP after surgery. Ultimately, 21 studies met the inclusion criteria. POP stage and recurrence of POP after surgery were strongly associated with pubovisceral avulsions. Contradictory results were found regarding the relation between pubovisceral avulsions and urinary symptoms and symptoms of anorectal dysfunction. Pubovisceral avulsions, as diagnosed by MR imaging or perineal ultrasonography, are associated with higher stages of POP and recurrence of POP after surgery.

Vulnerability of continence structures to injury by simulated childbirth.

The goal of this study was to examine acute morphological changes, edema, muscle damage, inflammation, and hypoxia in urethral and vaginal tissues with increasing duration of vaginal distension (VD) in a rat model. Twenty-nine virgin Sprague-Dawley rats underwent VD under anesthesia with the use of a modified Foley catheter inserted into the vagina and filled with saline for 0, 1, 4, or 6 h. Control animals were anesthetized for 4 h without catheter placement. Urogenital organs were harvested after intracardiac perfusion of fixative. Tissues were embedded, sectioned, and stained with Masson's trichrome or hematoxylin and eosin stains. Regions of hypoxia were measured by hypoxyprobe-1 immunohistochemistry. Within 1 h of VD, the urethra became vertically elongated and displaced anteriorly. Edema was most prominent in the external urethral sphincter (EUS) and urethral/vaginal septum within 4 h of VD, while muscle disruption and fragmentation of the EUS occurred after 6 h. Inflammatory damage was characterized by the presence of polymorphonuclear leukocytes in vessels and tissues after 4 h of VD, with the greatest degree of infiltration occurring in the EUS. Hypoxia localized mostly to the vaginal lamina propria, urethral smooth muscle, and EUS within 4 h of VD. Increasing duration of VD caused progressively greater tissue edema, muscle damage, and morphological changes in the urethra and vagina. The EUS underwent the greatest insult, demonstrating its vulnerability to childbirth injury.

Penile rehabilitation after prostate cancer treatment: outcomes and practical algorithm.

The number of patients diagnosed with prostate cancer was estimated to be 192,000 in 2009 according to the American Cancer Society. The prevalence of reported erectile dysfunction after radical prostatectomy has significant variance. Among the studies in which the nerve-sparing status was described, erectile function recovery adequate for sexual intercourse was achieved in 50% of patients. This article reviews the animal and human studies in this field and provides a useful penile rehabilitation algorithm.

Prospective, randomized study of conventional versus HybridKnife endoscopic submucosal dissection methods for the esophagus: an animal study.

BACKGROUND: Endoscopic submucosal dissection (ESD) increases en bloc and histologically complete resection rate of neoplastic mucosal tumors but is technically more demanding than EMR. Limited data are available comparing the efficacy and safety of a new ESD designed to overcome these limitations and conventional ESD (C-ESD) techniques. OBJECTIVE: To compare the safety, efficacy, and operation time of the new HybridKnife ESD (HK-ESD) with C-ESD in the esophagus. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Seventeen anesthetized Yorkshire pigs. INTERVENTIONS: Removal of a 4-cm length of half-circumference esophageal mucosa by C-ESD with Hook knife or Flexknife versus HK-ESD. MAIN OUTCOME MEASUREMENTS: Procedure time, en bloc and complete resection rate, and complications (bleeding and perforation). RESULTS: All resections were completed en bloc. Procedure time was shorter in C-ESD. However, it was similar after 12 procedures. Significantly more bleeding occurred during C-ESD (28 vs 12, P = .0007). Histological muscularis propria injuries occurred with equal frequency (16 vs 17) and were mostly seen during the first 11 procedures. There were 3 perforations (2 endoscopic, 1 histological), all with C-ESD. LIMITATIONS: Nonsurvival study, use of 2 conventional knives, no training period for a new procedure. CONCLUSIONS: The HK-ESD technique was equally effective as the C-ESD technique for successful en bloc resection and was safer with less bleeding and perforation. Although procedure time was longer in HK-ESD, the difference became nonsignificant after 12 procedures.

α-Smooth muscle actin and TGF-ß receptor I expression in the healing rabbit medial collateral and anterior cruciate ligaments.

The aim of our study was to advance the knowledge about the biological differences in the healing of the anterior cruciate ligament (ACL) versus the medial collateral ligament (MCL). We quantified α-smooth muscle actin (α-SMA) expression and TGF-ß receptor I (TGF-ßRI) expression in experimentally injured rabbit ligaments (from day 3 to 12 weeks post-injury). Myofibroblasts (α-SMA positive cells) were identified as early as the third day post-injury in MCL and their density increased steadily up to day 21. Myofibroblasts were also detected in injured ACL but their density remained very low at all time points. The percentage of positive TGF-ßRI area significantly increased in both injured ligaments compared to controls, with a peak expression at day 21; however, it remained constantly lower in ACL compared to MCL. A significant correlation was found between the percentage of TGF-ßRI positive cells and the percentage of α-SMA expression only in injured MCL. These results provide evidence that myofibroblasts are important players in MCL remodelling after injury. The combined presence of myofibroblasts and TGF-ßRI in the first 3 weeks post-MCL injury may partially explain the difference in the MCL and ACL healing process.

Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 µg/ml). The addition of 80 µg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

Ex vivo biomechanical, functional, and immunohistochemical alterations of adrenergic responses in the female urethra in a rat model of birth trauma.

Birth trauma and pelvic injury have been implicated in the etiology of stress urinary incontinence (SUI). This study aimed to assess changes in the biomechanical properties and adrenergic-evoked contractile responses of the rat urethra after simulated birth trauma induced by vaginal distension (VD). Urethras were isolated 4 days after VD and evaluated in our established ex vivo urethral testing system that utilized a laser micrometer to measure the urethral outer diameter at proximal, middle, and distal positions. Segments were precontracted with phenylephrine (PE) and then exposed to intralumenal static pressures ranging from 0 to 20 mmHg to measure urethral compliance. After active assessment, the urethra was rendered passive with EDTA and assessed. Pressure and diameter measurements were recorded via computer. Urethral thickness was measured histologically to calculate circumferential stress-strain response and functional contraction ratio (FCR), a measure of smooth muscle activity. VD proximal urethras exhibited a significantly increased response to PE compared with that in controls. Conversely, proximal VD urethras had significantly decreased circumferential stress and FCR values in the presence of PE, suggesting that VD reduced the ability of the proximal segment to maintain smooth muscle tone at higher pressures and strains. Circumferential stress values for VD middle urethral segments were significantly higher than control values. Histological analyses using antibodies against general (protein gene product 9.5) and sympathetic (tyrosine hydroxylase) nerve markers showed a significant reduction in nerve density in VD proximal and middle urethral segments. These results strongly suggest that VD damages adrenergic nerves and alters adrenergic responses of proximal and middle urethral smooth muscle. Defects in urethral storage mechanisms, involving changes in adrenergic regulation, may contribute to stress urinary incontinence induced by simulated birth trauma.

Multifocal iris sphincter ruptures: new sign of the lens-iris diaphragm retropulsion syndrome.

We describe the case of a 78-year-old highly myopic woman who had bilateral phacoemulsification with posterior chamber intraocular lens implantation. During surgery, the anterior chamber was extremely deep and the pupil was excessively dilated, consistent with lens-iris diaphragm retropulsion syndrome (LIDRS). Subsequent biomicroscopy revealed multifocal iris sphincter ruptures, a new finding associated with LIDRS.