Preclinical applications of high-definition manometry system to investigate pelvic floor muscle contribution to continence mechanisms in a rabbit model.

External anal sphincter (EAS), external urethral sphincters, and puborectalis muscle (PRM) have important roles in the genesis of anal and urethral closure pressures. In the present study, we defined the contribution of these muscles alone and in combination with the sphincter closure function using a rabbit model and a high-definition manometry (HDM) system. A total of 12 female rabbits were anesthetized and prepared to measure anal, urethral, and vaginal canal pressures using a HDM system. Pressure was recorded at rest and during electrical stimulation of the EAS and PRM. A few rabbits (n = 6) were subjected to EAS injury and the impact of EAS injury on the closure pressure profile was also evaluated. Anal, urethral, and vaginal canal pressures recorded at rest and during electrical stimulation of EAS and PRM demonstrated distinct pressure profiles. EAS stimulation induced anal canal pressure increase, whereas PRM stimulation increased the pressures in all the three orifices. Electrical stimulation of EAS after injury resulted in about 19% decrease in anal canal pressure. Simultaneous electrical stimulation of EAS and PRM resulted in an insignificant increase of individual anal canal pressures when compared with pressures recorded after EAS or PRM stimulations alone. Our data confirm that HDM is a viable system to measure dynamic pressure changes within the three orifices and to define the role of each muscle in the development of closure pressures within these orifices in preclinical studies.NEW & NOTEWORTHY We anticipate that with this new HDM technology, physiological changes within these orifices may be redefined using the extensive data that are generated from 96 sensors. When compared with conventional methods, HDM offers the advantages of an increased response rate, as well as the utilization of 96 circumferential sensors to simultaneously measure pressure along the three orifices. Our findings suggest a potential use of this technology to better define urinary leak point pressure.

Characterization of urethral fibrosis in a rabbit model: Potential roles of Wnt-ß catenin pathway and epithelial to mesenchymal transition.

AIM: To elucidate the precise cellular and molecular mechanisms that underlie urethral fibrogenesis. METHODS: Endoluminal electrocautery injury (using Karl Storz 10 Fr. Pediatric urethroscope) was employed in male rabbits (n = 6) to create mucosal injury. Retrograde urethrogram (RUG) and endoluminal ultrasound techniques were used to assess severity and changes in luminal cross-sectional area. Six control rabbits were subjected to sham injury, in which the electrocautery was inserted but not powered. Urethral tissues were harvested 30 days postinjury and subjected to RNA sequencing and quantitative polymerase chain reaction (qPCR) to determine changes in gene expression. Histological, immunostaining, and Western blot studies were used to determine changes in protein expression of known markers of fibrosis (eg, collagen, Integrinαv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and pSMAD1,2,3). RESULTS: Trichrome staining confirmed increased connective tissue in urethral scar tissues. Immunostaining revealed a potential role for epithelial to mesenchymal cell transition (EMT) and positive labeling for all fibrotic markers (eg, collagen-1, Integrin αv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and SMAD1,2,3). Western blot analysis confirmed increased protein levels of these fibrotic markers. CONCLUSION: Our RNA sequencing and qPCR studies, in conjunction with our protein data, suggest that urethral mucosal fibrogenesis may be mediated by novel fibrogenic signaling pathways involving Wnt-ß catenin, TGF-ß, GIV/Girdin, and EMT which lead to increased collagen deposition. Therapeutic strategies targeting these pathways may be beneficial in attenuating fibrogenesis and stricture progression.

Age and multiparity related urethral sphincter muscle dysfunction in a rabbit model: Potential roles of TGF-ß and Wnt-ß catenin signaling pathways.

AIMS: Prior studies demonstrate increased incidence of urinary incontinence (UI) in the geriatric population which affects their quality of life. Pathophysiology of UI in the geriatric population and the underlying molecular mechanisms are still unclear. To elucidate these mechanisms, we performed a pre-clinical study in a rabbit model and the objectives were to (i) determine the effect of aging as well as multiparity on urethral sphincter muscle thickness and urethral closing pressure (UCP); (ii) examine the role of fibrosis and atrophy; and (iii) elucidate the molecular pathways that mediate fibrosis and atrophy in the urethral tissue. METHODS: New Zealand White female rabbits (n = 6 each; young 6-12 months and old over 30 months of age) were anesthetized and urethral muscle thickness and sphincter closure function were measured. Rabbits were then sacrificed and urethral tissues (bladder neck and mid-urethra) were collected to process for immunostaining as well as for molecular studies for markers for fibrosis (ß-catenin which is an important mediator of Wnt signaling, Collagen-1, and TGF-ß) and atrophy (MuRF-1). RESULTS: Our studies showed a significant decrease in the urethral sphincter muscle thickness and closure function with age. Age-related increase in protein and mRNA expression levels of fibrosis, as well as atrophy markers were observed in the bladder neck and mid-urethral tissues. CONCLUSIONS: Age and multiparity related increase in fibrosis and atrophy of urethral sphincter muscles may contribute to impaired urethral closure function seen in old animals.

Exploration of male urethral sphincter complex using diffusion tensor imaging (DTI)-based fiber-tracking.

BACKGROUND: Urinary incontinence is a major clinical problem arising primarily from age-related degenerative changes to the sphincter muscles. However, the precise anatomy of the normal male sphincter muscles has yet to be established. Diffusion tensor imaging (DTI) may offer a unique insight into muscle microstructure and fiber architecture. PURPOSE: To explore the anatomy of the urethral sphincter muscles pertinent to urinary continence function using DT-MRI. STUDY TYPE: Prospective cohort study. SUBJECTS: Eleven normal male subjects (mean age: 25.4 years); two subjects were scanned in three separate sessions to assess reproducibility. FIELD STRENGTH/SEQUENCE: 3T; using a diffusion-weighted spin echo planar sequence. ASSESSMENT: DT parameters including fractional anisotropy (FA), primary (λ1 ), secondary (λ2 ), and tertiary (λ3 ) eigenvalues, Apparent diffusion coefficient and radial diffusivity were analyzed statistically, while tracked muscle fibers were assessed visually. STATISTICAL TESTS: Regional differences (sphincters and longitudinal muscle of the urethra) in the DTI indices were assessed by one-way analysis of variance. A Tukey post-hoc test was used to identify significant differences between muscle regions. RESULTS: Two sphincter muscles, one proximal near the base of the bladder, corresponding to the lisso-sphincter, and the other distal to the end of the prostate corresponding to the rhabdo-sphincter, surrounding a central urethral muscle fiber bundle, were clearly identified. FA was higher and λ3 lower in the proximal sphincter muscle compared to the central urethral muscle and the distal sphincter (P < 0.05). The average coefficient of variation ranged from 5-12% for the DTI indices. DATA CONCLUSION: Since DTI values are known to reflect underlying tissue microarchitecture, significant differences in DTI indices identified here between the muscles of the urethral complex may potentially arise from differences in tissue microarchitecture that may in turn be related to the specific function of the sphincter and other muscles. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;48:1002-1011.

Age-related external anal sphincter muscle dysfunction and fibrosis: possible role of Wnt/ß-catenin signaling pathways.

Studies show an age-related increase in the prevalence of anal incontinence and sphincter muscle atrophy. The Wnt/ß-catenin signaling pathway has been recently recognized as the major molecular pathway involved in age-related skeletal muscle atrophy and fibrosis. The goals of our study were to 1) evaluate the impact of normal aging on external anal sphincter (EAS) muscle length-tension (L-T) function and morphology and 2) specifically examine the role of Wnt signaling pathways in anal sphincter muscle fibrosis. New Zealand White female rabbits [6 young (6 mo of age) and 6 old (36 mo of age)] were anesthetized, and anal canal pressure was measured to determine the L-T function of EAS. Animals were killed at the end of the study, and the anal canal was harvested and processed for histochemical studies (Masson trichrome stain for muscle/connective tissue) as well as for molecular markers for fibrosis and atrophy [collagen I, ß-catenin, transforming growth factor-ß (TGF-ß), atrogin-1, and muscle-specific RING finger protein-1 (MuRF-1)]. The L-T was significantly impaired in older animals compared with young animals. Anal canal sections stained with trichrome showed a significant decrease in the muscle content (52% in old compared with 70% in young) and an increase in the connective tissue/collagen content in the old animals. An increased protein and mRNA expression of all the fibrosis markers was seen in the older animals. Aging EAS muscle exhibits impairment of function and increase in connective tissue. Upregulation of atrophy and profibrogenic proteins with aging may be the reason for the age-related decrease in anal sphincter muscle thickness and function.NEW & NOTEWORTHY Our studies using a female rabbit model show age-related alterations in the structure and function of the external anal sphincter (EAS) muscle. We used endoluminal ultrasound to measure age-related changes in EAS muscle thickness. We employed Western blot and quantitative PCR to demonstrate age-related changes in the levels of important fibrogenic as well as atrophy markers. Our findings may have significant clinical implications, i.e., use of specific antagonists to prevent age-related EAS muscle dysfunction.

Altered Penile Caveolin Expression in Diabetes: Potential Role in Erectile Dysfunction.

BACKGROUND: The pathophysiology of increased severity of erectile dysfunction in men with diabetes and their poor response to oral pharmacotherapy are unclear. Defective vascular endothelium and consequent impairment in the formation and action of nitric oxide (NO) are implicated as potential mechanisms. Endothelial NO synthase, critical for NO generation, is localized to caveolae, plasma membrane lipid rafts enriched in structural proteins, and caveolins. Type 2 diabetes mellitus (T2DM)-induced changes in caveolin expression are recognized to play a role in cardiovascular dysfunction. AIMS: To evaluate DM-related changes to male erectile tissue in a mouse model that closely resembles human T2DM and study the specific role of caveolins in penile blood flow and microvascular perfusion using mice lacking caveolin (Cav)-1 or Cav-3. METHODS: We used wild-type C57BL6 (control) and Cav-1 and Cav-3 knockout (KO) male mice. T2DM was induced by streptozotocin followed by a high-fat diet for 4 months. Penile expressions of Cav-1, Cav-3, and endothelial NO synthase were determined by western blot, and phosphodiesterase type 5 activity was measured using [3H] cyclic guanosine monophosphate as a substrate. For hemodynamic studies, Cav-1 and Cav-3 KO mice were anesthetized, and penile blood flow (peak systolic velocity and end-diastolic velocity; millimeters per second) was determined using a high-frequency and high-resolution digital imaging color Doppler system. Penile tissue microcirculatory blood perfusion (arbitrary perfusion units) was measured using a novel PeriCam PSI system. OUTCOMES: Penile erectile tissues were harvested for histologic studies to assess Cav-1, Cav-3, and endothelial NO synthase expression, phosphodiesterase type 5 activity, and blood flow, and perfusion measurements were assessed for hemodynamic studies before and after an intracavernosal injection of prostaglandin E1 (50 ng). RESULTS: In T2DM mice, decreased Cav-1 and Cav-3 penile protein expression and increased phosphodiesterase type 5 activity were observed. Decreased response to prostaglandin E1 in peak systolic velocity (33 ± 4 mm/s in Cav-1 KO mice vs 62 ± 5 mm/s in control mice) and perfusion (146 ± 12 AU in Cav-1 KO mice vs 256 ± 12 AU in control mice) was observed. Hemodynamic changes in Cav-3 KO mice were insignificant. CLINICAL TRANSLATION: Our findings provide novel mechanistic insights into erectile dysfunction severity and poor pharmacotherapy that could have potential application to patients with T2DM. STRENGTHS AND LIMITATIONS: Use of KO mice and novel hemodynamic techniques are the strengths. A limitation is the lack of direct evaluation of penile hemodynamics in T2DM mice. CONCLUSION: Altered penile Cav-1 expression in T2DM mice and impaired penile hemodynamics in Cav-1 KO mice suggests a regulatory role for Cav-1 in DM-related erectile dysfunction. Parikh J, Zemljic-Harpf A, Fu J, et al. Altered Penile Caveolin Expression in Diabetes: Potential Role in Erectile Dysfunction. J Sex Med 2017;14:1177-1186.

Circular and longitudinal muscles shortening indicates sliding patterns during peristalsis and transient lower esophageal sphincter relaxation.

Esophageal axial shortening is caused by longitudinal muscle (LM) contraction, but circular muscle (CM) may also contribute to axial shortening because of its spiral morphology. The goal of our study was to show patterns of contraction of CM and LM layers during peristalsis and transient lower esophageal sphincter (LES) relaxation (TLESR). In rats, esophageal and LES morphology was assessed by histology and immunohistochemistry, and function with the use of piezo-electric crystals and manometry. Electrical stimulation of the vagus nerve was used to induce esophageal contractions. In 18 healthy subjects, manometry and high frequency intraluminal ultrasound imaging during swallow-induced esophageal contractions and TLESR were evaluated. CM and LM thicknesses were measured (40 swallows and 30 TLESRs) as markers of axial shortening, before and at peak contraction, as well as during TLESRs. Animal studies revealed muscular connections between the LM and CM layers of the LES but not in the esophagus. During vagal stimulated esophageal contraction there was relative movement between the LM and CM. Human studies show that LM-to-CM (LM/CM) thickness ratio at baseline was 1. At the peak of swallow-induced contraction LM/CM ratio decreased significantly (<1), whereas the reverse was the case during TLESR (>2). The pattern of contraction of CM and LM suggests sliding of the two muscles. Furthermore, the sliding patterns are in the opposite direction during peristalsis and TLESR.

Purse-string morphology of external anal sphincter revealed by novel imaging techniques.

The external anal sphincter (EAS) may be injured in 25-35% of women during the first and subsequent vaginal childbirths and is likely the most common cause of anal incontinence. Since its first description almost 300 years ago, the EAS was believed to be a circular or a "donut-shaped" structure. Using three-dimensional transperineal ultrasound imaging, MRI, diffusion tensor imaging, and muscle fiber tracking, we delineated various components of the EAS and their muscle fiber directions. These novel imaging techniques suggest "purse-string" morphology, with "EAS muscles" crossing contralaterally in the perineal body to the contralateral transverse perineal (TP) and bulbospongiosus (BS) muscles, thus attaching the EAS to the pubic rami. Spin-tag MRI demonstrated purse-string action of the EAS muscle. Electromyography of TP/BS and EAS muscles revealed their simultaneous contraction and relaxation. Lidocaine injection into the TP/BS muscle significantly reduced anal canal pressure. These studies support purse-string morphology of the EAS to constrict/close the anal canal opening. Our findings have implications for the effect of episiotomy on anal closure function and the currently used surgical technique (overlapping sphincteroplasty) for EAS reconstructive surgery to treat anal incontinence.

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.

A novel pattern of longitudinal muscle contraction with subthreshold pharyngeal stimulus: a possible mechanism of lower esophageal sphincter relaxation.

A subthreshold pharyngeal stimulus induces lower esophageal sphincter (LES) relaxation and inhibits progression of ongoing peristaltic contraction in the esophagus. Recent studies show that longitudinal muscle contraction of the esophagus may play a role in LES relaxation. Our goal was to determine whether a subthreshold pharyngeal stimulus induces contraction of the longitudinal muscle of the esophagus and to determine the nature of this contraction. Studies were conducted in 16 healthy subjects. High resolution manometry (HRM) recorded pressures, and high frequency intraluminal ultrasound (HFIUS) images recorded longitudinal muscle contraction at various locations in the esophagus. Subthreshold pharyngeal stimulation was induced by injection of minute amounts of water in the pharynx. A subthreshold pharyngeal stimulus induced strong contraction and caudal descent of the upper esophageal sphincter (UES) along with relaxation of the LES. HFIUS identified longitudinal muscle contraction of the proximal (3-5 cm below the UES) but not the distal esophagus. Pharyngeal stimulus, following a dry swallow, blocked the progression of dry swallow-induced peristalsis; this was also associated with UES contraction and descent along with the contraction of longitudinal muscle of the proximal esophagus. We identify a unique pattern of longitudinal muscle contraction of the proximal esophagus in response to subthreshold pharyngeal stimulus, which we propose may be responsible for relaxation of the distal esophagus and LES through the stretch sensitive activation of myenteric inhibitory motor neurons.

Esophageal wall blood perfusion during contraction and transient lower esophageal sphincter relaxation in humans.

We recently reported that esophageal contraction reduces esophageal wall perfusion in an animal study. Our aim was to determine esophageal wall blood perfusion (EWBP) during esophageal contraction and transient lower esophageal sphincter relaxations (TLESRs) in humans. We studied 12 healthy volunteers. A custom-designed laser Doppler probe was anchored to the esophageal wall, 4-6 cm above the LES, by use of the Bravo pH system so that the laser light beam stay directed toward the esophageal mucosa. A high-resolution manometry equipped with impedance electrodes recorded esophageal pressures and reflux events. Synchronized pressure, impedance, pH, and EWBP recordings were obtained during dry and wet swallows and following a meal. Stable recordings of laser Doppler EWBP were only recorded when the laser Doppler probe was firmly anchored to the esophageal wall. Esophageal contractions induced by dry and wet swallows resulted in 46 ± 9% and 60 ± 10% reduction in the EWBP, respectively (compared to baseline). Reduction in EWBP was directly related to the amplitude (curvilinear fit) and duration of esophageal contraction. Atropine reduced the esophageal contraction amplitude and decreased the EWBP reduction associated with esophageal contraction. TLESRs were also associated with reduction in the EWBP, albeit of smaller amplitude (29 ± 3%) but longer duration (19 ± 2 s) compared with swallow-induced esophageal contractions. We report 1) an innovative technique to record EWBP for extended time periods in humans and 2) contraction of circular and longitudinal muscle during peristalsis and selective longitudinal muscle contraction during TLESR causes reduction in the EWBP; 3) using our innovative technique, future studies may determine whether esophageal wall ischemia is the cause of esophageal pain/heartburn.

Antireflux action of Nissen fundoplication and stretch-sensitive mechanism of lower esophageal sphincter relaxation.

BACKGROUND & AIMS: Surgical fundoplication is an effective treatment for gastroesophageal reflux disease. One of the proposed mechanisms for its antireflux action is that it reduces lower esophageal sphincter (LES) relaxation. We investigated whether fundoplication works through a stretch-sensitive mechanism of LES relaxation. METHODS: Studies were performed in rats. Intravenous and arterial lines were placed and tracheal intubation was performed. A midline laprotomy was performed to place sutures through the esophagus to exert axial stretch on the LES, and the vagus nerve was isolated in the neck for electrical stimulation. The LES pressure was monitored with a 2F solid-state pressure transducer placed through a gastrostomy. Cranial displacement of the LES was recorded using piezoelectric crystals. Data were recorded before and after 360-degree Nissen fundoplication. RESULTS: Axial stretch and vagus nerve stimulation induced cranial displacement of the LES as well as LES relaxation in a dose-dependent manner. LES relaxation and axial stretch were each significantly reduced after fundoplication (P < .01). Nitric-oxide-induced LES relaxation was not affected by fundoplication. Removal of fundoplication restored axial stretch- and vagus nerve-stimulated LES relaxation as well as LES cranial displacement. CONCLUSIONS: Fundoplication reduces LES relaxation by interfering with axial stretch on the LES. Based on this mechanism of the antireflux actions of fundoplication, it might be possible to design new surgical strategies to treat reflux disease and reduce complications of fundoplication surgery.

Role of puborectalis muscle in the genesis of urethral pressure.

PURPOSE: The internal (smooth muscle) and the external (rhabdosphincter striated muscle) urethral sphincters have important roles in the genesis of urethral closure pressure. The U-shaped pelvic floor puborectalis muscle is important in the closure of anal and vaginal orifices in humans. We defined the contribution of the puborectalis to urethral pressure. MATERIALS AND METHODS: A total of 11 female rabbits were anesthetized and prepared to measure urethral, vaginal and anal canal pressure using manometric methods. Pressure was recorded at rest, after administration of pharmacological agents and during electrical stimulation of the puborectalis and rhabdosphincter sphincter muscles. Phenylephrine, sodium nitroprusside (Sigma-Aldrich®) and rocuronium bromide (PharMEDium, Lake Forest, Illinois) were used to define the relative contribution of smooth and striated muscles to urethral pressure. Histology of the pelvic floor hiatus was also studied. RESULTS: At rest mean ± SEM maximum urethral pressure was 13 ± 6 mm Hg. Sodium nitroprusside (50 µg/kg) infusion resulted in a 30% to 40% decrease in resting urethral pressure (mean 7.2 ± 0.2 mm Hg). Phenylephrine produced a dose dependent increase in urethral pressure (mean 17 ± 6, 25 ± 5 and 29 ± 6 for 5, 10 and 50 µg/kg intravenously, respectively). Electrical stimulation of the puborectalis muscle induced a stimulus dependent increase in urethral, vaginal and anal canal pressure. On the other hand, rhabdosphincter stimulation induced a stimulus intensity dependent increase in urethral pressure only. The increase in urethral pressure after puborectalis muscle stimulation was more than twofold higher than after rhabdosphincter stimulation. CONCLUSIONS: Our data prove that the puborectalis, a component of the pelvic floor muscles, is an important contributor to urethral pressure in the rabbit.

Transpelvic Magnetic Stimulation Enhances Penile Microvascular Perfusion in a Rat Model: A Novel Interventional Strategy to Prevent Penile Fibrosis after Cavernosal Nerve Injury.

PURPOSE: Penile microvascular dysfunction is a known contributor to erectile dysfunction (ED) and penile fibrosis has been shown to impair microvascular perfusion (MVP). Our objectives were to: (i) determine beneficial effects of TPMS to modulate penile MVP, (ii) determine its mechanism, (iii) evaluate impact of cavernosal nerve injury (CNI) on penile MVP, and (iv) determine time-course of cavernosal tissue elastin changes after CNI in rats. MATERIALS AND METHODS: Adult male rats (n=5) were anesthetized and subjected to TPMS (13%, 15%, and 17%) and MVP changes were recorded using laser speckle contrast imaging (LSCI). Another group of male rats were subjected to either bilateral cavernosal nerve injury (CNI; n=7) or sham surgery (n=7). After recovery, animals were monitored for MVP using LSCI before and after TPMS. Rat penile tissues were harvested and analyzed for fibrosis using a marker for elastin. RESULTS: Rat TPMS resulted in a stimulus dependent increase in MVP; maximal perfusion was observed at 17%. L-N(G)-Nitroarginine methyl ester (L-NAME) resulted in a marked decrease in TPMS induced MVP increase (393.33 AU vs. 210.67 AU). CNI resulted in 40% to 50% decrease in MVP. CNI produced a remarkable increase in elastin deposits that are noticeable throughout the cavernosal tissues post injury. CONCLUSIONS: TPMS is a novel and non-invasive intervention to improve penile MVP after CNI. Potential application includes treatment of ED and sexual function preservation following cancer treatment, possibly through improved penile hemodynamics that might help prevent penile hypoxia and fibrosis.

Effect of esophageal contraction on esophageal wall blood perfusion.

Myocardial blood flow occurs during the diastolic phase of the cardiac cycle, because myocardial contraction during the systolic phase impedes myocardial perfusion. Using laser Doppler perfusion technique, we studied the effect of esophageal contraction on the esophageal wall perfusion. Studies were conducted in rats. A laser Doppler probe was anchored to the esophageal wall, and wall perfusion was studied under various experimental conditions. Increase and decrease in the systemic blood pressure induced by different pharmacological agents was associated with the increase and decrease in the esophageal wall perfusion, respectively. Esophageal contractions induced by electrical stimulation of the vagus nerve and electrical stimulation of the muscle directly resulted in a reduction in the esophageal wall perfusion, in a dose-dependent fashion. Esophageal wall perfusion could be monitored by placing the Doppler probe on the esophageal mucosa or on the outside of the esophageal wall. Esophageal contraction impedes entry of blood into the esophageal wall. Future studies may investigate if ischemia of the esophageal wall induced by sustained esophageal contractions/esophageal spasm is the cause of esophageal pain symptoms in humans.

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation.

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 µg/kg) in six rats (group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium (group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle (P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.

A unique esophageal motor pattern that involves longitudinal muscles is responsible for emptying in achalasia esophagus.

BACKGROUND & AIMS: Achalasia esophagus is characterized by loss of peristalsis and incomplete esophagogastric junction (EGJ) relaxation. We studied mechanisms of esophageal emptying in patients with achalasia using simultaneous high-resolution manometry, multiple intraluminal impedance, and high-frequency intraluminal ultrasonography image recordings. METHODS: Achalasia was categorized into 3 subtypes, based on the esophageal response to swallows: types 1 and 2 were defined by simultaneous pressure waves of <30 mm Hg and >30 mm Hg, respectively, and type 3 was defined by spastic simultaneous esophageal contractions. RESULTS: Based on high-resolution manometry, the predominant achalasia pattern of type 2 was characterized by a unique motor pattern that consisted of upper esophageal sphincter contraction, simultaneous esophageal pressure (pan-esophageal pressurization), and EGJ contraction following swallows. High-frequency intraluminal ultrasonography identified longitudinal muscle contraction of the distal esophagus as the cause of pan-esophageal pressurization in type 2 achalasia. Multiple intraluminal impedance revealed that esophageal emptying occurred intermittently (36% swallows) during periods of pan-esophageal pressurization. Patients with achalasia of types 1 and 3 had no emptying or relatively normal emptying during most swallows, respectively. CONCLUSIONS: In achalasia, esophageal emptying results from swallow-induced longitudinal muscle contraction of the distal esophagus, which increases esophageal pressure and allows flow across the nonrelaxed EGJ.

Sustained improvement in the anal sphincter function following surgical plication of rabbit external anal sphincter muscle.

BACKGROUND: We recently found that the anal canal function and external anal sphincter contraction can be enhanced by surgically adjusting the EAS muscle sarcomere length in rabbits. A 20% length plication of the external anal sphincter muscle results in significant increase in the anal canal pressure and EAS muscle stress without affecting its passive tension. The durability of the beneficial effect of external anal sphincter muscle plication on the anal canal function is not known. OBJECTIVE: We studied the long-term effects of optimal length external anal sphincter plication on the anal canal pressure, external anal sphincter sarcomere length, and anal canal histology. DESIGN: Female rabbits (n = 16) were anesthetized and either sham (n = 4) or external anal sphincter plication (n = 12) surgery was performed. MAIN OUTCOME MEASURES: The effect of external anal sphincter plication on the anal canal pressure was determined every 2 weeks for 6 months in 6 animals. Anal canal was harvested for sarcomere length and histological assessment. RESULTS: External anal sphincter plication resulted in 50% to 60% increase in the anal canal pressure, and 80% to 90% increase in external anal sphincter muscle stress (during maximum electrical stimulus). The effect of plication was durable for the entire study period of 24 weeks. Sarcomere length increased from 2.11 ± 0.08 µm to 2.59 ± 0.03 µm immediately after plication and was 2.35 ± 0.08 µm at the end of 6 months. Histology revealed no significant differences in the muscle (30% vs 29%) or connective tissue components (70% vs 71%) of the anal canal between control and chronically plicated animals. CONCLUSIONS: Normal external anal sphincter muscle plication results in long-term enhancement of the anal canal function without any untoward effects on the tissue architecture in the rabbit. External anal sphincter muscle plication could be an important strategy to improve the anal canal function in patients with anal incontinence.

The Bladder Microbiome Is Associated with Epithelial-Mesenchymal Transition in Muscle Invasive Urothelial Bladder Carcinoma.

The intra-tumor microbiome has recently been linked to epithelial-mesenchymal transition (EMT) in a number of cancers. However, the relationship between EMT and microbes in bladder cancer has not been explored. In this study, we profiled the abundance of individual microbe species in the tumor samples of over 400 muscle invasive bladder carcinoma (MIBC) patients. We then correlated microbe abundance to the expression of EMT-associated genes and genes in the extracellular matrix (ECM), which are key players in EMT. We discovered that a variety of microbes, including E. coli, butyrate-producing bacterium SM4/1, and a species of Oscillatoria, were associated with expression of classical EMT-associated genes, including E-cadherin, vimentin, SNAI2, SNAI3, and TWIST1. We also found significant correlations between microbial abundance and the expression of genes in the ECM, specifically collagens and elastin. Lastly, we found that a large number of microbes exhibiting significant correlations to EMT are also associated with clinical prognosis and outcomes. We further determined that the microbes we profiled were likely not environmental contaminants. In conclusion, we discovered that the intra-tumoral microbiome could potentially play a significant role in the regulation of EMT in MIBC.