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.

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.

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.

Evaluation of Age- and Radical-Prostatectomy Related Changes in Male Pelvic Floor Anatomy Based on Magnetic Resonance Imaging and 3-Dimensional Reconstruction.

PURPOSE: Puborectalis muscles (PRM) and ischiocavernosus muscles (ICM) play important roles in urinary continence and male erectile functions. Understanding of anatomy and surgical-injury related changes to these muscles is critical to monitor changes in continence or erectile function. Anatomical description of these muscles has undergone revisions because these conclusions were derived from cadavers. Our objectives were to: (i) elucidate male pelvic muscles by in-vivo magnetic resonance imaging (MRI) and 3-dimensional (3-D) reconstruction of these images and (ii) compare PRM and ICM thickness in healthy volunteers and symptomatic patients. MATERIALS AND METHODS: Healthy young male (mean age, 25 years; n=5), older male (age, 65-70 years; n=5), and post-prostatectomy patients with erectile dysfunction and urinary incontinence (age, 65-70 years; n=5) were scanned on a 3T-magnetic resonance scanner. Images were acquired from slices above urinary bladder base to urethra entry into penis. Pelvic bone, bladder/urethra, corpus cavernosum, ICM, PRM, and prostate were segmented. 3-D models of each structure were generated and assembled into composite images, and ICM and PRM thicknesses were calculated. RESULTS: We successfully reconstructed 3-D male pelvic floor anatomy including ICM, PRM, bladder, urethra, bulbospongiosus, corpus cavernosa, prostate and bones from the two groups. We documented significant reduction in PRM and ICM thickness in older men. CONCLUSIONS: This is perhaps the first 3-D reconstruction of male pelvic floor structures based on in-vivo MRI in healthy and symptomatic patients. Observed reduction in PRM and ICM thickness is possibly due to age-related atrophy.

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.

A Novel Endoluminal Ultrasound Imaging Technique to Determine Urethral Luminal Cross-Sectional Area.

INTRODUCTION AND OBJECTIVES: Retrograde urethrogram (RUG) and voiding cystourethrogram (VCUG) are currently the gold standard imaging technique for diagnosis of urethral stricture and determination of stricture location. However, RUG and VCUG have multiple limitations. These techniques require exposure to ionizing radiation, the quality is operator and patient dependent, there is a moderate degree of invasiveness with urethral catheterization, can have artifacts because of patient positioning that underestimates stricture length. The development of novel imaging modalities without ionizing radiation to accurately evaluate the presence, location, length, and lumen cross-sectional area (CSA) of the urethral stricture would be of great value. The objective of this study was to develop a novel endoluminal ultrasound (ELUS) imaging technique that permits the accurate quantitation of urethral stricture. METHODS: Urethral strictures were created in rabbits (n = 5) by electrocautery and an ELUS technique was developed for subsequent luminal imaging. A 3.2F 40 MHz ultrasound (US) probe was introduced transurethrally and infused with US contrast agent. Images were recorded as the catheter was pulled back at a constant speed to acquire tomographic images. Lumen CSA over the entire urethral length was calculated using a custom methodology and validated in our laboratory. RESULTS: Urethral luminal CSA over the entire length of urethra before and after experimental stricture development was quantified including the length of stenosis. Intra- and interobserver variability (r = 0.99 for both) was excellent. CONCLUSIONS: Feasibility of ELUS as a quantitative technique to determine healthy urethral lumen and stricture CSA was demonstrated. The translational potential for a nonionizing imaging modality to better describe CSA, length, location, and uninvolved urethral CSA of the stricture is a significant improvement over current methodology.

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.

Characterization of age-related penile microvascular hemodynamic impairment using laser speckle contrast imaging: possible role of increased fibrogenesis.

Current technology for penile hemodynamic evaluations in small animals is invasive and has limitations. We evaluated a novel laser speckle contrast imaging (LSCI) technique to determine age-related changes in penile microvascular perfusion (PMP) and tested the role of cavernosal muscle (CC) fibrosis mediated by Wnt-TGF ß1 signaling pathways in a mouse model. Ten young (2-3 months) and old (24-28 months) wild-type C57BL6 male mice were subjected to PMP measured using a LSCI system. Penile blood flow (PBF, peak systolic velocity, PSV) was also measured using a color Doppler ultrasound for comparison. Measurements were made before and after injection of vasoactive drugs: prostaglandin E1 (PGE1) and acetylcholine (ACh). CC was processed for immunohistochemical studies for markers of endothelium and fibrosis. Protein levels were quantified by Western blot.PMP and PBF increased significantly from baseline after injection of vasoactive drugs. Peak PMP after PGE1 and ACh was higher in young mice (225.0 ± 12.0 and 211.3 ± 12.1 AU) compared to old (155.9 ± 7.1 and 162.6 ± 5.1 AU, respectively). PSV after PGE1 was higher in young than old mice (112.7 ± 8.5 vs. 78.2 ± 4.6 mm/sec). PSV after ACh was also higher in young (112.7 ± 5.6 mm/sec) than older mice (69.2 ± 7.1 mm/sec). PMP positively correlated with PSV (r = 0.867, P = 0.001). Immunostaining and Western blot showed increased protein expression of all fibrosis markers with aging. LSCI is a viable technique for evaluating penile hemodynamics. Increased cavernosal fibrosis may cause impaired penile hemodynamics and increased incidence of erectile dysfunction in older men.

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.

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.

Wnt-ß Catenin Signaling Pathway: A Major Player in the Injury Induced Fibrosis and Dysfunction of the External Anal Sphincter.

Wnt-ß catenin is an important signaling pathway in the genesis of fibrosis in many organ systems. Our goal was to examine the role of Wnt pathway in the external anal sphincter (EAS) injury-related fibrosis and muscle dysfunction. New Zealand White female rabbits were subjected to surgical EAS myotomy and administered local injections of either a Wnt antagonist (sFRP-2; daily for 7 days) or saline. Anal canal pressure and EAS length-tension (L-T) were measured for 15 weeks after which the animals were sacrificed. Anal canal was harvested and processed for histochemical studies (Masson trichrome stain), molecular markers of fibrosis (collagen and transforming growth factor-ß) and immunostaining for ß catenin. Surgical myotomy of the EAS resulted in significant impairment in anal canal pressure and EAS muscle L-T function. Following myotomy, the EAS muscle was replaced with fibrous tissue. Immunostaining revealed ß catenin activation and molecular studies revealed 1.5-2 fold increase in the levels of markers of fibrosis. Local injection of sFRP-2 attenuated the ß catenin activation and fibrosis. EAS muscle content and function was significantly improved following sFRP-2 treatment. Our studies suggest that upregulation of Wnt signaling is an important molecular mechanism of injury related EAS muscle fibrosis and sphincter dysfunction.

Clinical Evaluation of Heart Failure: Agreement among Tests.

Methods commonly used clinically to assess cardiac function in patients with heart failure include ejection fraction (EF), exercise treadmill testing (ETT), and symptom evaluation. Although these approaches are useful in evaluating patients with heart failure, there are at times substantial mismatches between individual assessments. For example, ETT results are often discordant with EF, and patients with minimal symptoms sometimes have surprisingly low EFs. To better define the relationship of these methods of assessment, we studied 56 patients with heart failure with reduced EF (HFrEF) who underwent measurement of ETT duration, EF by echocardiography, quantitative symptom evaluation, and LV peak dP/dt (rate of left ventricular pressure development and decline, measured invasively). Correlations were determined among these four tests in order to assess the relationship of EF, ETT, and symptoms against LV peak dP/dt. In addition, we sought to determine whether EF, ETT, and symptoms correlated with each other. Overall, correlations were poor. Only 15 of 63 total correlations (24%) were significant (p < 0.05). EF correlated most closely with LV peak -dP/dt. Linear regression analysis indicated that EF, ETT, and symptoms taken together predicted LV peak dP/dt better than any one measure alone. We conclude that clinical tests used to assess LV function in patients with HFrEF may not be as accurate or correlate as well as expected. All three clinical measures considered together may be the best representation of cardiac function in HFrEF patients currently available.

Intracoronary Gene Transfer of Adenylyl Cyclase 6 in Patients With Heart Failure: A Randomized Clinical Trial.

IMPORTANCE: Gene transfer has rarely been tested in randomized clinical trials. OBJECTIVE: To evaluate the safety and efficacy of intracoronary delivery of adenovirus 5 encoding adenylyl cyclase 6 (Ad5.hAC6) in heart failure. DESIGN, SETTING, AND PARTICIPANTS: A randomized, double-blind, placebo-controlled, phase 2 clinical trial was conducted in US medical centers (randomization occurred from July 19, 2010, to October 30, 2014). Participants 18 to 80 years with symptomatic heart failure (ischemic and nonischemic) and an ejection fraction (EF) of 40% or less were screened; 86 individuals were enrolled, and 56 were randomized. Data analysis was of the intention-to-treat population. Participants underwent exercise testing and measurement of left ventricular EF (echocardiography) and then cardiac catheterization, where left ventricular pressure development (+dP/dt) and decline (-dP/dt) were recorded. Participants were randomized (3:1 ratio) to receive 1 of 5 doses of intracoronary Ad5.hAC6 or placebo. Participants underwent a second catheterization 4 weeks later for measurement of dP/dt. Exercise testing and EF were assessed 4 and 12 weeks after randomization. INTERVENTIONS: Intracoronary administration of Ad5.hAC6 (3.2 × 109 to 1012 virus particles) or placebo. MAIN OUTCOMES AND MEASURES: Primary end points included exercise duration and EF before and 4 and 12 weeks after randomization and peak rates of +dP/dt and -dP/dt before and 4 weeks after randomization. Fourteen placebo participants were compared (intention to treat) with 24 Ad5.hAC6 participants receiving the highest 2 doses (D4 + 5). RESULTS: Fifty-six individuals were randomized and monitored for up to 1 year. Forty-two participants (75%) received Ad5.hAC6 (mean [SE] age, 63 [1] years; EF, 30% [1%]), and 14 individuals (25%) received placebo (age, 62 [1] years; EF, 30% [2%]). Exercise duration showed no significant group differences (4 weeks, P = .27; 12 weeks, P = .47, respectively). The D4 + 5 participants had increased EF at 4 weeks (+6.0 [1.7] EF units; n = 21; P < .004), but not 12 weeks (+3.0 [2.4] EF units; n = 21; P = .16). Placebo participants showed no increase in EF at 4 weeks or 12 weeks. Exercise duration showed no between-group differences (4-week change from baseline: placebo, 27 [36] seconds; D4 + 5, 44 [25] seconds; P = .27; 12-week change from baseline: placebo, 44 [28] seconds; D4 + 5, 58 [29 seconds, P = .47). AC6 gene transfer increased basal left ventricular peak -dP/dt (4-week change from baseline: placebo, +93 [51] mm Hg/s; D4 + 5, -39 [33] mm Hg/s; placebo [n = 21]; P < .03); AC6 did not increase arrhythmias. The admission rate for patients with heart failure was 9.5% (4 of 42) in the AC6 group and 28.6% (4 of 14) in the placebo group (relative risk, 0.33 [95% CI, 0.08-1.36]; P = .10). CONCLUSIONS AND RELEVANCE: AC6 gene transfer safely increased LV function beyond standard heart failure therapy, attainable with one-time administration. Larger trials are warranted. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00787059.

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.

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.

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.

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.

Longitudinal muscle dysfunction in achalasia esophagus and its relevance.

Muscularis propria of the esophagus is organized into circular and longitudinal muscle layers. Goal of this review is to summarize the role of longitudinal muscle in physiology and pathophysiology of esophageal sensory and motor function. Simultaneous manometry and ultrasound imaging that measure circular and longitudinal muscle contraction respectively reveal that during peristalsis 2 layers of the esophagus contract in perfect synchrony. On the other hand, during transient relaxation of the lower esophageal sphincter (LES), longitudinal muscle contracts independently of circular muscle. Recent studies provide novel insights, i.e., longitudinal muscle contraction of the esophagus induces LES relaxation and possibly descending relaxation of the esophagus. In achalasia esophagus and other motility disorders there is discoordination between the 2 muscle layers. Longitudinal muscle contraction patterns are different in the recently described three types of achalasia identified by high-resolution manometry. Robust contraction of the longitudinal muscle in type II achalasia causes pan-esophageal pressurization and is the mechanism of whatever little esophageal emptying that take place in the absence of peristalsis and impaired LES relaxation. It may be that preserved longitudinal muscle contraction is also the reason for superior outcome to medical/surgical therapy in type II achalasia esophagus. Prolonged contractions of longitudinal muscles of the esophagus is a possible mechanism of heartburn and "angina like" pain seen in esophageal motility disorders and possibly achalasia esophagus. Novel techniques to record longitudinal muscle contraction are on the horizon. Neuro-pharmacologic control of circular and longitudinal muscles is different, which provides an important opportunity for the development of novel pharmacological therapies to treat sensory and motor disorders of the esophagus.

Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac fibrosis in mice.

BACKGROUND: Circulating subclinical lipopolysaccharide (LPS) occurs in health and disease. Ingesting high fatty meals increases LPS that cause metabolic endotoxemia. Subclinical LPS in periodontal disease may impair endothelial function. The heart may be targeted as cardiac cells express TLR4, the LPS receptor. It was hypothesized that recurrent exposure to subclinical LPS increases mortality and causes cardiac fibrosis. METHODS: C57Bl/6 mice were injected with intraperitoneal saline (control), low dose LPS (0.1 or 1 mg/kg), or moderate dose LPS (10 or 20 mg/kg), once a week for 3 months. Left ventricular (LV) function (echocardiography), hemodynamics (tail cuff pressure) and electrocardiograms (telemetry) were measured. Cardiac fibrosis was assessed by picrosirius red staining and LV expression of fibrosis related genes (QRT-PCR). Adult cardiac fibroblasts were isolated and exposed to LPS. RESULTS: LPS injections transiently increased heart rate and blood pressure (<6 hours) and mildly decreased LV function with full recovery by 24 hours. Mice tolerated weekly LPS for 2-3 months with no change in activity, appearance, appetite, weight, blood pressure, LV function, oximetry, or blood chemistries. Mortality increased after 60-90 days with moderate, but not low dose LPS. Arrhythmias occurred a few hours before death. LV collagen fraction area increased dose-dependently from 3.0±0.5% (SEM) in the saline control group, to 5.6±0.5% with low dose LPS and 9.7±0.9% with moderate dose LPS (P<0.05 moderate vs low dose LPS, and each LPS dose vs control). LPS increased LV expression of collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, periostin and IL-6 (P<0.05 moderate vs low dose LPS and vs control). LPS increased α-SMA immunostaining of myofibroblasts. LPS dose-dependently increased IL-6 in isolated adult cardiac fibroblasts. CONCLUSIONS: Recurrent exposure to subclinical LPS increases mortality and induces cardiac fibrosis.