M. Raj Rajasekaran

The external anal sphincter operates at short sarcomere length in humans.

Background  The length at which a muscle/sarcomere operates in vivo (operational length) and the length at which it generates maximal stress (optimal length) can be quite different. In a previous study, we found that the rabbit external anal sphincter (EAS) operates on the ascending limb of the length–tension curve, in other words at lengths shorter than its optimal length (short sarcomere length). In this study, we tested whether the human EAS muscle also operates at a short sarcomere length.

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.

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 &mgr;m to 2.59 ± 0.03 &mgr;m immediately after plication and was 2.35 ± 0.08 &mgr;m 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.

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.

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

Wnt-β 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-β) and immunostaining for β 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 β 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 β 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.

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

Studies show an age-related increase in the prevalence of anal incontinence and sphincter muscle atrophy. The Wnt/β-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, β-catenin, transforming growth factor-β (TGF-β), 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.

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 β1 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.

MP42-08 MALE URETHRAL SPHINCTER COMPLEX REVISITED USING NOVEL MR IMAGING TECHNIQUES

persists following the resolution of colitis, and that linaclotide, an FDA approved guanylate cyclase-C (GC-C) agonist, is able to attenuate these changes [3]. We hypothesise that these CCH-induced changes are the result of altered sensitivity of afferents both within the colon and bladder wall and within the dorsal root ganglion (DRG), and that oral linaclotide administration may act to reduce this hypersensitivity. METHODS: We investigated healthy C57BL/6J mice and mice with CCH, 28 days after intra-colonic TNBS administration. CCH mice were randomly assigned to either chronic linaclotide (3mg/kg/day) or placebo (water) administration, consisting of a once daily oral gavage for 2 weeks prior to experimentation. Ex-vivo electrophysiological recordings determined bladder afferent and contractile sensitivity to abMe-ATP (30mM), carbachol (1mM), and capsaicin (10mM) as well as whole cell patch clamp of retrogradely traced bladder DRG neurons in all four groups. RESULTS: Bladder DRG from mice with CCH display hyperexcitability, with a significant reduction in rheobase compared to controls (p 0.01). CCH mice also display significantly enhanced afferent responses to abMe-ATP (p 0.001), carbachol (p 0.001), and capsaicin (p 0.001). CCH mice treated with linaclotide display attenuated DRG hyperexcitability and normalised afferent responses to agonists (p 0.01) compared to placebo (p 0.01). CONCLUSIONS: Mice with CCH also display increased bladder afferent excitability within both the DRG and bladder wall, indicating cross-organ sensitisation. Chronic oral administration of linaclotide, a locally acting GC-C agonist that inhibits colonic nociceptors, reverses these colitis-induced changes in bladder afferent sensitivity. Common sensory pathways may allow agents that reduce abdominal pain to improve urological symptoms. 1. Lamb, K., et al. AJPGI, 2006. 2. Ustinova et al, Neurourology and Urodynamics, 2010. 3. Grundy, L., et al., MP28-06. The Journal of Urology, 2016.

MP81-16 THREE- DIMENSIONAL RECONSTRUCTION OF MALE PELVIC ANATOMY FROM MAGNETIC RESONANCE IMAGING

normalized to KCl). Data are presented as means SEM. Data were statistically analyzed using a one way ANOVA followed by Tukeys test(GraphPad Prism software). RESULTS: Vaginal wet weight was decreased (P<0.05) in OVXVV animals compared to SHVV, an effect reversed by local estrogen delivery. Qualitative analysis of vaginal cross sections indicated reversal of ovariectomy induced atrophy of the vaginal muscularis with estrogen treatment. In vitro contractility studies with carbachol demonstrated a trend of a lower EC50 (increased sensitivity) of vaginal strips obtained from OVXVV animals compared to SHVV and OVXVE. The amplitude of contraction to 10 uM carbachol was greater of proximal strips from OVXVV animals compared to SHVV and OVXVE (P<0.05). CONCLUSIONS: Our results indicate that vaginal estrogen is effective at reversing not only OVX induced atrophy of the epithelium but also the vaginal muscularis. We report an increased contractile response to carbachol in OVXVV animals, an effect also reversed by vaginal estrogen. Interestingly, previous studies have shown an increase in vaginal sensory innervation in ovariectomized rodents. More studies are needed to evaluate changes in autonomic innervation with this animal model to identify new therapeutic uses of vaginal estrogen treatment.

MP42-17 AGE RELATED URETHRAL SPHINCTER MUSCLE DYSFUNCTION AND FIBROSIS: POSSIBLE ROLE OF WNT SIGNALING PATHWAYS

identification of novel pathological regulators in this tissue. Oxidative stress is a fundamental pathological mediator; ROS generating enzyme NADPH oxidase (Nox enzyme) has attracted intense interest recently as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising normal biochemical oxidation. Our recent pilot study provided initial evidence for the presence of such system in bladder urothelium and its potential functional significance. This study aimed firstly to define the importance of urothelial superoxide production in the body and secondly to dissect the enzymatic sources of superoxide production in the bladder. METHODS: C57BL/6J mice were euthanized. Bladder and other types of tissue were isolated. Lucigenin-enhanced chemiluminescence quantified superoxide production in live tissue. Western blot determined Nox subtype expressions. RESULTS: Superoxide production in bladder mucosa (RLU/mg tissue: 536.8 104.8, mean SEM) was many folds as high as those in detrusor muscle (21.8 3.8, n1⁄415, p<0.01), aorta (67.2 26.6, n1⁄47, p<0.05), brain (9.4 1.7, n1⁄46, p<0.01), kidney (84.3 23.0, n1⁄46, p<0.05), ventricle (21.8 3.8, n1⁄46, p<0.01) and liver (80.8 12.9, n1⁄47, p<0.05). NADPH oxidase inhibitor diphenyleneiodonium (DPI, 20mM) reduced superoxide production to 10.8 3.4 % of control (n1⁄46, p<0.01) in bladder mucosa and to 30.8 8.4% of control (n1⁄46, p<0.01) in detrusor. Mitochondria de-coupler FCCP (10mM) suppressed superoxide production to 51.8 10.5 % of control (n1⁄46; p<0.01) in bladder mucosa and to 59.8 10.4 % of control (n1⁄46, p<0.05) in detrusor. Xanthine oxidase inhibitor oxypurinol (100mM) produced no significant effect in bladder mucosa (87.9 17.4 % of control, n1⁄46, p>0.05) but a small inhibition in detrusor (78.0 6.6% of control). Western blot showed specific bands for Nox1, Nox2 and Nox4 but no Nox3 expression in bladder mucosa and detrusor with significantly higher expression in bladder mucosa (p<0.05, n1⁄44-6). CONCLUSIONS: These data demonstrate for the first time that the urothelium is the most active tissue for superoxide production in the body. Nox enzymes are the main enzymatic source for superoxide in bladder. The main Nox subtypes are Nox1, Nox2 and Nox4, mainly located in the urothelium. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is prone to oxidative stress, inflammation and sensory dysfunction.