169 Investigation of High-Definition Anorectal Pressure Topography (HDM) in Patients With Constipation and Fecal Incontinence

AGA Abstracts

166 168 Successful Implantation of Intrinsically Innervated Physiologically Functional Bioengineered Human Internal Anal Sphincter (IAS) Shreya Raghavan, Eiichi A. Miyasaka, Robert R. Gilmont, Sita Somara, Shanthi Srinivasan, Daniel H. Teitelbaum, Khalil Bitar

Randomized Controlled Trial Comparing Biofeedback, Electrogalvanic Stimulation, and Massage for the Treatment of Levator Ani Syndrome Giuseppe Chiarioni, Adriana Nardo, Italo Vantini, Antonella Romito, William E. Whitehead

Background: We have bioengineered intrinsically innervated IAS tissue by co-culturing human IAS circular smooth muscle and immorto mouse fetal enteric neurons (IM-FENs) Objective: We provide proof of concept that intrinsically innervated bioengineered human IAS rings implanted in RAG1-/- mice preserve physiological functionality of constituent myogenic and neuronal components. Methods: Intrinsically innervated human IAS rings were bioengineered by embedding human IAS smooth muscle cells and de-differentiated IM-FENs on a collagen/laminin matrix. IM-FENs were allowed to differentiate at 39C for 12 days. Bioengineered rings were implanted for 25 days under the skin of RAG1-/- mice. Results: Histology of harvested post-implant IAS revealed neovascularization. Real-time force acquisition from these constructs revealed that physiological responses of post-implant innervated IAS were similar to control non-implanted innervated IAS rings: (1)Spontaneous basal tone generated was tetrodotoxin (TTX)-insensitive and hence purely myogenic; (2)Vasoactive Intestinal Peptide(VIP) caused a relaxation of basal tone upto 300μN. VIPinduced relaxation in the presence of TTX was only 20% of the original, indicating dual involvement of neuronal and myogenic component in VIP-induced relaxation;(3)Electrical Field Stimulation(10Hz,0.3ms) caused relaxation of basal tone upto 153μN. EFS response was abolished in presence of TTX indicating selective stimulation of neuron mediated relaxation pathway; (4)Acetylcholine(Ach) response was dose-dependent and elicited a rapidrising, sustained contraction of 477±77μN. Contraction was attenuated by 90% in the presence of TTX, implying synergistic involvement of both myogenic and neuronal components in cholinergic contraction; (5)30mM KCl elicited a rapid rising spike contraction, indicating preservation of physiologically relevant ion channels. Summary: Implanted intrinsically innervated IAS maintained: 1-Spontaneous generation of myogenic basal tone, a constitutive property of IAS. 2- Neurotransmitter-mediated contraction and relaxation, and their TTX sensitivity displaying distinct contributions of viable intrinsic innervation (IM-FEN) and constituent myogenic component (human IAS smooth muscle).Conclusion: Intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiological functionality of both myogenic and neuronal components, upon implantation into RAG1-/- mice. This is the first report of implantation of intrinsically innervated human IAS bioengineered construct, with vast potential to create functional autologous graft tissue to repair degenerated IAS. Supported by NIHRO1DK071614 and 1RC1DK087151

Uncontrolled trials suggest that chronic proctalgia/levator ani syndrome (LAS) can be treated with biofeedback to teach pelvic floor relaxation, electrogalvanic stimulation (EGS), or massage of levator muscles. Aims: This prospective, randomized controlled trial compared the effectiveness of these techniques, assessed physiological mechanisms for treatment, and identified patient characteristics that predict success. Methods: Inclusion/exclusion criteria were Rome II symptom criteria, weekly pain, negative medical evaluation, and no psychiatric disorder. Patients were categorized as “highly likely” LAS if they reported tenderness with traction on the levator muscles, or as “possible” LAS if they did not. All 157 patients received nine sessions of psychological counseling plus biofeedback, EGS, or massage. Clinical and physiology outcomes were reassessed at 1, 3, 6, and 12 months. Results: Among patients with “highly likely” LAS, biofeedback was more effective than EGS, which was more effective that massage. At one month, adequate pain relief was reported by 87% for biofeedback, 45% for EGS, and 22% for massage. Pain days per month decreased from an average of 14.69 to 3.32 with biofeedback, 8.87 with EGS, and 13.25 with massage. Pain intensity decreased from an average of 6.78 (0-10 VAS scale) to 1.76 with biofeedback, 4.69 with EGS, and 6.01 with massage. Improvements were maintained throughout follow-up. Patients with only a “possible” diagnosis of LAS did not benefit from any treatment. Biofeedback and EGS improved LAS by increasing the ability to relax pelvic floor muscles and evacuate a water-filled balloon, and by reducing the urge and pain thresholds. Significant predictors of success were baseline inability to evacuate the balloon and tenderness on rectal examination. Conclusions: Biofeedback is the most effective of the evaluated treatments for chronic proctalgia, but it only benefits patients with a “highly likely” diagnosis. The pathophysiology of LAS is similar to that of dyssynergic defecation. 169 Investigation of High-Definition Anorectal Pressure Topography (HDM) in Patients With Constipation and Fecal Incontinence Michelle Nguyen, Gregory Cheeney, Kasaya Tantiphlachiva, Jessica Valestin, Ashok Attaluri, Satish S. Rao Introduction: Anorectal manometry (ARM) and anal ultrasound (AUS) are routinely performed in patients with constipation or fecal incontinence. Whether a single test can simultaneously evaluate both structure and function, provide more information, and minimize costs has not been examined. Hypothesis: HDM simultaneously evaluates anal sphincter morphology and function. Aim: To examine anorectal structure and function with HDM and compare with ARM and AUS. Methods: During HDM (Sierra Scientific, CA) and ARM, anal sphincter and rectal pressures were recorded during rest, squeeze, valsalva, and bear down. Rectal sensations and rectoanal inhibitory reflexes (RAIR) were also assessed. Anal sphincter defects were evaluated with HDM and AUS. Anal sphincter lengths were assessed using all three techniques. 10 healthy (F:M 4:6, mean age 35 ± 4 yrs), 10 constipated (F:M 9:1, mean age 48 ± 4 yrs), and 10 fecal incontinent (F:M 8:2, mean age 58 ± 4 yrs) subjects were enrolled. Statistical Analysis: Intraclass correlation (ICC) and Kappa coefficients were used. Results: Mean procedure level of discomfort rated on visual analog scales were 2.9, 2.8, and 3.4 (10=worse discomfort) for ARM, AUS, and HDM, respectively. Intraclass correlations comparing HDM and ARM variables are shown in Table. Sphincter pressures were comparable, but more subjects showed features of dyssynergia with HDM. There was good agreement in 26/ 30 (87%) subjects for sphincter defects; 16 (53%) were negative and 10 (33%) were positive with both AUS and HDM (Kappa coefficient=0.72). Anal canal length showed good correlation between AUS and HDM (ICC=0.64). Conclusions: HDM is feasible, safe, well-tolerated and provides comparable information regarding sphincter defects when compared to AUS and regarding manometric functions when compared to ARM. The circumferential array gives superior definition of anal sphincter length, contraction, relaxation, and paradoxical contraction. Correlation of Anal sphincter and rectal pressure profiles measured by ARM and HDM

167 Pelvic Floor Injury and Dysfunctions in Fecal Incontinence (FI): A PopulationBased Case-Control Study Adil E. Bharucha, Joel Fletcher, Lee J. Melton, Alan R. Zinsmeister Background: The precise contribution of pelvic floor injury to fecal incontinence (FI) in women is unclear because (i) most studies emanate from selected patients in tertiary centers; (ii) FI often begins several decades after vaginal delivery; and (iii) imaging reveals clinicallyoccult sphincter defects even after uncomplicated vaginal delivery. Aims: To compare the morphology of the internal (IAS) and external (EAS) anal sphincters and puborectalis (PR), and, pelvic floor motion among randomly selected community women with (“cases”) and without FI (“controls”). Methods: Through the Rochester Epidemiology Project, an agestratified randomized sample of 5200 women in Olmsted County, MN was surveyed; 2800 women responded and 507 had FI (GE 2005). In this case-control study, 66 cases (mean age 57y, range 25-82y) and 66 controls (57y, 25-82y) matched for age and nested within that population-based cohort had pelvic floor imaging by magnetic resonance imaging (MRI). Anal sphincter and puborectalis morphology, characterized as normal or abnormal (i.e., tears, scars or atrophy), and pelvic floor motion during voluntary contraction (squeeze) and defecation were compared by univariate and multiple variable analyses (i.e., conditional logistic regression models) of the matched pairs. Results. The severity of FI was mild (28%), moderate (64%) or severe (8%). 31 cases (47%) and 12 controls (18%) had sphincter or puborectalis abnormalities; IAS and EAS abnormalities were more prevalent in FI (Table). 9 cases (14%) had both IAS and EAS injury. The anorectal angle change from rest to squeeze was smaller (p = 0.0001), signifying weaker PR contraction during squeeze, in FI (13 ± 4°) than in controls (30 ± 2°). Perineal descent during defecation was also lower (p = 0.01) in FI (2.6 ± 0.2 cm) than controls (3.1 ± 0.2 cm). Abnormal IAS morphology (OR 4.9, 95% CI 1.2-19.8), a smaller reduction in the anorectal angle during squeeze (OR 1.03, 95% CI 1.003-1.05), and reduced perineal descent during defecation (OR 1.5, 95% CI 1.02-2.2) increased the risk for FI. Conclusions. Among community women, FI is associated with anal sphincter injury and disordered pelvic floor motion, not only during squeeze but also during evacuation. Ongoing analyses are evaluating the etiology of pelvic floor injury.

AGA Abstracts

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Microvascular and Micro-Architectural Spectral Markers Having Synergistic Diagnostic Capabilities for Colon Neoplasia Risk Stratification Hemant K. Roy, Nikhil Mutyal, Michael J. Goldberg, Jeremy D. Rogers, Ashish K. Tiwari, Andrew Radosevich, Ramesh K. Wali, Laura K. Bianchi, Eugene F. Yen, Mohammed Jameel, Andrej Bogojevic, Vadim Backman Introduction: Our group has developed a suite of novel light scattering technologies for identification field carcinogenesis. In the histologically-normal mucosa, micro-architectural alterations can be identified with low-coherence enhanced backscattering spectroscopy (LEBS) (Clin Cancer Res 2006) whereas micro-circulatory dysregulation was detectable with polarization-gated spectroscopy(PGS) (Gastro 2008). Micro-architectural and microvascular markers obtained from the endoscopically-normal rectal mucosa had an AUROC of 0.90 (Cancer Res 2009) and 0.88 (Clin Cancer Res), respectively for advanced adenomas but only 0.65-0.75 for adenomas 5-9 mm, lesions with arguably some clinical relevance. We now investigate whether these distinct field effect markers provides synergistic or redundant diagnostic information. Methods: In order to simultaneously measure both marker types, we developed a novel fiber-optic probe. We first tested this in the azoxymethane (AOM)treated rat model at a premalignant time point (10 weeks) evaluating 10 sites throughout the colon. We then performed a trial on patients immediately prior to screening colonoscopy. Using a “blind” insertion of the fiber-optic probe, 5 rectal readings were taken. Spectral marker analysis was performed by investigators blinded to the diagnosis. Results: Both the micro-architectural and micro-vascular marker were elevated in the microscopically normal ex vivo colonic mucosa of the AOM-treated rat when compared with age-matched salinetreated rats. As detailed in table, the AUROC for combined markers was better than each separately. The effect was more dramatic in the clinical studies that were performed In Vivo. Indeed, rectal performance with the novel dual functional fiberoptic probe for a more challenging endpoint (≥5 mm) was markedly superior to our previously published larger scale advanced adenomas studies performed with each marker type individually. Conclusion: Our technological breakthrough enables simultaneous detection of micro-architectural (LEBS) and micro-vascular markers. We demonstrate, for the first time, that the combination of two distinct spectral marker categories was synergistic for identification of field carcinogenesis. Indeed, the marker combination obtained from the uninvolved rectum was outstanding for all significant adenomas (≥5 mm). Multicenter trials are planned for validation of this colonoscopic “pre-screen” through a minimally-intrusive, simple rectal test. Diagnostic ability of Spectral Marker Type (AUROC)

170 Topographic and Manometric Characterization of the Recto-Anal Inhibitory Reflex (RAIR) Gregory Cheeney, Ashok Attaluri, Michelle Nguyen, Jessica Valestin, Satish S. Rao Introduction: RAIR is an integral part of normal defecation. The degree of RAIR relaxation along the anal length and anterior-posterior axis is unknown. Aim: To perform topographic and vectorgraphic evaluation of RAIR along the anal canal using high definition manometry (HDM), and examine role of various muscle components. Methods: Anorectal topographic and manometric data were evaluated in 10 healthy volunteers (6 males) using a HDM probe (Sierra Scientific, CA) with 256 sensors. The RAIR data were analyzed for topographic, baseline, lowest relaxation, and plateau pressures during 5 volumes of balloon inflation (15cc, 40cc, 70cc, 100cc, 170cc) every mm along the length of anal canal, and in 3D by dividing the anal canal into 4x4 mm grids. Results: Figure 1 shows progressive increase in relaxation pressure along the anal canal with increasing balloon distention that plateau after 70 cc. Higher volume produces greater anal relaxation. Figure 2 shows the 3D analysis with 2 volumes, and reveals that RAIR is maximally seen at the middle and upper portions of anal canal (levels 1.2-3.2 cm) and in the posterior quadrants. Conclusions: RAIR is characterized by a differential relaxation along the A-P axis, the length of anal canal, and at each vector with maximum change at level of Internal anal sphincter. The detection of pressure changes at 3-4 cm and 0.5 to 1 cm suggests longitudinal elongation of the anal sphincter and circumferential relaxation. Our findings emphasis importance of sensor location and orientation, i.e. an anterior and more distal location is less likely to detect RAIR. HDM facilitates more accurate characterization of RAIR and aids detection of dysganglionosis.

173 Engagement of Shc-Dependent Signals by RTK Promotes In Vivo Tumorigenic and Metastatic Behaviors in Normal Intestinal Epithelial Cells Walid Chababi, Véronique Pomerleau, Jimmy Bernier, Caroline Saucier

171 Physiology of Intrinsically Innervated Bioengineered Construct From Human Internal Anal Sphincter Smooth Muscle Cells Shreya Raghavan, Robert R. Gilmont, Sita Somara, Shanthi Srinivasan, Khalil Bitar

Evidence entailing a role for deregulation of receptors tyrosine kinase (RTK) in human colorectal cancers (CRC) are abundant and compelling, and RTK-targeting agents are viewed as a promising therapeutic approach in CRC. Given the heterogeneity of RTKs deregulated in CRC, a valuable therapeutic approach for the development of new CRC therapies could be to target RTK signaling effectors engaged by all, or at least several RTKs, which are involved in the regulation of biological responses critical for the progression of CRC. Taking into account that all RTKs deregulated in CRC recruit the adaptor protein Shc and/or engage Shc-dependent signaling pathways, we hypothesized that Shc may represent such target. To test this hypothesis, we have assessed whether the aberrant activation of Shc-dependent signals by RTKs was sufficient to drive neoplastic transformation in normal intestinal epithelial cells. For this, oncogenic forms of the Met/hepatocyte growth factor RTK engineered to recruit only the adaptor protein Shc, thereby engaging specifically its signaling pathways, were expressed in a normal-derived crypt intestinal epithelial cell line, the IEC-6 cells. The biological consequence of oncogenic activation of Shc-dependent signals was then assessed in cell-based and In Vivo assays. Herein, we showed that the sole engagement of Shc by RTKs was sufficient to induce a variety of cancer behaviors in normal IEC-6 cells. This included morphological transformation, proliferation, focus formation, anchorage-independent growth and motility. The morphological alterations observed in IEC-6 cells expressing the Shc-specific RTK oncoproteins were found to correlate with hallmarks of EMT, a process viewed as being a prerequisite for metastatic dissemination of epithelial tumor cells. EMTlike phenotypic changes seen in IEC-6 cells expressing Shc-specific RTK oncoproteins were associated with downregulation of the epithelial markers E-cadherin and occludin, along with an upregulation of the mesenchymal marker vimentin. Notably, IEC-6 cells expressing the Shc-RTK oncoproteins were forming tumors in nude mice with short latency. Moreover, whereas mice injected into the tail vein with control IEC-6 cells did not display any signs of lung colonization, mice injected with these cells developed extensive lung metastases within 25 days. These data provide the first evidence that oncogenic engagement of Shcdependent signals by RTKs is sufficient to endow normal IECs with tumorigenic and metastatic behaviors. This identifies Shc and/or Shc-dependent signaling pathways as attractive targets for the development of drugs for the treatment of human colorectal malignancies.

Background: Physiological functionality of Internal Anal Sphincter (IAS) is a complex synergy of neural and myogenic components. We have developed a co-culture of human IAS smooth muscle cells with immorto-mouse fetal enteric neuronal (IM-FEN) cell line that differentiate and intrinsically innervate bioengineered human IAS rings. Hypothesis: Intrinsically innervated human IAS smooth muscle bioengineered ring constructs: 1) maintain functionality of both myogenic and neural cell populations, each of which can be selectively activated, and 2) improves the sensitivity of IAS constructs to physiologically relevant contractile and relaxant neurotransmitters. Methods: Smooth muscle cells were isolated from human IAS. IMFEN cells were expanded under permissive tissue-culture conditions. Intrinsically innervated human IAS rings were bioengineered by embedding the two cell types on a double-layered collagen/laminin matrix. Rings formed due to intrinsic contractility of smooth muscle cells. IM-FENs in the ring were allowed to differentiate at 39C for 12 days. Force generation was measured in response to Acetylcholine (Ach), Vasoactive Intestinal Peptide (VIP) or Electrical Field Stimulation (EFS). Results: (1) Intrinsically innervated rings generated spontaneous basal tone in a tetrodotoxin (TTX)-insensitive manner; (2) Ach-induced (1μM) force was 33% higher in innervated IAS rings versus non-innervated rings; Ach-induced peak contraction was attenuated by 37% in innervated IAS rings in the presence of TTX; (3) VIP (1μM) caused relaxation of basal tone up to 132.8±48μN. VIP-induced relaxation of basal tone was reduced to 72±15.9μN, in the presence of TTX, estimating the myogenic contribution to total VIP response to be ~55%; (4) Pre-treatment with 30mM KCl augmented Ach-induced contraction in innervated rings by an extra 50%; (5) EFS (10Hz, 0.3ms) relaxed basal tone up to 120μN. EFS response was totally abolished in the presence of TTX. Summary: 1Bioengineered intrinsically innervated human IAS rings maintained integrity of neural (IMFEN) and myogenic (human IAS smooth muscle) components of IAS physiology. 2-Spontaneous basal tone was identified to be entirely myogenic. 3-Myogenic and neural components worked in tandem to produce a combined physiological response. 4-Cholinergic stimulation and VIP treatment elicited a combined neural and myogenic response. Conclusion: This is the first successful bioengineering of intrinsically innervated human IAS rings similar to In Vivo IAS, representing great advancement for replacement therapy. Supported by NIHRO1DK071614

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AGA Abstracts

AGA Abstracts

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