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M2027 Impairment of Mitochondrial Function Alters Global Ca2+ Mobilization but Not Contractility in Aged Mouse Colon
M2025
Importance of Abdominal Pain as a Symptom in Gastroparesis: Relation to Clinical Factors, Disease Severity, Quality of Life, Gastric Retention, and Medication use William L. Hasler, Laura Wilson, Henry P. Parkman, Kenneth L. Koch, Thomas L. Abell, Pankaj J. Pasricha, Linda Nguyen, William J. Snape, Gianrico Farrugia, James Tonascia, Aynur Unalp-Arida, Linda A. Lee, Frank A. Hamilton
Investigation of Roles of 5-HT2B Receptors in Modulation of Colonic Motility in the Anaesthetized Rat Katsuyo Ohashi-Doi, Billie Hunne, Dorota Ferens, Nobuyuki Takahashi, John B. Furness Tegaserod is a prokinetic agent for constipation dominant irritable bowel syndrome whose effect was initially attributed to 5-HT4 receptor activation. However tegaserod is both a 5HT4 receptor agonist and a 5-HT2B receptor antagonist. Recently, we reported that RS127445, a selective 5-HT2B receptor antagonist, inhibited defecation and visceral hypersensitivity induced by TNBS in rats (Ohashi-Doi et al., 2009). This study aimed to characterise the effects of 5-HT2B receptor antagonism on colonic motility in the naïve rat. Male SpragueDawley rats (300 - 400g) were anaesthetized, the colon was cannulated in the region of the colonic flexure and at the anus for measurement of colonic motility and propulsion. The body wall was closed with the oral cannula protruding. The oral cannula was connected to a Marriotte bottle filled with PBS, and the distal cannula was connected to a pressure transducer, one way value and fluid outlet. RS-127445 was dissolved in 5% well-solve solution and given intravenously. The data are presented as mean ± S.E. (n=5-6). Other rats were used in the PCR study. Segments of fundus, proximal colon and distal colon were dissected to give EM/MP (external muscle/ myenteric plexus) and mucosal samples. RNA was extracted from the samples and reverse transcribed to cDNA, and then amplified by PCR using primers specific for 5HT2B (forward-ATAGCCATCAAAAAGCCAA, reverseTGTTAGGCGTTGAGGTGG) In naïve control rats, when the basal intraluminal pressure in the colon was 6.46 ± 0.03 mmHg, small pressure oscillations were observed (maximum intraluminal pressure, 8.4±0.1 mmHg). The single contractions had durations of 2.1±0.1 min; the number of contractions was 9.0±1.6 in 20 min periods. RS-127445 (1 mg/kg/hr, i.v.) significantly reduced the cycle of colonic contraction (number of contractions, 5.3±0.9; interval, 3.8±0.5 min; P<0.05). However, RS-127445 did not affect either basal intraluminal or maximum pressure of the colon in the rat. L-NAME (10 mg/kg, i.v.) significantly increased the number of contractions (16 ± 1.1 / 20 min, P<0.01) and maximum intraluminal pressure (44±1 vs. 23±2 %, P<0.01). Pre-treatment with RS-127445 partially inhibited increased colonic contractions induced by L-NAME in rats (11±0.9 / 20 min, P<0.05). The duration of single contractions was reduced 2.2±0.47 min (P<0.01). PCR results indicated the presence of 5-HT2B receptor transcripts in the fundus, proximal and distal colon in the rats. These results suggest that 5-HT2B receptors are involved in modulation of colonic motility, and that a selective 5-HT2B receptor antagonist might be a useful therapeutic approach for the treatment of functional gut disorders.
Background: While gastroparesis usually presents with nausea or vomiting, abdominal pain also may be part of the presenting illness. The importance of pain in gastroparesis and its associated factors are largely unexplored. Aims: Define abdominal pain prevalence in gastroparesis and relate to demography, etiology, severity, quality of life, gastric retention, and analgesic vs. prokinetic/antiemetic drug use. Methods: 339 gastroparesis patients were enrolled from 6 centers of the NIDDK Gastroparesis Clinical Research Consortium from 1/ 07-11/09. Survey, exam, and gastric emptying data were compared in those with pain vs. no pain and pain as the predominant symptom vs. predominant nausea or vomiting. Results: 243 patients (72%) noted abdominal pain; pain was the predominant symptom in 65 (19%) vs. nausea or vomiting in 194 (57%). On Patient Assessment of GI Symptoms surveys, upper abdominal pain scores were higher in those with pain (3.5±1.4) vs. no pain (1.9±1.8, P<0.0001). Lower abdominal pain scores also were higher with (2.3±1.7) vs. no pain (1.4±1.5, P<0.0001). Higher percentages of those with pain were women (86 vs. 76%, P= 0.04) and had idiopathic etiology (70 vs. 54%, P=0.007). Infection-like prodromes were similar with (17%) vs. no pain (19%, P=0.75). Investigator-rated gastroparesis severity was similar with vs. no pain (P=0.25). Patient Assessment of GI Quality of Life (PAGI-QOL) scores were lower with pain (2.3±1.1 vs. 2.8±1.1, P<0.0007). SF-36 physical function (P<0.0001), energy/fatigue (P=0.004), emotional wellbeing (P=0.02), social function (P= 0.0004), pain (P<0.0001), and general health (P=0.02) scores were lower with pain. 4 hr gastric retention (normal <10%) was similar with (32±24%) vs. no pain (33±22%, P=0.91). Opiate use was higher with pain (P<0.0001), while antidepressant (P=0.22), neuropathic pain modulator (P=0.10), prokinetic (P=0.40), and antiemetic (P=0.13) use did not significantly differ. Compared to those with predominant nausea or vomiting, patients with predominant pain had higher upper abdominal pain scores (P<0.0001), but similar severity (P= 0.91), lower abdominal pain scores (P=0.21), 4 hr gastric retention (P=0.96), PAGI-QOL and SF-36 scores (P=NS). Conclusions: Abdominal pain is prevalent in gastroparesis and is predominant in 1 in 5 patients. Pain, like nausea and vomiting, impairs quality of life. Pain is associated with female gender and idiopathic etiology but not gastric retention. Increased opiate use reflects its health care impact and underscores a need to investigate new treatment options for pain. Our findings provide insight into this important symptom and emphasize its negative influence in gastroparesis.
M2026 Appetite Reduction and Weight Loss During Antiviral Therapy for Hepatitis C are Associated With Impaired Gastric Emptying and Altered Gastrointestinal Hormone Secretion. Mark Ellrichmann, Ina D. Boehm, Jens J. Holst, Wolfgang E. Schmidt, Jan-Michel Otte, Juris J. Meier
M2024 Ghrelin Stimulates Gastric Contraction in a Specific Physiological Condition In Vivo and In Vitro in the House Musk Shrew (Suncus Murinus), a GhrelinAnd Motilin-Producing Laboratory Animal Zuoyun Xie, Satoshi Sakahara, Satoya Hoshino, Yuko Ishida, Airi Suzuki, Kanako Koike, Yuki Miyano, Yuta Kodaira, Toku Takahashi, Sen-ichi Oda, Takafumi Sakai
Background and Aims: Weight loss and inappetence occur frequently during antiviral combination treatment for hepatitis C, thereby leading to impaired adherence and lowered response rates. The mechanisms underlying these side effects are unknown. We therefore investigated the effects of Peg-Interferon alpha 2a and Ribavirin on gastrointestinal hormone regulation, satiety ratings and gastric motility. Methods: 35 patients with chronic hepatitis C were studied before, during (at 12 week intervals over 48 weeks) and 24 weeks after antiviral therapy with Peg-Interferon alpha 2a and Ribavirin. Gastric emptying was assessed using a 13C octanoate breath test, and plasma levels of ghrelin, CCK, PYY, GLP-1 and PP were determined by radioimmunoassay over 240 min after mixed meal ingestion. Hunger and satiety were assessed using visual analogue scales (VAS). Results: Antiviral therapy resulted in a significant weight loss in all patients (mean 6.3 ± 3.1 kg; p=0.04), while hunger ratings decreased (p<0.001) and satiety increased (p<0.001). This was associated with an increase in the postprandial concentrations of CCK, PYY and GLP-1 (p<0.05, respectively), whereas fasting levels were unaffected. In contrast, fasting ghrelin and PP concentrations increased by 134,9% and 325,3%, respectively (p<0.0001). Of note, the changes in gastric emptying, hunger ratings and GI hormone concentrations were completely normalised 24 weeks after cessation of antiviral treatment. The treatment-related changes in body weight were highly correlated to the respective changes in the integrated postprandial concentrations of CCK, PYY and GLP-1 (p<0.05). Conclusion: Changes in body weight and appetite sensations are associated with alterations in gastric emptying and GI hormone levels. Most likely, increased concentrations of the anorexigenic peptides CCK, PYY and GLP-1 augment postprandial satiety leading to reduced caloric intake. Elevations in fasting ghrelin levels may represent an attempted compensation to raise hunger levels. Modulators of GI hormone secretion, such as CCK antagonists, may therefore potentially improve the adherence and outcome of antiviral therapy for chronic hepatitis C.
Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is released by the stomach during fasting and has recently been shown to stimulate gastrointestinal motility and gastric emptying. It has been reported that ghrelin and motilin and their receptors represent a family of gastrointestinal hormones and that these hormones have complementary physiological effects. However, the mechanism by which gastric contraction is induced by ghrelin administration and the effects of coadministration of motilin and ghrelin on gastric motility are still obscure. To clarify these points, we studied the effects of ghrelin/motilin family on gastric contraction In Vivo and In Vitro by using the musk shrew (Suncus murinus), a ghrelin- and motilin-producing laboratory animal. For the In Vivo experiment, a strain gauge transducer was sutured on the suncus stomach to monitor gastric contraction activity. In a fasted state, clear phase I, phase II and phase III contractions, almost the same as these found in humans and dogs, were observed in the conscious suncus gastric body, and typical gastric phase III contractions at 80 -150 min intervals were found. Ten-minute i.v. infusion of motilin (50 ng kg-1 min-1) or erythromycin (80 μg kg-1 min-1) provoked strong gastric contraction in the suncus stomach, and these contractions were blocked by pretreatment with atropine. Then ghrelin was administrated at the timing of different phases of migrating motor complex (MMC), and it was found that ten-minute i.v. infusion of ghrelin (0.1, 0.5, 1, 5 μg kg-1 min-1) did not stimulate gastric contraction in the first half of phase I but that each dose of ghrelin significantly evoked gastric contraction in the latter half of phase I. Ghrelin administration (0.1, 0.2 μg kg-1 min-1) in phase II also stimulated gastric contraction in a dose-dependent manner. For the In Vitro experiment using an organ bath, although even a high dose of ghrelin did not stimulate contraction of suncus stomach preparations, ghrelin administration (10-10 M ~ 10-7 M) after pretreatment with a low dose of motilin (10-10 M) caused dose-related stimulation of gastric contraction. Moreover, we determined the cDNA sequences of suncus GPR-38 and GHS-R and demonstrated that mRNAs of these receptors are expressed in the suncus stomach. Considering that the plasma concentrations of motilin in fasted dogs and humans gradually increased from later phase I, these results suggest that, ghrelin-induced gastric contraction in the suncus is dependent on the intrinsic motilin concentration.
M2027 Impairment of Mitochondrial Function Alters Global Ca2+ Mobilization but Not Contractility in Aged Mouse Colon Francisco E. Martín-Cano, Luz Fatima Barriga Sanchez, Raquel Ronco, Ignacio Morales, Pedro J. Camello, Maria J. Pozo Background: Functional gastrointestinal (GI) disorders are common in the elderly and the idea that many age-related disorders are due to motility abnormalities is attracting attention. Mitochondrial dysfunction and associated oxidant stress have been linked with aging. In smooth muscle cells, as well as other cell types, mitochondrial Ca2+ sequestration and release influences spatial and temporal patterns of Ca2+ transients in the cytoplasm. Aim: To determine the effects of aging on mitochondrial participation in Ca2+ mobilization and contractility of mouse colon smooth muscle and the effects of melatonin treatment. Methods: Young (3 months old) and senescent (22-24 months old) Swiss OF1 mice were used in the study. In a group of senescent animals, melatonin treatment (10 mg/Kg/day, during the dark phase) started at the age of 13 months. Colonic contractility was assessed by isometric
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AGA Abstracts
AGA Abstracts
M2023
AGA Abstracts
tension recordings of KCl- and bethanechol-induced contractions in circular orientated myenteric strips. Intracellular calcium was determined by fluorescence microscopy in isolated smooth muscle cells loaded with fura-2-AM (ExWL 340/380nm, EmWL 510nm) and calcium changes were expressed as the ratio between elicited fluorescence at 340nm and 380nm (F340/380). Mitochondrial membrane potential was measured in isolated colonic smooth muscle cells using the cell-permeant green fluorescent dye rhodamine 123 Results: Aging caused an increase in caffeine-induced Ca2+ release (140 % respect to adult cells) and a decrease in mitochondrial membrane potential, as indicated by the reduction of the heterogeneity of the rhodamine123 fluorescence signal in isolated cells. When mitochondria function was impaired using rotenone (1 μM) plus oligomycin (5 μM) the amplitude of Ca2+ release was significantly reduced (by 45%) and the [Ca2+]i decay showed a slower rate in senescent cells. Melatonin treatment protected from the deleterious effects of these agents. These changes in Ca2+ homeostasis did not translate into alterations in contractility, since pretreatment of both proximal and distal circular myenteric strips with rotenone, oligomicin of both for 30 min did not modify significantly the contractile response to neither KCl (60 mM) nor bethanechol (100 μM). Conclusion: Mitochondria play a more important role in Ca2+ homeostasis in aged than adult cells, however these changes are not enough to modify contractility, which suggest that in colonic smooth muscle mitochondrial respiratory chain is not essencial for contraction and that more sources of ATP than ATP synthase coexist in these cells. Supported by BFU2007-60563 and JEX
M2030 A High-Resolution View of Fasting Antroduodenal Motor Function Claudia P. Sanmiguel, Jeffrey L. Conklin, Mane Arabyan, Robert Tabrizi, Edy E. Soffer Fasting motor activity of the stomach and small bowel (SB) is characterized by a stereotyped, cyclical pattern. Most studies of antroduodenal motility were done using manometry catheters with a relatively wide separation between consecutive sensors. Contractile activity between sensors is not seen, making interpretation of contraction patterns and propagation difficult. High-resolution manometry (HRM) allows better spatial discrimination of GI motor patterns. Aim: To characterize fasting antroduodenal motor activity in healthy volunteers using HRM. Methods: 15 subjects, 9 females, age 33.8±6.7 yr, underwent antroduodenal manometry using a catheter with 36 solid-state sensors positioned at 1 cm intervals. It spanned from distal stomach to proximal SB. Propagated gastric contractions were classified as: 1) propagated (P), if they reached the pyloric region; 2) interrupted propagation (IP) if they arrested before reaching the pyloric region and 3) propagated to SB (SBP) if they traversed the pyloric region into the SB. Visual and computer-assisted analyses of all recordings were performed. Data are mean±SD. Results: Length of the intragastric catheter segment was 15.3 (range 430 cm), and the intraduodenal segment 20.7 (range 6-32 cm). In 3 subjects, the intragastric segment was too short to evaluate antral motility. In phase II, 63% of gastric contractions stopped 9.1±1.2 cm above the pyloric region, 33% were P and 4.2% were SBP, which propagated 27.2±9.5 cm into the SB. An almost constant low-amplitude (<10mmHg), 3/ minute, gastric activity propagated towards the pylorus in 10 subjects during phase II. It was also seen during phase I in 4, and in 3 during phase III isolated to the SB. This pattern could not be discerned from recordings in line mode. In phase II, gastric, pyloric region and duodenal contractions differed in velocity (0.4±0.1 vs. 0.7±0.1 vs. 2.1±1.4 cm/s respectively) and amplitude (19.1±4.1 vs. 60.1±14 vs. 19.1±7, respectively). Eight subjects had typical SB phase III activity, but in only 1 was there an accompanying typical gastric component. Gastric contractions during phase III, for the most part, did not propagate into the SB. SB phase III lasted 6.1±2.9 min, propagated 15±7.5 cm into SB, with a mean pressure of 38.8±15 mmHg, frequency of 11.4±0.7/min and velocity of 2.1±0.3 cm/s. Conclusions: HRM permits a more detailed characterization of antroduodenal motility, specifically patterns of propagation. We observed previously undescribed low amplitude 3/min cyclic gastric contractions during phases I and II of the MMC. The typical gastric phase III was uncommonly seen.
M2028 The use of a New Wireless Motility Device (SmartPill®) for Measurement of Gastrointestinal Transit Time After Dietary Fiber Intervention Derek A. Timm, Holly Willis, William Thomas, David Willis, Lisa Sanders, Thomas Boileau, Dan Holmberg, Joanne L. Slavin Although it is generally accepted that one of the beneficial health effects of dietary fiber is decreasing gastrointestinal transit time, few recent studies have measured transit time with diet intervention. Historically, measurement of gastrointestinal transit time has required collection of fecal samples for up to 7 days after swallowing radio-opaque pellets and xraying collected fecal samples; a tedious, labor-intensive technique for both subjects and investigators. Recently, a wireless motility capsule (Smartpill®) capable of measuring gut pH, pressure, and temperature in real time was developed. The Smartpill® device is able to measure gastric emptying time by pH change and whole gut transit time by temperature change. Intestinal transit time is the difference between whole gut transit time and gastric emptying time. This device, however, has not been validated with dietary interventions. Therefore, we conducted a crossover trial to determine if the device could detect a significant difference in gastric emptying, intestinal and whole gut transit time after subjects consumed 20 grams of wheat bran fiber or an equal volume, fiber-free control. This dose of wheat bran has previously been shown to decrease whole gut transit time using traditional methods. In this study, ten healthy subjects (5 men, 5 women) swallowed the Smartpill® after consuming a standard breakfast with or without 20 grams of wheat bran. A paired t-test was used to determine differences in transit times. Whole gut transit time decreased by 9.5 ± 2.9 hours (p=0.0092) after consumption of the wheat bran. This decrease was due entirely to changes in intestinal transit time as gastric emptying time did not differ between the treatments. In addition, no differences were observed between genders. Results show, with minimal participant burden, the Smartpill® technology appears to be a useful tool for assessing transit time after dietary intervention. In conclusion, wheat bran significantly reduces transit time and the Smartpill® technology shows promise for digestive studies with novel fibers and other ingredients that are promoted for gut health.
M2031 Activation of SST1 and SST2 Receptors Decreases Neurogenic Chloride Secretion via VIP Secretomotor Neurons in Guinea Pig Small Intestine Jaime Pei Pei Foong, Laura J. Parry, Joel C. Bornstein Purpose: Vasoactive intestinal peptide (VIP) submucosal neurons are, major regulators of gut water and salt secretion. These neurons display inhibitory postsynaptic potentials that are mediated by somatostatin (SOM) acting on SST1 and SST2 receptors in guinea-pig small intestine. As SOM is anti-secretory, the present study aimed to determine if the inhibitory SST1 and SST2 receptors found in VIP neurons have a functional role in controlling gut secretion. Methods: Mucosal-submucosal preparations obtained from guinea-pig jejunum were mounted on Ussing chamber set-ups to measure chloride (Cl-) secretion across the intestine, which is reflected by an increase in short circuit current (Isc). All drugs were added serosally. Veratridine (1 μM) induced a robust secretory response for subsequent pharmacological investigation. 5-HT (300 nM) was used to specifically activate non-cholinergic secretomotor neurons, while DMPP (nicotinic agonist, 10 μM) stimulated all secretomotor neurons. Results: SOM (50 nM) induced a TTX (1 μM)-sensitive decrease in basal secretion. SOM also significantly reduced the veratridine-induced increase in Isc (p < 0.001), an effect abolished by blocking SST1 receptors (SRA 880 3 μM) and partially diminished by blocking SST2 receptors (CYN 154806 100 nM). Blocking these receptors also significantly reduced the SOM-induced decrease in basal secretion. Quantitiative PCR demonstrated that SST1 and SST2 receptor were more highly expressed in the submucous plexus compared to the mucosal layer. However, SST1 receptor mRNA levels were significantly (p < 0.01) higher than SST2 within both layers. Serosal application of DMPP produced a biphasic response. The first phase was a rapid rise in Isc that was abolished by hyoscine (1 μM), the second phase was a slower, more sustained response that was not significantly affected by hyoscine indicating it was due to activation of the VIP neurons. Responses to DMPP and 5-HT (300 nM) were TTX-sensitive. The 5-HT-induced increase in Isc, and the second more sustained phase evoked by DMPP were significantly reduced by SOM. Conclusion: These data suggest that SOM exerts its antisecretory effects indirectly by supressing firing of VIP non-cholinergic secretomotor neurons, rather than via a direct action on mucosal enterocytes. SOM acts predominately on SST1 receptors, which suggests a greater role of SST1 receptors in the regulation of gut secretion. SST1 receptors could be potential therapeutic targets for treating secretory diarrhea resulting from overactivity of the enteric nervous system, like that produced by cholera and other bacterial toxins.
M2029 Relaxatory Responses of Mouse Lower Gastrointestinal Tract Mediated to Transient Receptor Potential Melastatin Type-8 (TRPM8) Channel Activation Takuji Hosoya, Kenjiro Matsumoto, Kimihito Tashima, Toshihiko Murayama, Syunji Horie BACKGROUND & AIMS: Transient receptor potential melastatin type-8 (TRPM8) is a nonselective cation channels expressed in primary sensory neurons, and is activated by innocuous cold ( or cool, < 27 degrees C), icilin, menthol and a number of other odorants and cooling compounds. In recent clinical paper, it has reported that peppermint oil containing menthol is more effective than placebo in the treatment of irritable bowel syndrome. However, the distribution and the role of TRPM8 in motor function of lower gastrointestinal tract have hardly examined. In the present study, we investigated the localization of TRPM8 channels and effect of TRPM8 agonists on smooth muscle tension in lower gastrointestinal tract. METHODS: TRPM8-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification in both transverse and horizontal sections of mouse lower gastrointestinal tract from C57BL/6J mice. Calretinin, c-Kit and neuronal nitric oxide synthase (nNOS)-immunoreactivity were studied by normal indirect staining with the corresponding specific antibodies. The rectum and distal, transverse and proximal colon were isolated. The longitudinal change in smooth muscle tone was isometrically measured using Magnus apparatus. RESULTS: In immunohistochemical study, TRPM8immunoreactivties were found in the mucosal, submucosal and muscle layers. Especially, TRPM8-immunoreactivities were abundant in deep muscular plexus, circular muscle, myenteric plexus and longidutinal muscle. Next, double labaling studies on TRPM8 were carried out using the intrinsic neuron marker calretinin, interstetial cells of Cajal (ICC) marker cKit and nNOS. Double-labeling for TRPM8/calretinin and TRPM8/nNOS was slightly detected in muscle layer. However, double-labeling for TRPM8/c-Kit was not observed. In the Magnus experiment, menthol (30-300 μM) and icilin (10-100 μM) induced a concentration-dependent relaxation in rectum and distal, transverse and proximal colon preparations. The selective TRPM8 agonist, WS-12 (3-30 μM) also induced a long-lasting relaxation in rectum and colon contracted by 40 mM KCl. DISCUSSION: These results suggest that TRPM8 channels are largely localized in lower gastrointestinal tract of mucosa, submucosa and muscle layers and are involved in inhibition of colonic and rectal motor function. TRPM8 channels may be localized in intrinsic neuron, but not in ICC. It is probably that TRPM8 neurons are correlated with nitrergic neurons.
AGA Abstracts
M2032 Longitudinal Muscle Physiological Specificity Correlates With the Differences in Expression and Phosphorylation of Contractile Proteins Sita Somara, Daniela Musaka, Shreya Raghavan, Robert R. Gilmont, Khalil Bitar Background: Colonic motility is a collective response of circular and longitudinal smooth muscle contraction. Circular smooth muscle cells (CSMC) are aligned concentrically around the lumen while longitudinal smooth muscle cell (LSMC) are aligned parallel to the length of the colon. Differences in innervation and myogenic characteristic of LSMC and CSMC allow the regulation of the coordinated contraction required for peristalsis. Hypothesis: The myogenic differences between basal tone of longitudinal and circular muscles of the colon could be due to the fundamental differences in the basal level of expression and phosphorylation of contractile proteins in each cell type. Objective: To correlates the differences in basal tone to the differences in the expression and phosphorylation of contractile proteins at basal level. Methods: Whole cell lysates from confluent cultured CSMC and LSMC isolated
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Importance of Abdominal Pain as a Symptom in Gastroparesis: Relation to Clinical Factors, Disease Severity, Quality of Life, Gastric Retention, and Medication use William L. Hasler, Laura Wilson, Henry P. Parkman, Kenneth L. Koch, Thomas L. Abell, Pankaj J. Pasricha, Linda Nguyen, William J. Snape, Gianrico Farrugia, James Tonascia, Aynur Unalp-Arida, Linda A. Lee, Frank A. Hamilton
Investigation of Roles of 5-HT2B Receptors in Modulation of Colonic Motility in the Anaesthetized Rat Katsuyo Ohashi-Doi, Billie Hunne, Dorota Ferens, Nobuyuki Takahashi, John B. Furness Tegaserod is a prokinetic agent for constipation dominant irritable bowel syndrome whose effect was initially attributed to 5-HT4 receptor activation. However tegaserod is both a 5HT4 receptor agonist and a 5-HT2B receptor antagonist. Recently, we reported that RS127445, a selective 5-HT2B receptor antagonist, inhibited defecation and visceral hypersensitivity induced by TNBS in rats (Ohashi-Doi et al., 2009). This study aimed to characterise the effects of 5-HT2B receptor antagonism on colonic motility in the naïve rat. Male SpragueDawley rats (300 - 400g) were anaesthetized, the colon was cannulated in the region of the colonic flexure and at the anus for measurement of colonic motility and propulsion. The body wall was closed with the oral cannula protruding. The oral cannula was connected to a Marriotte bottle filled with PBS, and the distal cannula was connected to a pressure transducer, one way value and fluid outlet. RS-127445 was dissolved in 5% well-solve solution and given intravenously. The data are presented as mean ± S.E. (n=5-6). Other rats were used in the PCR study. Segments of fundus, proximal colon and distal colon were dissected to give EM/MP (external muscle/ myenteric plexus) and mucosal samples. RNA was extracted from the samples and reverse transcribed to cDNA, and then amplified by PCR using primers specific for 5HT2B (forward-ATAGCCATCAAAAAGCCAA, reverseTGTTAGGCGTTGAGGTGG) In naïve control rats, when the basal intraluminal pressure in the colon was 6.46 ± 0.03 mmHg, small pressure oscillations were observed (maximum intraluminal pressure, 8.4±0.1 mmHg). The single contractions had durations of 2.1±0.1 min; the number of contractions was 9.0±1.6 in 20 min periods. RS-127445 (1 mg/kg/hr, i.v.) significantly reduced the cycle of colonic contraction (number of contractions, 5.3±0.9; interval, 3.8±0.5 min; P<0.05). However, RS-127445 did not affect either basal intraluminal or maximum pressure of the colon in the rat. L-NAME (10 mg/kg, i.v.) significantly increased the number of contractions (16 ± 1.1 / 20 min, P<0.01) and maximum intraluminal pressure (44±1 vs. 23±2 %, P<0.01). Pre-treatment with RS-127445 partially inhibited increased colonic contractions induced by L-NAME in rats (11±0.9 / 20 min, P<0.05). The duration of single contractions was reduced 2.2±0.47 min (P<0.01). PCR results indicated the presence of 5-HT2B receptor transcripts in the fundus, proximal and distal colon in the rats. These results suggest that 5-HT2B receptors are involved in modulation of colonic motility, and that a selective 5-HT2B receptor antagonist might be a useful therapeutic approach for the treatment of functional gut disorders.
Background: While gastroparesis usually presents with nausea or vomiting, abdominal pain also may be part of the presenting illness. The importance of pain in gastroparesis and its associated factors are largely unexplored. Aims: Define abdominal pain prevalence in gastroparesis and relate to demography, etiology, severity, quality of life, gastric retention, and analgesic vs. prokinetic/antiemetic drug use. Methods: 339 gastroparesis patients were enrolled from 6 centers of the NIDDK Gastroparesis Clinical Research Consortium from 1/ 07-11/09. Survey, exam, and gastric emptying data were compared in those with pain vs. no pain and pain as the predominant symptom vs. predominant nausea or vomiting. Results: 243 patients (72%) noted abdominal pain; pain was the predominant symptom in 65 (19%) vs. nausea or vomiting in 194 (57%). On Patient Assessment of GI Symptoms surveys, upper abdominal pain scores were higher in those with pain (3.5±1.4) vs. no pain (1.9±1.8, P<0.0001). Lower abdominal pain scores also were higher with (2.3±1.7) vs. no pain (1.4±1.5, P<0.0001). Higher percentages of those with pain were women (86 vs. 76%, P= 0.04) and had idiopathic etiology (70 vs. 54%, P=0.007). Infection-like prodromes were similar with (17%) vs. no pain (19%, P=0.75). Investigator-rated gastroparesis severity was similar with vs. no pain (P=0.25). Patient Assessment of GI Quality of Life (PAGI-QOL) scores were lower with pain (2.3±1.1 vs. 2.8±1.1, P<0.0007). SF-36 physical function (P<0.0001), energy/fatigue (P=0.004), emotional wellbeing (P=0.02), social function (P= 0.0004), pain (P<0.0001), and general health (P=0.02) scores were lower with pain. 4 hr gastric retention (normal <10%) was similar with (32±24%) vs. no pain (33±22%, P=0.91). Opiate use was higher with pain (P<0.0001), while antidepressant (P=0.22), neuropathic pain modulator (P=0.10), prokinetic (P=0.40), and antiemetic (P=0.13) use did not significantly differ. Compared to those with predominant nausea or vomiting, patients with predominant pain had higher upper abdominal pain scores (P<0.0001), but similar severity (P= 0.91), lower abdominal pain scores (P=0.21), 4 hr gastric retention (P=0.96), PAGI-QOL and SF-36 scores (P=NS). Conclusions: Abdominal pain is prevalent in gastroparesis and is predominant in 1 in 5 patients. Pain, like nausea and vomiting, impairs quality of life. Pain is associated with female gender and idiopathic etiology but not gastric retention. Increased opiate use reflects its health care impact and underscores a need to investigate new treatment options for pain. Our findings provide insight into this important symptom and emphasize its negative influence in gastroparesis.
M2026 Appetite Reduction and Weight Loss During Antiviral Therapy for Hepatitis C are Associated With Impaired Gastric Emptying and Altered Gastrointestinal Hormone Secretion. Mark Ellrichmann, Ina D. Boehm, Jens J. Holst, Wolfgang E. Schmidt, Jan-Michel Otte, Juris J. Meier
M2024 Ghrelin Stimulates Gastric Contraction in a Specific Physiological Condition In Vivo and In Vitro in the House Musk Shrew (Suncus Murinus), a GhrelinAnd Motilin-Producing Laboratory Animal Zuoyun Xie, Satoshi Sakahara, Satoya Hoshino, Yuko Ishida, Airi Suzuki, Kanako Koike, Yuki Miyano, Yuta Kodaira, Toku Takahashi, Sen-ichi Oda, Takafumi Sakai
Background and Aims: Weight loss and inappetence occur frequently during antiviral combination treatment for hepatitis C, thereby leading to impaired adherence and lowered response rates. The mechanisms underlying these side effects are unknown. We therefore investigated the effects of Peg-Interferon alpha 2a and Ribavirin on gastrointestinal hormone regulation, satiety ratings and gastric motility. Methods: 35 patients with chronic hepatitis C were studied before, during (at 12 week intervals over 48 weeks) and 24 weeks after antiviral therapy with Peg-Interferon alpha 2a and Ribavirin. Gastric emptying was assessed using a 13C octanoate breath test, and plasma levels of ghrelin, CCK, PYY, GLP-1 and PP were determined by radioimmunoassay over 240 min after mixed meal ingestion. Hunger and satiety were assessed using visual analogue scales (VAS). Results: Antiviral therapy resulted in a significant weight loss in all patients (mean 6.3 ± 3.1 kg; p=0.04), while hunger ratings decreased (p<0.001) and satiety increased (p<0.001). This was associated with an increase in the postprandial concentrations of CCK, PYY and GLP-1 (p<0.05, respectively), whereas fasting levels were unaffected. In contrast, fasting ghrelin and PP concentrations increased by 134,9% and 325,3%, respectively (p<0.0001). Of note, the changes in gastric emptying, hunger ratings and GI hormone concentrations were completely normalised 24 weeks after cessation of antiviral treatment. The treatment-related changes in body weight were highly correlated to the respective changes in the integrated postprandial concentrations of CCK, PYY and GLP-1 (p<0.05). Conclusion: Changes in body weight and appetite sensations are associated with alterations in gastric emptying and GI hormone levels. Most likely, increased concentrations of the anorexigenic peptides CCK, PYY and GLP-1 augment postprandial satiety leading to reduced caloric intake. Elevations in fasting ghrelin levels may represent an attempted compensation to raise hunger levels. Modulators of GI hormone secretion, such as CCK antagonists, may therefore potentially improve the adherence and outcome of antiviral therapy for chronic hepatitis C.
Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is released by the stomach during fasting and has recently been shown to stimulate gastrointestinal motility and gastric emptying. It has been reported that ghrelin and motilin and their receptors represent a family of gastrointestinal hormones and that these hormones have complementary physiological effects. However, the mechanism by which gastric contraction is induced by ghrelin administration and the effects of coadministration of motilin and ghrelin on gastric motility are still obscure. To clarify these points, we studied the effects of ghrelin/motilin family on gastric contraction In Vivo and In Vitro by using the musk shrew (Suncus murinus), a ghrelin- and motilin-producing laboratory animal. For the In Vivo experiment, a strain gauge transducer was sutured on the suncus stomach to monitor gastric contraction activity. In a fasted state, clear phase I, phase II and phase III contractions, almost the same as these found in humans and dogs, were observed in the conscious suncus gastric body, and typical gastric phase III contractions at 80 -150 min intervals were found. Ten-minute i.v. infusion of motilin (50 ng kg-1 min-1) or erythromycin (80 μg kg-1 min-1) provoked strong gastric contraction in the suncus stomach, and these contractions were blocked by pretreatment with atropine. Then ghrelin was administrated at the timing of different phases of migrating motor complex (MMC), and it was found that ten-minute i.v. infusion of ghrelin (0.1, 0.5, 1, 5 μg kg-1 min-1) did not stimulate gastric contraction in the first half of phase I but that each dose of ghrelin significantly evoked gastric contraction in the latter half of phase I. Ghrelin administration (0.1, 0.2 μg kg-1 min-1) in phase II also stimulated gastric contraction in a dose-dependent manner. For the In Vitro experiment using an organ bath, although even a high dose of ghrelin did not stimulate contraction of suncus stomach preparations, ghrelin administration (10-10 M ~ 10-7 M) after pretreatment with a low dose of motilin (10-10 M) caused dose-related stimulation of gastric contraction. Moreover, we determined the cDNA sequences of suncus GPR-38 and GHS-R and demonstrated that mRNAs of these receptors are expressed in the suncus stomach. Considering that the plasma concentrations of motilin in fasted dogs and humans gradually increased from later phase I, these results suggest that, ghrelin-induced gastric contraction in the suncus is dependent on the intrinsic motilin concentration.
M2027 Impairment of Mitochondrial Function Alters Global Ca2+ Mobilization but Not Contractility in Aged Mouse Colon Francisco E. Martín-Cano, Luz Fatima Barriga Sanchez, Raquel Ronco, Ignacio Morales, Pedro J. Camello, Maria J. Pozo Background: Functional gastrointestinal (GI) disorders are common in the elderly and the idea that many age-related disorders are due to motility abnormalities is attracting attention. Mitochondrial dysfunction and associated oxidant stress have been linked with aging. In smooth muscle cells, as well as other cell types, mitochondrial Ca2+ sequestration and release influences spatial and temporal patterns of Ca2+ transients in the cytoplasm. Aim: To determine the effects of aging on mitochondrial participation in Ca2+ mobilization and contractility of mouse colon smooth muscle and the effects of melatonin treatment. Methods: Young (3 months old) and senescent (22-24 months old) Swiss OF1 mice were used in the study. In a group of senescent animals, melatonin treatment (10 mg/Kg/day, during the dark phase) started at the age of 13 months. Colonic contractility was assessed by isometric
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M2023
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tension recordings of KCl- and bethanechol-induced contractions in circular orientated myenteric strips. Intracellular calcium was determined by fluorescence microscopy in isolated smooth muscle cells loaded with fura-2-AM (ExWL 340/380nm, EmWL 510nm) and calcium changes were expressed as the ratio between elicited fluorescence at 340nm and 380nm (F340/380). Mitochondrial membrane potential was measured in isolated colonic smooth muscle cells using the cell-permeant green fluorescent dye rhodamine 123 Results: Aging caused an increase in caffeine-induced Ca2+ release (140 % respect to adult cells) and a decrease in mitochondrial membrane potential, as indicated by the reduction of the heterogeneity of the rhodamine123 fluorescence signal in isolated cells. When mitochondria function was impaired using rotenone (1 μM) plus oligomycin (5 μM) the amplitude of Ca2+ release was significantly reduced (by 45%) and the [Ca2+]i decay showed a slower rate in senescent cells. Melatonin treatment protected from the deleterious effects of these agents. These changes in Ca2+ homeostasis did not translate into alterations in contractility, since pretreatment of both proximal and distal circular myenteric strips with rotenone, oligomicin of both for 30 min did not modify significantly the contractile response to neither KCl (60 mM) nor bethanechol (100 μM). Conclusion: Mitochondria play a more important role in Ca2+ homeostasis in aged than adult cells, however these changes are not enough to modify contractility, which suggest that in colonic smooth muscle mitochondrial respiratory chain is not essencial for contraction and that more sources of ATP than ATP synthase coexist in these cells. Supported by BFU2007-60563 and JEX
M2030 A High-Resolution View of Fasting Antroduodenal Motor Function Claudia P. Sanmiguel, Jeffrey L. Conklin, Mane Arabyan, Robert Tabrizi, Edy E. Soffer Fasting motor activity of the stomach and small bowel (SB) is characterized by a stereotyped, cyclical pattern. Most studies of antroduodenal motility were done using manometry catheters with a relatively wide separation between consecutive sensors. Contractile activity between sensors is not seen, making interpretation of contraction patterns and propagation difficult. High-resolution manometry (HRM) allows better spatial discrimination of GI motor patterns. Aim: To characterize fasting antroduodenal motor activity in healthy volunteers using HRM. Methods: 15 subjects, 9 females, age 33.8±6.7 yr, underwent antroduodenal manometry using a catheter with 36 solid-state sensors positioned at 1 cm intervals. It spanned from distal stomach to proximal SB. Propagated gastric contractions were classified as: 1) propagated (P), if they reached the pyloric region; 2) interrupted propagation (IP) if they arrested before reaching the pyloric region and 3) propagated to SB (SBP) if they traversed the pyloric region into the SB. Visual and computer-assisted analyses of all recordings were performed. Data are mean±SD. Results: Length of the intragastric catheter segment was 15.3 (range 430 cm), and the intraduodenal segment 20.7 (range 6-32 cm). In 3 subjects, the intragastric segment was too short to evaluate antral motility. In phase II, 63% of gastric contractions stopped 9.1±1.2 cm above the pyloric region, 33% were P and 4.2% were SBP, which propagated 27.2±9.5 cm into the SB. An almost constant low-amplitude (<10mmHg), 3/ minute, gastric activity propagated towards the pylorus in 10 subjects during phase II. It was also seen during phase I in 4, and in 3 during phase III isolated to the SB. This pattern could not be discerned from recordings in line mode. In phase II, gastric, pyloric region and duodenal contractions differed in velocity (0.4±0.1 vs. 0.7±0.1 vs. 2.1±1.4 cm/s respectively) and amplitude (19.1±4.1 vs. 60.1±14 vs. 19.1±7, respectively). Eight subjects had typical SB phase III activity, but in only 1 was there an accompanying typical gastric component. Gastric contractions during phase III, for the most part, did not propagate into the SB. SB phase III lasted 6.1±2.9 min, propagated 15±7.5 cm into SB, with a mean pressure of 38.8±15 mmHg, frequency of 11.4±0.7/min and velocity of 2.1±0.3 cm/s. Conclusions: HRM permits a more detailed characterization of antroduodenal motility, specifically patterns of propagation. We observed previously undescribed low amplitude 3/min cyclic gastric contractions during phases I and II of the MMC. The typical gastric phase III was uncommonly seen.
M2028 The use of a New Wireless Motility Device (SmartPill®) for Measurement of Gastrointestinal Transit Time After Dietary Fiber Intervention Derek A. Timm, Holly Willis, William Thomas, David Willis, Lisa Sanders, Thomas Boileau, Dan Holmberg, Joanne L. Slavin Although it is generally accepted that one of the beneficial health effects of dietary fiber is decreasing gastrointestinal transit time, few recent studies have measured transit time with diet intervention. Historically, measurement of gastrointestinal transit time has required collection of fecal samples for up to 7 days after swallowing radio-opaque pellets and xraying collected fecal samples; a tedious, labor-intensive technique for both subjects and investigators. Recently, a wireless motility capsule (Smartpill®) capable of measuring gut pH, pressure, and temperature in real time was developed. The Smartpill® device is able to measure gastric emptying time by pH change and whole gut transit time by temperature change. Intestinal transit time is the difference between whole gut transit time and gastric emptying time. This device, however, has not been validated with dietary interventions. Therefore, we conducted a crossover trial to determine if the device could detect a significant difference in gastric emptying, intestinal and whole gut transit time after subjects consumed 20 grams of wheat bran fiber or an equal volume, fiber-free control. This dose of wheat bran has previously been shown to decrease whole gut transit time using traditional methods. In this study, ten healthy subjects (5 men, 5 women) swallowed the Smartpill® after consuming a standard breakfast with or without 20 grams of wheat bran. A paired t-test was used to determine differences in transit times. Whole gut transit time decreased by 9.5 ± 2.9 hours (p=0.0092) after consumption of the wheat bran. This decrease was due entirely to changes in intestinal transit time as gastric emptying time did not differ between the treatments. In addition, no differences were observed between genders. Results show, with minimal participant burden, the Smartpill® technology appears to be a useful tool for assessing transit time after dietary intervention. In conclusion, wheat bran significantly reduces transit time and the Smartpill® technology shows promise for digestive studies with novel fibers and other ingredients that are promoted for gut health.
M2031 Activation of SST1 and SST2 Receptors Decreases Neurogenic Chloride Secretion via VIP Secretomotor Neurons in Guinea Pig Small Intestine Jaime Pei Pei Foong, Laura J. Parry, Joel C. Bornstein Purpose: Vasoactive intestinal peptide (VIP) submucosal neurons are, major regulators of gut water and salt secretion. These neurons display inhibitory postsynaptic potentials that are mediated by somatostatin (SOM) acting on SST1 and SST2 receptors in guinea-pig small intestine. As SOM is anti-secretory, the present study aimed to determine if the inhibitory SST1 and SST2 receptors found in VIP neurons have a functional role in controlling gut secretion. Methods: Mucosal-submucosal preparations obtained from guinea-pig jejunum were mounted on Ussing chamber set-ups to measure chloride (Cl-) secretion across the intestine, which is reflected by an increase in short circuit current (Isc). All drugs were added serosally. Veratridine (1 μM) induced a robust secretory response for subsequent pharmacological investigation. 5-HT (300 nM) was used to specifically activate non-cholinergic secretomotor neurons, while DMPP (nicotinic agonist, 10 μM) stimulated all secretomotor neurons. Results: SOM (50 nM) induced a TTX (1 μM)-sensitive decrease in basal secretion. SOM also significantly reduced the veratridine-induced increase in Isc (p < 0.001), an effect abolished by blocking SST1 receptors (SRA 880 3 μM) and partially diminished by blocking SST2 receptors (CYN 154806 100 nM). Blocking these receptors also significantly reduced the SOM-induced decrease in basal secretion. Quantitiative PCR demonstrated that SST1 and SST2 receptor were more highly expressed in the submucous plexus compared to the mucosal layer. However, SST1 receptor mRNA levels were significantly (p < 0.01) higher than SST2 within both layers. Serosal application of DMPP produced a biphasic response. The first phase was a rapid rise in Isc that was abolished by hyoscine (1 μM), the second phase was a slower, more sustained response that was not significantly affected by hyoscine indicating it was due to activation of the VIP neurons. Responses to DMPP and 5-HT (300 nM) were TTX-sensitive. The 5-HT-induced increase in Isc, and the second more sustained phase evoked by DMPP were significantly reduced by SOM. Conclusion: These data suggest that SOM exerts its antisecretory effects indirectly by supressing firing of VIP non-cholinergic secretomotor neurons, rather than via a direct action on mucosal enterocytes. SOM acts predominately on SST1 receptors, which suggests a greater role of SST1 receptors in the regulation of gut secretion. SST1 receptors could be potential therapeutic targets for treating secretory diarrhea resulting from overactivity of the enteric nervous system, like that produced by cholera and other bacterial toxins.
M2029 Relaxatory Responses of Mouse Lower Gastrointestinal Tract Mediated to Transient Receptor Potential Melastatin Type-8 (TRPM8) Channel Activation Takuji Hosoya, Kenjiro Matsumoto, Kimihito Tashima, Toshihiko Murayama, Syunji Horie BACKGROUND & AIMS: Transient receptor potential melastatin type-8 (TRPM8) is a nonselective cation channels expressed in primary sensory neurons, and is activated by innocuous cold ( or cool, < 27 degrees C), icilin, menthol and a number of other odorants and cooling compounds. In recent clinical paper, it has reported that peppermint oil containing menthol is more effective than placebo in the treatment of irritable bowel syndrome. However, the distribution and the role of TRPM8 in motor function of lower gastrointestinal tract have hardly examined. In the present study, we investigated the localization of TRPM8 channels and effect of TRPM8 agonists on smooth muscle tension in lower gastrointestinal tract. METHODS: TRPM8-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification in both transverse and horizontal sections of mouse lower gastrointestinal tract from C57BL/6J mice. Calretinin, c-Kit and neuronal nitric oxide synthase (nNOS)-immunoreactivity were studied by normal indirect staining with the corresponding specific antibodies. The rectum and distal, transverse and proximal colon were isolated. The longitudinal change in smooth muscle tone was isometrically measured using Magnus apparatus. RESULTS: In immunohistochemical study, TRPM8immunoreactivties were found in the mucosal, submucosal and muscle layers. Especially, TRPM8-immunoreactivities were abundant in deep muscular plexus, circular muscle, myenteric plexus and longidutinal muscle. Next, double labaling studies on TRPM8 were carried out using the intrinsic neuron marker calretinin, interstetial cells of Cajal (ICC) marker cKit and nNOS. Double-labeling for TRPM8/calretinin and TRPM8/nNOS was slightly detected in muscle layer. However, double-labeling for TRPM8/c-Kit was not observed. In the Magnus experiment, menthol (30-300 μM) and icilin (10-100 μM) induced a concentration-dependent relaxation in rectum and distal, transverse and proximal colon preparations. The selective TRPM8 agonist, WS-12 (3-30 μM) also induced a long-lasting relaxation in rectum and colon contracted by 40 mM KCl. DISCUSSION: These results suggest that TRPM8 channels are largely localized in lower gastrointestinal tract of mucosa, submucosa and muscle layers and are involved in inhibition of colonic and rectal motor function. TRPM8 channels may be localized in intrinsic neuron, but not in ICC. It is probably that TRPM8 neurons are correlated with nitrergic neurons.
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M2032 Longitudinal Muscle Physiological Specificity Correlates With the Differences in Expression and Phosphorylation of Contractile Proteins Sita Somara, Daniela Musaka, Shreya Raghavan, Robert R. Gilmont, Khalil Bitar Background: Colonic motility is a collective response of circular and longitudinal smooth muscle contraction. Circular smooth muscle cells (CSMC) are aligned concentrically around the lumen while longitudinal smooth muscle cell (LSMC) are aligned parallel to the length of the colon. Differences in innervation and myogenic characteristic of LSMC and CSMC allow the regulation of the coordinated contraction required for peristalsis. Hypothesis: The myogenic differences between basal tone of longitudinal and circular muscles of the colon could be due to the fundamental differences in the basal level of expression and phosphorylation of contractile proteins in each cell type. Objective: To correlates the differences in basal tone to the differences in the expression and phosphorylation of contractile proteins at basal level. Methods: Whole cell lysates from confluent cultured CSMC and LSMC isolated
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