Long Term Gastric Electrical Stimulation has an Anti-inflammatory Effect

TNFa and Inner Muscle Mast Cells Over Time

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Mo1596

LONG TERM GASTRIC ELECTRICAL STIMULATION HAS AN ANTIINFLAMMATORY EFFECT Thomas Abell, Archana Kedar, Abigail Stocker, Karen Beatty, Lindsay McElmurray, Michael G. Hughes, Hani Rashed, William Kennedy, Gwen Wendelschafer-Crabb, Xiu Yang, Mostafa Fraig, Gerald W. Dryden, Ed Miller, Michael Griswold, Christina Pinkston

OPTOGENETIC ACTIVATION OF CENTRAL AMYGDALOID CIRCUITRY INDUCES VISCERAL PAIN IN FREELY MOVING RATS Anthony Johnson, Casey O. Ligon, Rocco Latorre, Beverley Greenwood-Van Meerveld

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of FGID. In the present study, we investigated the intestinal motility and mast cell behavior in mice after DSS-induced colitis. Methods: C57BL/6J (8 weeks-age, female) mice were treated with dextran sulfate sodium (DSS; 2% w/v) in a drinking water for 5 days and followed up to 24 weeks later. The gastrointestinal tissues were obtained at various time points and investigated to evaluate the inflammation score and lymphoid follicle formation by histopathology. The population of mast cells were evaluated by immunohistochemistry using anti-mast cell tryptase antibody. Gastrointestinal transit time (GITT) was measured by oral administration of 6% carmine red solution. Results: The inflammation scores in the colon and small intestine were peaked at one week after DSS treatment and thereafter, decreased but sustained at low levels up to 24 weeks later. The number of lymphoid follicles was significantly increased in the small intestine from 12 weeks after DSS treatment. The infiltration of mast cells was observed in the lamina propria in not only colonic but also small intestinal mucosa with DSS-induced colitis. The number of mast cells in the small intestine was significantly greater in mice with DSS treatment than in control from 12 weeks after colitis induction. GITT became shorter in mice with DSS treatment than in controls at 24 weeks after the start of experiment. GITT was negatively correlated with both the severity of inflammation and the number of lymphoid follicles in the small intestine. Conclusions: Small intestinal inflammation with mast cell infiltration is associated with the acceleration of the intestinal motility after acute colitis.

Background: Optogenetics identifies brain circuits controlling behaviors in conscious animals by using light to alter neuronal function. Currently, in vivo optogenetics offers a novel tool to study the brain-gut axis, which is dysregulated in irritable bowel syndrome (IBS). Experimental evidence suggests signaling from the central nucleus of the amygdala (CeA) to the bed nucleus of the stria terminalis (BNST) modulates stress-induced colonic hypersensitivity. The goal of study was to test the hypothesis that directly activating the CeA-BNST pathway via optogenetic stimulation, in the absence of a stressor, increases colonic sensitivity. Methods: In anesthetized male rats, stereotaxic surgical procedures were used to infect the CeA on both sides of the brain with viral vectors to express channelrhodopsin (ChR2) or halorhodopsin (HR3.0) in neurons. Bilateral fiber optic cannulae were also implanted on the BNST. After 8-10 weeks, the response to graded (0-60 mmHg), isobaric colorectal distension (CRD) was measured via a visceromotor behavioral response (VMR) quantified as the number of abdominal muscle contractions to CRD in freely moving rats, first without laser stimulation and then during laser stimulation of CeA fibers at the BNST. Immunohistochemistry and histology were used to evaluate vector expression and neuronal integrity. A repeated measure two-way ANOVA was used to compare the VMR to distension followed by a Tukey's post-hoc analysis to compare differences in laser activation at individual distension pressures (mean ± standard error). Results: We found that adenoviral infection of the CeA does not alter neuronal morphology and neuronal number, or induce a central inflammatory response. Additionally, the fiber optic cannula at the BNST were correctly positioned to activate ChR2 or HR3.0 in transfected CeA terminals. Inhibiting CeA fibers at the BNST via 532 nm laser stimulation of HR3.0 infected CeA nerve fibers did not change colonic sensitivity to luminal distension (20 mmHg: 10.0 ± 1.6 vs. 8.0 ± 2.0, P = 0.706; 40 mmHg: 23.3 ± 3.2 vs. 17.0 ± 2.5, P = 0.029; 60 mmHg: 28.7 ± 2.4 vs. 26.0 ± 3.6, P = 0.444, n = 6). In contrast, activation of CeA fibers in the BNST with ChR2 via 473 nm laser light stimulation induced a significant increase in the VMR to CRD, (20 mmHg: 19.5 ± 2.8 vs. 8.5 ± 1.9, P = 0.001; 40 mmHg: 30.5 ± 2.8 vs. 18.7 ± 2.1, P = 0.001; 60 mmHg: 38.0 ± 3.4 vs. 24.8 ± 2.0, P < 0.001; n = 6) indicating that activation of CeA terminals at the BNST promotes colonic hypersensitivity. Conclusions & Inferences: This study demonstrated that in a free moving rodent model, optogenetic activation of the CeA-BNST pathway induced colonic hypersensitivity to luminal distension. Thus, we have shown for the first time that in vivo optogenetics can be used to investigate the brain-gut axis in a freely moving rat model.

Introduction: Emerging evidence suggests that patients with gastroparesis may have systemic inflammation. We investigated the hypothesis that gastric electrical stimulation (GES) might change inflammation by following a group of patients with the symptoms (Sx) of gastroparesis (Gp) studied at baseline, after temporary, then permanent stimulation in the majority of patients (pts). Patients: From a baseline group of 43 consecutive eligible pts with the Sx of Gp (15 M, 28 F, mean age 46.3 yrs; 23 with Diabetes (DM) and 20 idiopathic (ID), we studied 41 pts (13 M, 28 F, mean 45.7 yrs; 21 DM and 20 ID ) who underwent temporary GES for 5-7 days, followed by 30 patients (9 M, 21 F, age 43.1 yrs; 14 DM and 16 ID) who completed 6 months with permanent GES. Methods: GI Sx (GCSI and PRO), gastric emptying (solid/liquid GET), and inflammatory cytokine levels (IL6 & TNF alpha=a) were measured at baseline (visit 1), after temporary GES (visit 2) and permanent GES (visit 3). Full thickness gastric biopsies, obtained at with permanent stimulation were stained for ICC (CD117), neural fibers (S100) and mast cells (tryptase), for inner (In) and outer (Out) muscle layers. Results were reported as mean and standard deviation and compared by ttest as well as via regression analysis using a mixed model adjusted (adj) for multiple comparisons. Findings: Patients' GI Sx at baseline were abnormal: GCSI (total score 29.9/ 45) and PRO (total score 14.5/20) and decreased with temporary GES (total GCSI 14.83 ± 10.09, adj p<0.001; total PRO 6.61 ± 4.64; adj p<0.001), but rebounded with permanent stimulation (GCSI 20.53 9.34 , adj p=0.03; PRO 10.05 ± 5.04; adj p=0.003 ). GET were abnormal for solid and/or liquid in 25/43 pts. Full thickness biopsies were abnormal for ICC (97.4% In &100 % Out) and/or S100 (31.6% In & 52.6 % Out) compared to normal controls. Mast cell values for In were 2.57 ± 1.33 and Out 2.08 ± 1.09. Patients with the symptoms of Gp had elevated levels of IL6 (46.64 ± 53.01) and TNFa (22.18 ± 7.46) at baseline (normal IL-6 < 10.1 pg/ml and TFNa <7.1 pg/ml). After temporary GES, IL-6 increased (141.74 ± 133.04) and TNFa began to decrease (19.84 ± 8.50). At 6 months with permanent GES, IL6 (15.34 ± 20.51) and TNFa (6.58 ± 2.58) were decreased to near normal ranges. (figure 1). TNFa did not correlate with mast cell stains in full thickness biopsy at baseline, or after temporary GES, but did correlate at 6 months (p=0.001 for mast and ICC IN; p=0.03 for mast and ICC OUT.) (figure 2). Conclusions: In this group of patients with the symptoms of gastroparesis, inflammatory cytokines are increased at baseline, have some changes with temporary GES, and are markedly decreased at 6-months with permanent GES. Mast cells in the GI tract may plan a role the mechanism of the inflammatory modulation seen with long-term GES and deserve further evaluation.

Mo1597 ROLES OF ENDOGENOUS OPIOID ACTIVITY IN THE ANTERIOR CINGULATE CORTEX IN A NEWLY-ESTABLISHED OF RAT MODEL OF VISCERAL PLACEBO ANALGESIA Pei-Yi Liu, Yen-Po Wang, Chia-Fen Tsai, Ming-Tsung Kuo, Ming-Chih Hou, Ching-Liang Lu Background Placebo effects of up to 50% are commonly observed when treating chronic visceral pain of irritable bowel syndrome (IBS). Neuroimaging studies of placebo analgesia have demonstrated placebo-induced brain changes and the involvement of opioid receptors in the anterior cingulated cortex (ACC) during analgesia (Science 2002; 295:1737-40). Without a reliable animal model, the molecular mechanisms underlying the visceral placebo effect remain unclear. Aim To establish an animal model of visceral placebo analgesia with which to re-examine the role of the ACC's endogenous opioid system. Methods (1) 5 groups of male Sprague-Dawley rats were studied. All groups received daily colorectal distension (CRD), conducted stepwise from 10mg to 80mg. Pain thresholds were determined by simultaneous electromyography recording at external oblique muscles. This was done for 11 days (Table 1). (2) Group 1 measures the visceral placebo analgesia induced by conditioning. Intra-peritoneal (IP) injection of morphine (10 mg/kg) was administered on days 3, 4, 7 and 8. On day10 and 30 minutes after saline injection, pain threshold was measured while a cue (blue light) and CRD were administered. (3) Group 2 determines the role of an opioid pathway in visceral placebo analgesia. On day10 and 30 minutes after naloxone (5mg/kg) IP injection, the pain threshold was measured. (4) Group 3 was the natural history control and received daily CRD. Group 4 and 5 were controls treated with either saline or naloxone on days 3, 4, 7, 8 and 10. (5) Role of an endogenous opioid pathway in ACC was evaluated by microinjection of cytotoxic ribosome inhibitor dermorphic-saporin (DermSAP) (1.5 pmol) to deplete neurons expressing the mu-opioid receptors (MOR). Results (1) Comparing pain thresholds of day1 and 10 show a significant increase for group 1 (n = 14; 25.48 ± 1.73 mmHg vs. 33.57 ± 1.46 mmHg, P < 0.001), which demonstrates visceral

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