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Sa2064 Genes Regulating Cyclooxygenase Pathway of Arachidonic Acid Metabolism Are Highly Mechanosensitive in the Gut
AGA Abstracts
and humans. We developed a novel method of electrical stimulation, Q-StimTM, a truncated decaying exponential stimulation method, designed to generate only the charge needed to deliver stimulation. This method maximizes energy efficiency and extends battery life for implantable neurostimulators. Q-stimTM poses as a potential treatment for STC patients. In this study, we aimed at studying the effects of CES using Q-StimTM to accelerate colon transit in normal rats. Materials and Methods Fourteen adult male Sprague Dawley® rats underwent surgical implantation of serosal electrodes for CES and a cannula in the proximal colon. Following recovery and acclimatization to daily restraint for 3 hours, colon transit studies were carried out. Following a gavage meal of 10ml/Kg Ensure, rats were placed in a restrainer where they received CES or sham CES via an external stimulator using the QStimTM technology. A methylcellulose solution mixed with phenol red, as marker, was injected via the colon cannula into the proximal colon. Saline (0.1ml/min) was continuously infused via the cannula for the duration of the study. The experiment continued until the observation of first appearance of phenol red from the anus, reflecting the colon transit time. CES or sham CES was applied throughout the duration of the entire experiment and each rat served as its own control. Q-stimTM stimulation parameters were based on our previous studies and pulse trains were used: train duration of 2s and 12 trains/min. In each train, there were pulses with a frequency of 40 Hz, width of 6ms and amplitude of 1-6 mA. We also tested various numbers of trains per minutes (5, 10 and 14.6 trains/min). Q-stimTM was compared to the regular constant current stimulation. Results We found that 1) CES with all tested parameter sets accelerated colon transit compared to control in transit time (88.5±11 min for control; p ≤0.05 each). 2) Using Q-stimTM at 5 trains/min was most effective in accelerating transit; transit times were 32.9±12 min, 34.6±6 min and 63.8±15 min for 5, 10 and 14.6 trains/min, respectively. 3) Q-stimTM was marginally superior to constant current stimulation in accelerating colon transit at 5 trains/min (45.6±15.2 min for constant current, 32.9±12 min for Q-Stim TM; p=0.07). Conclusion Colon electrical stimulation using Q-stimTM accelerates colon transit in rats. A lower number of trains per min (5/min) is superior in accelerating transit. Q-stimTM may represent a better tool for CES as it seems more effective than the constant current stimulation. Further studies in a constipation animal model are warranted.
Sa2062 Interstitial Cells of Cajal (ICC) and Smooth Muscle Actin (SMA) Activity After Non-Ablative Radiofrequency Energy Application to the Internal Anal Sphincter (IAS): An Animal Study Roman M. Herman, Maciej Murawski, Janusz Rys, Michal Nowakowski, Tomasz Schwarz, Dorota Wojtysiak, Roma B. Herman, Dorota A. Zieba Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscles of the gut. ICC have been shown to be involved in neurotransmission of the lower oesophageal sphincter, pylorus, and IAS. ICC deterioration has been confirmed in IAS Achalasia (IASA) and Hirshprung's disease. The activity of smooth muscle actin within the muscle represents its potential contractile properties. METHODS; An experimental model of fecal incontinence (FI) was created in 10 pigs by pudendal nerve destruction+ external anal sphincter (EAS) and IAS sphincterotomy. 6 weeks later the animals underwent RF application; 10 weeks after treatment, they were euthanized and the IAS harvested for pathologic evaluation. A control group of 6 age and weight matched pigs was also sacrificed. Sections from the IAS were examined using both a routine H&E and Masson trichrome staining to evaluate the architectural features of the IAS. Semi quantitative assessment of smooth muscle(SM) cells and myofibroblasts was performed basing on image analysis of immunohistochemical stained (SMA-positive) cells. IAS specimens were also examined for the presence for CD117 and antiperipherin-positive cells. Density of the ICCs was graded by computerized image analysis. RESULTS: Microscopic evaluation of the IAS in the control animals revealed characteristic circular muscle bundles separated by connective tissue septae. The basic architecture of the IAS was maintained following RF treatment. In all groups the circular muscle bundles greatly varied although the cross section area occupied by smooth muscle within the bundles increased after RF. There was strong peripherin immunoreactivity in the ganglia cells and nerve fibers in the control animals' IASs. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. The number of peripherin-positive nerve fibers was markedly reduced in the IAS in subjects after RF treatment. There was a complete lack of peripherin immunoreactivity in the IAS, but hypertrophic nerve trunks stained strongly. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. In the FI group after RF, ICCs were absent or markedly reduced. Statistically significant increase of smooth muscle actin within the IAS and fibroblasts have been observed suggesting phenotype switch of fibroblasts into myofibroblasts. CONCLUSION .Non-ablative RF application to the IAS significantly influences the structural arrangement of IAS smooth muscle and Interstitial Cell of Cajal distribution. Deterioration of the ICC network within the IAS may be responsible for increased contraction or lack of relaxation similar to IAS achalasia. The increase of IAS smooth muscle actin and myofibroblast contents within septa are potentially responsible for IAS remodeling.
Sa2061 Peri-Capillary Interstitial Cells of Cajal in the Human Colon Muscle Layers Yuan-An Liu, Yuan-Chiang Chung, Shien-Tung Pan, Pankaj J. Pasricha, Shiue-Cheng Tang Background: Interstitial cells of Cajal (ICC) integrate with nerves and smooth muscles to generate and regulate motility of the gastrointestinal (GI) tract. Beyond the GI tract, regulation of smooth muscles by ICC may occur in locations in which rhythmic contraction is required including the arteries, veins, and lymph vessels. However, the interaction between ICC and capillaries in the GI tract has not been described. In this research, we applied 3-dimensional (3-D) histology with optical clearing to simultaneously reveal the ICC and vascular networks, assessing their morphological association in the muscle layers of human colon. Methods: Human transverse and sigmoid colons were obtained from colectomies performed for carcinoma. Transverse sections of muscularis were prepared away from tumor site. Capillaries and ICC were labeled by CD31 and c-kit immunostaining, respectively. Optical clearing was used to enhance photon penetration in the tissue for 3-D confocal microscopy and indepth projection of the tissue networks in space. ICC were defined as the c-kit-positive cells with at least two processes extending from the cell body for quantitation. Results: In addition to the classic ICC subgroups (ICC-SM, ICC-CM, ICC-MY, and ICC-LM) that associate with the muscles and nerves at different layers of the gut wall, we found close association of ICC with muscular capillaries in the human colon as well (3 patients; 10 specimens). Through joint projection of ICC and capillaries, extensions of the ICC network reach the capillaries in connection with a subset of ICC on the vessel wall, in which their cell bodies reside. The processes of these ICC encircle and elongate along the intricate capillaries. In morphological analysis, we ruled out these c-kit-positive cells as pericytes based on the shapes of the cell bodies and processes and their connections to the ICC network. In quantitative analysis, these peri-capillary ICC account for 17% of the overall ICC in the human colon circular muscle. Conclusion: Taking advantage of the in-depth ICC image data, we propose a new subclass of ICC that closely associates with capillaries in the human colon. According to the morphological proximity, our finding suggests functional relationship between these ICC and capillaries.
Sa2063 The Role of Nerve Growth Factor in the Ameliorating Effect of Gastric Electrical Stimulation on Visceral Hypersensitivity Fei Dai, Jiande Chen Background: Nerve growth factor (NGF) is a key signaling protein in the sensitization of visceral afferent neurons during inflammation. The role of NGF in a rodent model of gastric hypersensitivity has not yet been explored. We hypothesize that NGF contributes to acetic acid-induced gastric hypersensitivity and gastric electrical stimulation (GES) improves visceral hypersensitivity by down-regulating the expression of NGF in the gastric wall. Aims: This study was to investigate the involvement of NGF in the ameliorating effect of GES on gastric hypersensitivity. Methods: Twenty-six rats with gastric hyperalgesia induced by the injection of acetic acid into submucosal layer of the gastric wall and fourteen controls were used. The rats were chronically placed with an intragastric balloon, one pair of electrodes at the acromiotrapezius muscle for electromyogram (EMG) recordings and one pair of serosal electrodes in the stomach for GES. Visceromotor response (VMR) to gastric distention (GD) was calculated as a percentage compared with baseline at distention pressures of 20, 40, 60 and 80mmHg. NGF protein expressions in the gastric wall and DRG were measured by enzyme-linked immunosorbent assay. The expression of NGF in gastric wall was also confirmed by Western blotting. Results: (1) GES (0.1s on, 0.4s off, 0.25ms, 100Hz, 6mA) reduced EMG activity; the VMR (% baseline) was 116.1± 4.6% with GES and 139.1±6.4% with sham-GES (p=0.008) at GD of 60mmHg and 130.9±8.1% vs. 162.3 ±7.4% (p ,0.001) at GD of 80 mmHg. (2) Compared with vehicle treated rats and isotype control antibodytreated rats, anti-NGF antibody (16μg/kg, in 0.5 mL PBS, i.p. , 30 min before GD) significantly reduced VMR at GD of 60mmHg and 80mmHg: 115.9±6.0% vs. 142.1±3.58% (p=0.002) and 132.2±5.12% vs. 161.7±4.96% (p=0.008), respectively. (3) Exogenous NGF significantly increased VMR in comparison with the vehicle treatment: 149.2±6.6% vs. 114.8±4.8% (p= 0.002) at GD of 60 mmHg and 171.4±8.9% vs. 133.3±4.5% (p=0.007) at GD of 80 mmHg. The NGF-induced gastric hypersensitivity was normalized by GES with VMR (% baseline) 116.2±6.1% (p=0.9, vs. vehicle) at 60 mmHg and 138.7±9.67% (p=0.6 vs. vehicle) at 80mmHg. (4) The NGF concentration of gastric mucosa/submucosa was elevated in acetic acid-treated animals but normalized with GES: 18.8±2. 9 pg/g in control, 42.7±4.1 pg/g in acetic acid rats with sham-GES, (p ,0.001) and 24.4±3.0 pg/g in acetic acid rats with GES (p=0.483). Similarly, the protein expression of NGF in the gastric tissue was also elevated in acetic acid-treated rats but normalized with GES. Conclusions: GES reduces gastric hypersensitivity in a rodent model of hyperalgesia via the NGF pathway and may have a therapeutic potential for the treatment of visceral hypersensitivity.
(A and B) Overlay of ICC (green: c-kit), capillary (red: CD31), and nuclear (blue) signals in the circular muscle of human colon. Arrows indicate the cell bodies of the peri-capillary ICC. (C) 3-D illustration of the peri-capillary ICC. Nuclei were segmented from panel B. Dimensions: 225 x 225 x 30 (depth) μm.
Sa2064 Genes Regulating Cyclooxygenase Pathway of Arachidonic Acid Metabolism Are Highly Mechanosensitive in the Gut You Min Lin, Feng Li, Chester C. Wu, Xuan-Zheng P. Shi Background and aims: The cyclooxygenase (COX) pathway of arachidonic acid metabolism plays a critical role in gastrointestinal physiology and pathophysiology. The pathway involves COX-1, COX-2, and various prostaglandin (PG) synthases, i.e. PGD synthase (PGDS), PGES,
AGA Abstracts
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volumes (95% CI). More women had abnormal WLT compared with men (70.5% vs 29.5%, p,0.01). Tachygastria was the most common dysrhythmia evoked by the WLT, and patients with normal or abnormal WLT had similar incidences of gastric dysrhythmias overall (73.5% vs 71.1). However, patients with abnormal WLT are more likely to have tachygastria with a normal GES than with abnormal gastric emptying (64% vs. 49%, p=0.04). Additionally, 63% of the patients with abnormal WLTs reported increased nausea compared with 42% of patients who had normal WLTs (p=0.028). The incidence of delayed gastric emptying was similar in the two WLT groups (43% vs 40%). Conclusion: 1. Ingestion of a normal or abnormal water load test volume evoked gastric dysrhythmias in 70% of these patients. 2. Patients who ingested low WLT volumes are more likely to have tachygastria with normal gastric emptying. 3. Patients who ingested abnormally low volumes during the WLT experienced increased nausea, suggesting hypersensitivity to gastric distention. 4. Increased nausea and the onset of gastric dysrhythmias after the WLT indicates the stomach is the site of origin of nausea in these patients with or without gastroparesis. Sa2067 Obese Patients Have Higher Circulating Levels of Dipeptidyl Peptidase IV That May Impact on Satiety Signaling Under These Conditions Andreas Stengel, Tobias Hofmann, Anne Ahnis, Miriam Goebel-Stengel, Peter Kobelt, Burghard F. Klapp Background: Dipeptidyl peptidase IV (DPP IV) is a serine protease with broad distribution in tissue and circulation known to be involved in various homeostatic processes such as immune defense, psychoneuroendocrine functions as well as nutrition. Despite the fact that DPP IV levels have been investigated in patients with hyporectic disorders, the levels under conditions of obesity, a major health hazard in industrialized countries around the world, are not well studied. Aims: To investigate DPP IV concentration and total enzyme activity in obese subjects and a possible dependency on body weight across a broad range of body mass index (BMI). Methods: Obese (BMI .30 kg/m2, n=4 women and 5 men, age 46.2 ± 4.4 years), anorexic (BMI ,17.5 kg/m2, n=9 women, age 25.4 ± 2.7 years) and normal weight (BMI 19-25 kg/m2, n=4 women and 5 men, age 48.4 ± 3.8 years) hospitalized patients were recruited from the Division of Psychosomatic Medicine and gave informed consent to participate in the study. Blood was withdrawn from overnight fasted subjects between 07:00 and 08:00 am, plasma processed for Western blot, stained with an antihuman-DPP IV polyclonal antibody and semiquantitative analysis of pixel intensity was performed. DPP IV activity in serum was assessed using Gly-Pro p-nitroanilide as substrate. Results: DPP IV protein expression was detected in human plasma as indicated by a strong band at the expected size of 110 kDa. In normal weight patients (mean BMI 22.7 ± 0.8 kg/ m2) the intensity of the 110 kDa band was 9947.6 ± 1724.4. The expression was not different in anorexic patients (mean BMI 12.6 ± 0.6 kg/m2, intensity: 10418.5 ± 1348.8, P.0.05), while it was higher in the plasma of obese patients (mean BMI 68.9 ± 2.7 kg/m2, intensity: 21208.6 ± 1336.6, P,0.001). Plasma DPP IV protein expression showed a significant positive correlation with BMI (r=0.64, P ,0.001). Serum activity of DPP IV did not differ between normal weight (66.5 ± 4.7 U/l), anorexic (71.9 ± 5.0 U/l) and obese patients (69.1±5.5 U/l, P.0.05) and showed no correlation with BMI (r=0.05, P .0.05). The concentration/activity ratio was not different in anorexic patients (153.1 ± 27.5) compared to normal weight controls (153.5 ± 38.1, P .0.05) but was higher in obese (305.7 ± 34.9, P,0.05) and showed a positive correlation with BMI (r=0.63, P ,0.001). Conclusions: DPP IV protein levels, unlike serum activity, depend on the metabolic condition with increased levels in morbidly obese patients resulting in an increased concentration/activity ratio. Since DPP IV is involved in the deactivation of food intake inhibitory hormones such as glucagonlike peptide-1 these alterations of the concentration/activity ratio may reduce anorexigenic signaling under conditions of obesity and therefore contribute to the development or maintenance of a greatly elevated BMI.
Sa2065 Purinergic Pathway Is the Major Mechanism of Inhibitory Effect of Motilitone on the Rat Gastric Fundus Relaxation Yang Won Min, Kyu Choi, Eun-ju Ko, Ji Yeon Lee, Eun Ran Kim, Byung-Hoon Min, Jun Haeng Lee, Jae J Kim, Jong Chul Rhee, Sang D. Koh, Poong-Lyul Rhee Background/Aims: Motilitone, a new prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, has strong prokinetic effects. In a previous animal study, motilitone significantly improved gastric accommodation by increasing the postprandial gastric volume. In this study, we investigated how motilitone affects the muscle relaxation in the rat gastric fundus. Methods: Gastric fundus smooth muscle strips (12 longitudinal muscles and 9 circular muscles) were obtained from rats. Isometric force measurements were performed by using both longitudinal and circular muscle strips in response to the electrical field stimulation (EFS, 0.3 ms in trains of 5 Hz for 10 s, 150 V). Peak (the highest value) and nadir (the lowest value) were observed during the first 1 minute after the initiation of EFS in the control state and after administration of atropine (1 μM), motilitone (5 μg, 10 μg, 50 μg, and 100 μg), N-nitro-L-arginine (L-NNA, 100 μM), and MRS 2500 (1 μM), which were added in sequential order to the organ bath. Then tetrodotoxin (TTX, 1 μM) was administered to inhibit nerve-mediated relaxation. Results: EFS induced contractions in the control state, which were relaxed by atropine in the both longitudinal and circular muscles. When motilitone was added to the atropine group, the nadir dropped in a dose-dependent manner in the both longitudinal and circular muscles. The nadir did not increase significantly when L-NNA was added to the atropine and motilitone (100 μg) group in the both longitudinal and circular muscles. After addition of MRS 2500, the nadir increased to the level of atropine group (before administration of motilitone) in the circular muscles. In the longitudinal muscles, the nadir also increased but was not fully reversed to the level of atropine group after addition of MRS 2500. Addition of TTX increased the nadir further and completely abolished EFS-induced relaxation in the both longitudinal and circular muscles. However, there were no significant changes in the peak after administration of motilitone regardless of its dosage. Conclusion: Our data suggest that the inhibitory effect of motilitone on the rat gastric relaxation is largely mediated by purinergic rather than nitrergic pathway, and non-nitrergic non-purinergic component also appears to be involved in the EFS-induced relaxation in the presence of motilitone.
Sa2068 Prevention of Fructose-Induced Obesity by Intestinal Alkaline Phosphatase Konstantinos P. Economopoulos, Kanakaraju Kaliannan, Nur Muhammad, Abeba Teshager, Sulaiman R. Hamarneh, Mussa Mohamed, Palak Patel, Qingsong Tao, Seyed M. Abtahi, Madhu S. Malo, Richard A. Hodin Introduction: The average consumption of fructose per capita in the U.S. has increased by approximately 200% over the past 30 years, mainly due to the dramatic increase in the consumption of fructose-sweetened beverages. It has been proposed that chronic fructose ingestion leads to intestinal bacterial dysbiosis and increased gut permeability. Intestinal Alkaline Phosphatase (IAP) is a brush border enzyme known to preserve intestinal epithelial barrier function and maintain the normal gut microbial homeostasis. Our aim in this study was to examine if IAP could prevent fructose-induced obesity and its deleterious effects. Methods: Three groups of eight-week old, C57BL/6 male mice (n=5) with unrestricted access to regular chow diet were administered either regular autoclaved water, or water enriched with 30% of fructose ± oral IAP (50 units/ml drinking water) for 16 weeks. Body weight and food intake were monitored weekly. The effects of IAP on serum glucose, intestinal permeability and normal flora were assessed. Data were expressed as mean ± standard error and were analyzed by one-way analysis of variance with Tukey's multiple comparison posttests. A significant difference was considered when p ,0.05. Results: The mice that received fructose gained significantly more weight compared to the controls (14.4±1.93 vs 7.2±0.35 g; p=0.016). Food intake was similar between mice receiving fructose+IAP and fructose alone (p=0.897). Fructose+IAP compared with fructose alone was associated with significantly less weight gain (8.3±1.81 vs 14.4±1.93 g; p=0.004), less visceral (1.1±0.10 vs 1.9±0.32 g; p=0.032) and subcutaneous fat weight (1.4±0.15 vs 2.4±0.26 g; p=0.011), lower pancreas weight (0.16±0.01 vs 0.22±0.02 g; p=0.022), lower fasting blood glucose levels (130±6.92 vs 181±15.71 mg/dl; p=0.017), lower area under the curve during glucose tolerance test (21,210±771.57 vs 24,394.5±668.72; p=0.011) and decreased intestinal permeability (0.3±0.08 vs 1.3±0.35; p=0.032). Mice receiving fructose+IAP compared to those receiving only fructose had increased number of aerobic bacteria (46.1E+05±9.75E+05 vs 4.48E+05±0.75E+05; p=0.001) and decreased number of gram negative bacteria (80±58.3 vs 2.77E+04±0.69E+04; p=0.001) in their stool. Conclusions: Oral supplementation with
Sa2066 The Provocative Water Load Test Evokes Nausea and Gastric Dysrhythmias in Patients With Unexplained Chronic Nausea and Vomiting Marcum Gillis, Kenneth L. Koch Background: The origins of nausea in patients with chronic nausea and vomiting syndromes can be difficult to elucidate. Ingestion of solids or liquids frequently precipitates symptomatic fullness and nausea. Aims: 1. To determine the incidence of abnormal water load tests (WLT) in patients with unexplained nausea and vomiting. 2. To determine the incidence of gastric dysrhythmias and severity of nausea symptoms evoked by the WLT. 3. To determine the relationship of water load volume ingested, gastric dysrhythmias, and gastric emptying results. Methods: Patients from Wake Forest Baptist Medical Center Gastroenterology Clinics who had an electrogastrogram (EGG) with WLT and solid phase gastric emptying studies were identified retrospectively. Baseline EGG recordings before and 10, 20, and 30 minutes after the WLT were evaluated. Patients drank water until they were "full" over a 5-minute period. The total volume of water ingested and visual analog scales for nausea, bloating, fullness, and hunger were completed at the time points above and overall EGG diagnoses were determined. An abnormal WLT volume was defined as less than 550 ml ingested in 5 minutes. Chi square analyses were used for statistical significance. Results: 113 patients were identified (72 women, 41 men, average age 50 years). 68/113 (60%) of patients had abnormal WLTs (average volume ingested 360 ± 29 ml) vs 702 ± 27 ml for normal WLT
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AGA Abstracts
AGA Abstracts
PGFS, and PGIS leading to the production of PGD, PGE2, PGF2 α, and PGI, respectively. Previous studies found that mechanical stretch in bowel obstruction markedly induced gene expression of COX-2, but not COX-1, in colonic smooth muscle cells (SMCs). The aims of the present study were to determine whether genes encoding individual prostaglandin synthases are mechanically responsive, and whether the composition of prostaglandins resulting from obstruction differs from that in inflammation. Methods: Partial colon obstruction was induced with a silicon band implanted surgically in the distal colon of male SpragueDawley rats. Static mechanical stretch (18% elongation) was mimicked in vitro in the culture of rat colonic circular SMCs with a Flexercell FX-4000 System. Rat colon inflammation was induced by manipulation of the colonic surface with wet cotton applicators. Results: 1. Quantitative RT-PCR found that the expression of PGDS and PGIS genes was not significantly changed on day 3 of obstruction. However, the membrane bound PGES-1 (mPGES-1) mRNA was significantly up-regulated in the obstructed segment, whereas mRNAs for other two types of PGES, cytosolic PGES and mPGES-2, were not changed. On the contrary, the PGFS mRNA was down-regulated in the obstructed colon. 2. PGE2, but not PGF2 α, was significantly increased in the colonic muscularis externae in obstruction. The PGE2 level increased to 4577±1249 pg/mg at day 3 of obstruction, compared to 1264±259 in sham (n=6, p,0.05). The PGF2 α level was 5718±910 pg /mg in obstruction compared to 6550±2178 pg/mg in the sham (n=5, p .0.05). 3. Direct stretch of rat colonic circular SMCs in vitro significantly up-regulated the mPGES-1 mRNA, but down-regulated that of PGFS. After stretch for 3 hrs, the mRNA levels of PGES and PGFS changed by 2.61±0.42 fold and 0.82±0.03 fold, respectively (n = 4 or 5, p ,0.05). 4. In colonic inflammation, both PGE2 and PGF2α were significantly increased in the colonic smooth muscle (5.2(±1.5)-fold and 4.7(±1.3)-fold of control, respectively, p ,0.05, n = 3). 5. Rat colonic circular muscle contractility was inhibited by exogenous PGE2 (10-9 ~10-6 M), but enhanced by PGF2 α (10-9 ~10-6 M) in a concentration-dependent manner (n = 4). Conclusions: Genes involved in the COX pathway of arachidonic acid metabolism are highly mechanosensitive in colonic SMCs, and play a critical role in motility function. Mechano-regulation of prostaglandin synthases accounts for increased PGE2 and unchanged PGF2 α in bowel obstruction, a pathological feature different from inflammation in which both PGE2 and PGF2 α are increased.
and humans. We developed a novel method of electrical stimulation, Q-StimTM, a truncated decaying exponential stimulation method, designed to generate only the charge needed to deliver stimulation. This method maximizes energy efficiency and extends battery life for implantable neurostimulators. Q-stimTM poses as a potential treatment for STC patients. In this study, we aimed at studying the effects of CES using Q-StimTM to accelerate colon transit in normal rats. Materials and Methods Fourteen adult male Sprague Dawley® rats underwent surgical implantation of serosal electrodes for CES and a cannula in the proximal colon. Following recovery and acclimatization to daily restraint for 3 hours, colon transit studies were carried out. Following a gavage meal of 10ml/Kg Ensure, rats were placed in a restrainer where they received CES or sham CES via an external stimulator using the QStimTM technology. A methylcellulose solution mixed with phenol red, as marker, was injected via the colon cannula into the proximal colon. Saline (0.1ml/min) was continuously infused via the cannula for the duration of the study. The experiment continued until the observation of first appearance of phenol red from the anus, reflecting the colon transit time. CES or sham CES was applied throughout the duration of the entire experiment and each rat served as its own control. Q-stimTM stimulation parameters were based on our previous studies and pulse trains were used: train duration of 2s and 12 trains/min. In each train, there were pulses with a frequency of 40 Hz, width of 6ms and amplitude of 1-6 mA. We also tested various numbers of trains per minutes (5, 10 and 14.6 trains/min). Q-stimTM was compared to the regular constant current stimulation. Results We found that 1) CES with all tested parameter sets accelerated colon transit compared to control in transit time (88.5±11 min for control; p ≤0.05 each). 2) Using Q-stimTM at 5 trains/min was most effective in accelerating transit; transit times were 32.9±12 min, 34.6±6 min and 63.8±15 min for 5, 10 and 14.6 trains/min, respectively. 3) Q-stimTM was marginally superior to constant current stimulation in accelerating colon transit at 5 trains/min (45.6±15.2 min for constant current, 32.9±12 min for Q-Stim TM; p=0.07). Conclusion Colon electrical stimulation using Q-stimTM accelerates colon transit in rats. A lower number of trains per min (5/min) is superior in accelerating transit. Q-stimTM may represent a better tool for CES as it seems more effective than the constant current stimulation. Further studies in a constipation animal model are warranted.
Sa2062 Interstitial Cells of Cajal (ICC) and Smooth Muscle Actin (SMA) Activity After Non-Ablative Radiofrequency Energy Application to the Internal Anal Sphincter (IAS): An Animal Study Roman M. Herman, Maciej Murawski, Janusz Rys, Michal Nowakowski, Tomasz Schwarz, Dorota Wojtysiak, Roma B. Herman, Dorota A. Zieba Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscles of the gut. ICC have been shown to be involved in neurotransmission of the lower oesophageal sphincter, pylorus, and IAS. ICC deterioration has been confirmed in IAS Achalasia (IASA) and Hirshprung's disease. The activity of smooth muscle actin within the muscle represents its potential contractile properties. METHODS; An experimental model of fecal incontinence (FI) was created in 10 pigs by pudendal nerve destruction+ external anal sphincter (EAS) and IAS sphincterotomy. 6 weeks later the animals underwent RF application; 10 weeks after treatment, they were euthanized and the IAS harvested for pathologic evaluation. A control group of 6 age and weight matched pigs was also sacrificed. Sections from the IAS were examined using both a routine H&E and Masson trichrome staining to evaluate the architectural features of the IAS. Semi quantitative assessment of smooth muscle(SM) cells and myofibroblasts was performed basing on image analysis of immunohistochemical stained (SMA-positive) cells. IAS specimens were also examined for the presence for CD117 and antiperipherin-positive cells. Density of the ICCs was graded by computerized image analysis. RESULTS: Microscopic evaluation of the IAS in the control animals revealed characteristic circular muscle bundles separated by connective tissue septae. The basic architecture of the IAS was maintained following RF treatment. In all groups the circular muscle bundles greatly varied although the cross section area occupied by smooth muscle within the bundles increased after RF. There was strong peripherin immunoreactivity in the ganglia cells and nerve fibers in the control animals' IASs. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. The number of peripherin-positive nerve fibers was markedly reduced in the IAS in subjects after RF treatment. There was a complete lack of peripherin immunoreactivity in the IAS, but hypertrophic nerve trunks stained strongly. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. In the FI group after RF, ICCs were absent or markedly reduced. Statistically significant increase of smooth muscle actin within the IAS and fibroblasts have been observed suggesting phenotype switch of fibroblasts into myofibroblasts. CONCLUSION .Non-ablative RF application to the IAS significantly influences the structural arrangement of IAS smooth muscle and Interstitial Cell of Cajal distribution. Deterioration of the ICC network within the IAS may be responsible for increased contraction or lack of relaxation similar to IAS achalasia. The increase of IAS smooth muscle actin and myofibroblast contents within septa are potentially responsible for IAS remodeling.
Sa2061 Peri-Capillary Interstitial Cells of Cajal in the Human Colon Muscle Layers Yuan-An Liu, Yuan-Chiang Chung, Shien-Tung Pan, Pankaj J. Pasricha, Shiue-Cheng Tang Background: Interstitial cells of Cajal (ICC) integrate with nerves and smooth muscles to generate and regulate motility of the gastrointestinal (GI) tract. Beyond the GI tract, regulation of smooth muscles by ICC may occur in locations in which rhythmic contraction is required including the arteries, veins, and lymph vessels. However, the interaction between ICC and capillaries in the GI tract has not been described. In this research, we applied 3-dimensional (3-D) histology with optical clearing to simultaneously reveal the ICC and vascular networks, assessing their morphological association in the muscle layers of human colon. Methods: Human transverse and sigmoid colons were obtained from colectomies performed for carcinoma. Transverse sections of muscularis were prepared away from tumor site. Capillaries and ICC were labeled by CD31 and c-kit immunostaining, respectively. Optical clearing was used to enhance photon penetration in the tissue for 3-D confocal microscopy and indepth projection of the tissue networks in space. ICC were defined as the c-kit-positive cells with at least two processes extending from the cell body for quantitation. Results: In addition to the classic ICC subgroups (ICC-SM, ICC-CM, ICC-MY, and ICC-LM) that associate with the muscles and nerves at different layers of the gut wall, we found close association of ICC with muscular capillaries in the human colon as well (3 patients; 10 specimens). Through joint projection of ICC and capillaries, extensions of the ICC network reach the capillaries in connection with a subset of ICC on the vessel wall, in which their cell bodies reside. The processes of these ICC encircle and elongate along the intricate capillaries. In morphological analysis, we ruled out these c-kit-positive cells as pericytes based on the shapes of the cell bodies and processes and their connections to the ICC network. In quantitative analysis, these peri-capillary ICC account for 17% of the overall ICC in the human colon circular muscle. Conclusion: Taking advantage of the in-depth ICC image data, we propose a new subclass of ICC that closely associates with capillaries in the human colon. According to the morphological proximity, our finding suggests functional relationship between these ICC and capillaries.
Sa2063 The Role of Nerve Growth Factor in the Ameliorating Effect of Gastric Electrical Stimulation on Visceral Hypersensitivity Fei Dai, Jiande Chen Background: Nerve growth factor (NGF) is a key signaling protein in the sensitization of visceral afferent neurons during inflammation. The role of NGF in a rodent model of gastric hypersensitivity has not yet been explored. We hypothesize that NGF contributes to acetic acid-induced gastric hypersensitivity and gastric electrical stimulation (GES) improves visceral hypersensitivity by down-regulating the expression of NGF in the gastric wall. Aims: This study was to investigate the involvement of NGF in the ameliorating effect of GES on gastric hypersensitivity. Methods: Twenty-six rats with gastric hyperalgesia induced by the injection of acetic acid into submucosal layer of the gastric wall and fourteen controls were used. The rats were chronically placed with an intragastric balloon, one pair of electrodes at the acromiotrapezius muscle for electromyogram (EMG) recordings and one pair of serosal electrodes in the stomach for GES. Visceromotor response (VMR) to gastric distention (GD) was calculated as a percentage compared with baseline at distention pressures of 20, 40, 60 and 80mmHg. NGF protein expressions in the gastric wall and DRG were measured by enzyme-linked immunosorbent assay. The expression of NGF in gastric wall was also confirmed by Western blotting. Results: (1) GES (0.1s on, 0.4s off, 0.25ms, 100Hz, 6mA) reduced EMG activity; the VMR (% baseline) was 116.1± 4.6% with GES and 139.1±6.4% with sham-GES (p=0.008) at GD of 60mmHg and 130.9±8.1% vs. 162.3 ±7.4% (p ,0.001) at GD of 80 mmHg. (2) Compared with vehicle treated rats and isotype control antibodytreated rats, anti-NGF antibody (16μg/kg, in 0.5 mL PBS, i.p. , 30 min before GD) significantly reduced VMR at GD of 60mmHg and 80mmHg: 115.9±6.0% vs. 142.1±3.58% (p=0.002) and 132.2±5.12% vs. 161.7±4.96% (p=0.008), respectively. (3) Exogenous NGF significantly increased VMR in comparison with the vehicle treatment: 149.2±6.6% vs. 114.8±4.8% (p= 0.002) at GD of 60 mmHg and 171.4±8.9% vs. 133.3±4.5% (p=0.007) at GD of 80 mmHg. The NGF-induced gastric hypersensitivity was normalized by GES with VMR (% baseline) 116.2±6.1% (p=0.9, vs. vehicle) at 60 mmHg and 138.7±9.67% (p=0.6 vs. vehicle) at 80mmHg. (4) The NGF concentration of gastric mucosa/submucosa was elevated in acetic acid-treated animals but normalized with GES: 18.8±2. 9 pg/g in control, 42.7±4.1 pg/g in acetic acid rats with sham-GES, (p ,0.001) and 24.4±3.0 pg/g in acetic acid rats with GES (p=0.483). Similarly, the protein expression of NGF in the gastric tissue was also elevated in acetic acid-treated rats but normalized with GES. Conclusions: GES reduces gastric hypersensitivity in a rodent model of hyperalgesia via the NGF pathway and may have a therapeutic potential for the treatment of visceral hypersensitivity.
(A and B) Overlay of ICC (green: c-kit), capillary (red: CD31), and nuclear (blue) signals in the circular muscle of human colon. Arrows indicate the cell bodies of the peri-capillary ICC. (C) 3-D illustration of the peri-capillary ICC. Nuclei were segmented from panel B. Dimensions: 225 x 225 x 30 (depth) μm.
Sa2064 Genes Regulating Cyclooxygenase Pathway of Arachidonic Acid Metabolism Are Highly Mechanosensitive in the Gut You Min Lin, Feng Li, Chester C. Wu, Xuan-Zheng P. Shi Background and aims: The cyclooxygenase (COX) pathway of arachidonic acid metabolism plays a critical role in gastrointestinal physiology and pathophysiology. The pathway involves COX-1, COX-2, and various prostaglandin (PG) synthases, i.e. PGD synthase (PGDS), PGES,
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volumes (95% CI). More women had abnormal WLT compared with men (70.5% vs 29.5%, p,0.01). Tachygastria was the most common dysrhythmia evoked by the WLT, and patients with normal or abnormal WLT had similar incidences of gastric dysrhythmias overall (73.5% vs 71.1). However, patients with abnormal WLT are more likely to have tachygastria with a normal GES than with abnormal gastric emptying (64% vs. 49%, p=0.04). Additionally, 63% of the patients with abnormal WLTs reported increased nausea compared with 42% of patients who had normal WLTs (p=0.028). The incidence of delayed gastric emptying was similar in the two WLT groups (43% vs 40%). Conclusion: 1. Ingestion of a normal or abnormal water load test volume evoked gastric dysrhythmias in 70% of these patients. 2. Patients who ingested low WLT volumes are more likely to have tachygastria with normal gastric emptying. 3. Patients who ingested abnormally low volumes during the WLT experienced increased nausea, suggesting hypersensitivity to gastric distention. 4. Increased nausea and the onset of gastric dysrhythmias after the WLT indicates the stomach is the site of origin of nausea in these patients with or without gastroparesis. Sa2067 Obese Patients Have Higher Circulating Levels of Dipeptidyl Peptidase IV That May Impact on Satiety Signaling Under These Conditions Andreas Stengel, Tobias Hofmann, Anne Ahnis, Miriam Goebel-Stengel, Peter Kobelt, Burghard F. Klapp Background: Dipeptidyl peptidase IV (DPP IV) is a serine protease with broad distribution in tissue and circulation known to be involved in various homeostatic processes such as immune defense, psychoneuroendocrine functions as well as nutrition. Despite the fact that DPP IV levels have been investigated in patients with hyporectic disorders, the levels under conditions of obesity, a major health hazard in industrialized countries around the world, are not well studied. Aims: To investigate DPP IV concentration and total enzyme activity in obese subjects and a possible dependency on body weight across a broad range of body mass index (BMI). Methods: Obese (BMI .30 kg/m2, n=4 women and 5 men, age 46.2 ± 4.4 years), anorexic (BMI ,17.5 kg/m2, n=9 women, age 25.4 ± 2.7 years) and normal weight (BMI 19-25 kg/m2, n=4 women and 5 men, age 48.4 ± 3.8 years) hospitalized patients were recruited from the Division of Psychosomatic Medicine and gave informed consent to participate in the study. Blood was withdrawn from overnight fasted subjects between 07:00 and 08:00 am, plasma processed for Western blot, stained with an antihuman-DPP IV polyclonal antibody and semiquantitative analysis of pixel intensity was performed. DPP IV activity in serum was assessed using Gly-Pro p-nitroanilide as substrate. Results: DPP IV protein expression was detected in human plasma as indicated by a strong band at the expected size of 110 kDa. In normal weight patients (mean BMI 22.7 ± 0.8 kg/ m2) the intensity of the 110 kDa band was 9947.6 ± 1724.4. The expression was not different in anorexic patients (mean BMI 12.6 ± 0.6 kg/m2, intensity: 10418.5 ± 1348.8, P.0.05), while it was higher in the plasma of obese patients (mean BMI 68.9 ± 2.7 kg/m2, intensity: 21208.6 ± 1336.6, P,0.001). Plasma DPP IV protein expression showed a significant positive correlation with BMI (r=0.64, P ,0.001). Serum activity of DPP IV did not differ between normal weight (66.5 ± 4.7 U/l), anorexic (71.9 ± 5.0 U/l) and obese patients (69.1±5.5 U/l, P.0.05) and showed no correlation with BMI (r=0.05, P .0.05). The concentration/activity ratio was not different in anorexic patients (153.1 ± 27.5) compared to normal weight controls (153.5 ± 38.1, P .0.05) but was higher in obese (305.7 ± 34.9, P,0.05) and showed a positive correlation with BMI (r=0.63, P ,0.001). Conclusions: DPP IV protein levels, unlike serum activity, depend on the metabolic condition with increased levels in morbidly obese patients resulting in an increased concentration/activity ratio. Since DPP IV is involved in the deactivation of food intake inhibitory hormones such as glucagonlike peptide-1 these alterations of the concentration/activity ratio may reduce anorexigenic signaling under conditions of obesity and therefore contribute to the development or maintenance of a greatly elevated BMI.
Sa2065 Purinergic Pathway Is the Major Mechanism of Inhibitory Effect of Motilitone on the Rat Gastric Fundus Relaxation Yang Won Min, Kyu Choi, Eun-ju Ko, Ji Yeon Lee, Eun Ran Kim, Byung-Hoon Min, Jun Haeng Lee, Jae J Kim, Jong Chul Rhee, Sang D. Koh, Poong-Lyul Rhee Background/Aims: Motilitone, a new prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, has strong prokinetic effects. In a previous animal study, motilitone significantly improved gastric accommodation by increasing the postprandial gastric volume. In this study, we investigated how motilitone affects the muscle relaxation in the rat gastric fundus. Methods: Gastric fundus smooth muscle strips (12 longitudinal muscles and 9 circular muscles) were obtained from rats. Isometric force measurements were performed by using both longitudinal and circular muscle strips in response to the electrical field stimulation (EFS, 0.3 ms in trains of 5 Hz for 10 s, 150 V). Peak (the highest value) and nadir (the lowest value) were observed during the first 1 minute after the initiation of EFS in the control state and after administration of atropine (1 μM), motilitone (5 μg, 10 μg, 50 μg, and 100 μg), N-nitro-L-arginine (L-NNA, 100 μM), and MRS 2500 (1 μM), which were added in sequential order to the organ bath. Then tetrodotoxin (TTX, 1 μM) was administered to inhibit nerve-mediated relaxation. Results: EFS induced contractions in the control state, which were relaxed by atropine in the both longitudinal and circular muscles. When motilitone was added to the atropine group, the nadir dropped in a dose-dependent manner in the both longitudinal and circular muscles. The nadir did not increase significantly when L-NNA was added to the atropine and motilitone (100 μg) group in the both longitudinal and circular muscles. After addition of MRS 2500, the nadir increased to the level of atropine group (before administration of motilitone) in the circular muscles. In the longitudinal muscles, the nadir also increased but was not fully reversed to the level of atropine group after addition of MRS 2500. Addition of TTX increased the nadir further and completely abolished EFS-induced relaxation in the both longitudinal and circular muscles. However, there were no significant changes in the peak after administration of motilitone regardless of its dosage. Conclusion: Our data suggest that the inhibitory effect of motilitone on the rat gastric relaxation is largely mediated by purinergic rather than nitrergic pathway, and non-nitrergic non-purinergic component also appears to be involved in the EFS-induced relaxation in the presence of motilitone.
Sa2068 Prevention of Fructose-Induced Obesity by Intestinal Alkaline Phosphatase Konstantinos P. Economopoulos, Kanakaraju Kaliannan, Nur Muhammad, Abeba Teshager, Sulaiman R. Hamarneh, Mussa Mohamed, Palak Patel, Qingsong Tao, Seyed M. Abtahi, Madhu S. Malo, Richard A. Hodin Introduction: The average consumption of fructose per capita in the U.S. has increased by approximately 200% over the past 30 years, mainly due to the dramatic increase in the consumption of fructose-sweetened beverages. It has been proposed that chronic fructose ingestion leads to intestinal bacterial dysbiosis and increased gut permeability. Intestinal Alkaline Phosphatase (IAP) is a brush border enzyme known to preserve intestinal epithelial barrier function and maintain the normal gut microbial homeostasis. Our aim in this study was to examine if IAP could prevent fructose-induced obesity and its deleterious effects. Methods: Three groups of eight-week old, C57BL/6 male mice (n=5) with unrestricted access to regular chow diet were administered either regular autoclaved water, or water enriched with 30% of fructose ± oral IAP (50 units/ml drinking water) for 16 weeks. Body weight and food intake were monitored weekly. The effects of IAP on serum glucose, intestinal permeability and normal flora were assessed. Data were expressed as mean ± standard error and were analyzed by one-way analysis of variance with Tukey's multiple comparison posttests. A significant difference was considered when p ,0.05. Results: The mice that received fructose gained significantly more weight compared to the controls (14.4±1.93 vs 7.2±0.35 g; p=0.016). Food intake was similar between mice receiving fructose+IAP and fructose alone (p=0.897). Fructose+IAP compared with fructose alone was associated with significantly less weight gain (8.3±1.81 vs 14.4±1.93 g; p=0.004), less visceral (1.1±0.10 vs 1.9±0.32 g; p=0.032) and subcutaneous fat weight (1.4±0.15 vs 2.4±0.26 g; p=0.011), lower pancreas weight (0.16±0.01 vs 0.22±0.02 g; p=0.022), lower fasting blood glucose levels (130±6.92 vs 181±15.71 mg/dl; p=0.017), lower area under the curve during glucose tolerance test (21,210±771.57 vs 24,394.5±668.72; p=0.011) and decreased intestinal permeability (0.3±0.08 vs 1.3±0.35; p=0.032). Mice receiving fructose+IAP compared to those receiving only fructose had increased number of aerobic bacteria (46.1E+05±9.75E+05 vs 4.48E+05±0.75E+05; p=0.001) and decreased number of gram negative bacteria (80±58.3 vs 2.77E+04±0.69E+04; p=0.001) in their stool. Conclusions: Oral supplementation with
Sa2066 The Provocative Water Load Test Evokes Nausea and Gastric Dysrhythmias in Patients With Unexplained Chronic Nausea and Vomiting Marcum Gillis, Kenneth L. Koch Background: The origins of nausea in patients with chronic nausea and vomiting syndromes can be difficult to elucidate. Ingestion of solids or liquids frequently precipitates symptomatic fullness and nausea. Aims: 1. To determine the incidence of abnormal water load tests (WLT) in patients with unexplained nausea and vomiting. 2. To determine the incidence of gastric dysrhythmias and severity of nausea symptoms evoked by the WLT. 3. To determine the relationship of water load volume ingested, gastric dysrhythmias, and gastric emptying results. Methods: Patients from Wake Forest Baptist Medical Center Gastroenterology Clinics who had an electrogastrogram (EGG) with WLT and solid phase gastric emptying studies were identified retrospectively. Baseline EGG recordings before and 10, 20, and 30 minutes after the WLT were evaluated. Patients drank water until they were "full" over a 5-minute period. The total volume of water ingested and visual analog scales for nausea, bloating, fullness, and hunger were completed at the time points above and overall EGG diagnoses were determined. An abnormal WLT volume was defined as less than 550 ml ingested in 5 minutes. Chi square analyses were used for statistical significance. Results: 113 patients were identified (72 women, 41 men, average age 50 years). 68/113 (60%) of patients had abnormal WLTs (average volume ingested 360 ± 29 ml) vs 702 ± 27 ml for normal WLT
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PGFS, and PGIS leading to the production of PGD, PGE2, PGF2 α, and PGI, respectively. Previous studies found that mechanical stretch in bowel obstruction markedly induced gene expression of COX-2, but not COX-1, in colonic smooth muscle cells (SMCs). The aims of the present study were to determine whether genes encoding individual prostaglandin synthases are mechanically responsive, and whether the composition of prostaglandins resulting from obstruction differs from that in inflammation. Methods: Partial colon obstruction was induced with a silicon band implanted surgically in the distal colon of male SpragueDawley rats. Static mechanical stretch (18% elongation) was mimicked in vitro in the culture of rat colonic circular SMCs with a Flexercell FX-4000 System. Rat colon inflammation was induced by manipulation of the colonic surface with wet cotton applicators. Results: 1. Quantitative RT-PCR found that the expression of PGDS and PGIS genes was not significantly changed on day 3 of obstruction. However, the membrane bound PGES-1 (mPGES-1) mRNA was significantly up-regulated in the obstructed segment, whereas mRNAs for other two types of PGES, cytosolic PGES and mPGES-2, were not changed. On the contrary, the PGFS mRNA was down-regulated in the obstructed colon. 2. PGE2, but not PGF2 α, was significantly increased in the colonic muscularis externae in obstruction. The PGE2 level increased to 4577±1249 pg/mg at day 3 of obstruction, compared to 1264±259 in sham (n=6, p,0.05). The PGF2 α level was 5718±910 pg /mg in obstruction compared to 6550±2178 pg/mg in the sham (n=5, p .0.05). 3. Direct stretch of rat colonic circular SMCs in vitro significantly up-regulated the mPGES-1 mRNA, but down-regulated that of PGFS. After stretch for 3 hrs, the mRNA levels of PGES and PGFS changed by 2.61±0.42 fold and 0.82±0.03 fold, respectively (n = 4 or 5, p ,0.05). 4. In colonic inflammation, both PGE2 and PGF2α were significantly increased in the colonic smooth muscle (5.2(±1.5)-fold and 4.7(±1.3)-fold of control, respectively, p ,0.05, n = 3). 5. Rat colonic circular muscle contractility was inhibited by exogenous PGE2 (10-9 ~10-6 M), but enhanced by PGF2 α (10-9 ~10-6 M) in a concentration-dependent manner (n = 4). Conclusions: Genes involved in the COX pathway of arachidonic acid metabolism are highly mechanosensitive in colonic SMCs, and play a critical role in motility function. Mechano-regulation of prostaglandin synthases accounts for increased PGE2 and unchanged PGF2 α in bowel obstruction, a pathological feature different from inflammation in which both PGE2 and PGF2 α are increased.