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T1779 Enhanced Ghrelin Secretion and Gastric Excitatory Neural Network in Rats with Gastric Outlet Obstruction
AGA Abstracts
T1778
We examined effects of various chemical compounds on adaptive relaxation using this model. Methods: A latex balloon was inserted through the gastric distal body and placed at the fundus of the guinea pig. Ten to 14 days after this procedure, water was injected at a speed of 1mL/min (1-10 mL, maximum 10mL) into the balloon, and intragastric pressure was recorded for each volume. Either the nitric oxide synthetase inhibitor L-arginine methyl ester (L-NAME), the muscarinic receptor antagonist atropine, or the serotonin (5-HT) 1B/ D receptor agonist sumatriptan was administered intraperitoneally 15 minutes before starting gastric distention. Results: Stepwise distension increased intragastric pressure, reaching a plateau at 6 to 10 mL. Administration of L-NAME (100 mg/kg) increased intragastric pressure on each volume when compared to control (saline). However atropine (3mg/kg) decreased intragastric pressure, and administration of sumatriptan (0.3 or 1.0 mg/kg) induced a dosedependent relaxation of the gastric fundus (Fig.1, *p < .05). Conclusions: Gastric tone was assessed in a conscious guinea pig, which can be a new animal model for measurement of gastric accommodation.
Reciprocal Modulation of Smooth Muscle Cell Contractility in TH1 and Th2 Dominant Environments Using Murine Model of Early Post Inflammatory Gut Dysfunction Hiroyuki Murao, Hirotada Akiho, Takahiro Mizutani, Mariko Yamada, Noriko Tokunaga, Akira Aso, Haruei Ogino, Kenji Kanayama, Yorinobu Sumida, Yoichiro Iboshi, Soichi Itaba, Kazuhiko Nakamura, Ryoichi Takayanagi, Waliul I. Khan Background & Aim: In the enteric-infection-induced model of inflammation, Th1 and Th2 cytokines have opposing effect on intestinal muscle function and 5-HT signaling (Motomura et al. Gut 2008; Gastroenterology 128;Suppl A 626,2005), but the role of Th1 and Th2 dominant environments for non-infective post-inflammatory (PI) gut dysfunction remains to be determined. Here we investigated the mechanism for gut dysfunction that persists after acute inflammation in Th1 and Th2 dominant environments utilizing mouse model of T cell-induced enteropathy. Methods: BALB/c (Th2 dominant response) and AKR (Th1 dominant response) mice were treated with an anti-CD3 antibody (100 μg), and sacrificed at days 0, 1, 3, 7, and 14 post-treatment to investigate the histological changes, longitudinal smooth muscle cell contraction, various cytokines (Th1, Th2, Th17 cytokines), chemokines, and mediators mRNA /protein expressions and 5-HT-expressing enterochromaffin (EC) cells numbers in the small intestine. Results: In BALB/c mice small intestinal tissue damage was observed from 24 hours after the anti-CD3 antibody injection, but had resolved by day 5. Carbachol-induced smooth muscle cell contractility was significantly increased (p<0.05) from 4 hours after injection, and this muscle hypercontractility was evident (p<0.05) in the early PI phase (at day 7). Th2 cytokines (IL-4, IL-13) were significantly increased (p<0.05) from 4 hours to day 7 in the small intestine. EC cells in the intestine were significantly increased from day 1 to day 7 (p<0.05). On the contrary in AKR mice carbachol-induced smooth muscle cell contractility was significantly decreased from 4 hours after injection (p<0.05), and this muscle hypocontractility was evident until day 14. Th1 cytokines (IFNγ) were increased from 4 hours to day 7 in the small intestine. Conclusions: Intestinal muscle dysfunction in the early PI phase is maintained at the smooth muscle cell level. Th1 and Th2 cytokines have opposing effect on intestinal muscle contraction via 5-HT signaling in the early PI phase in this model. The gut dysfunctions in the early PI phase are influenced by Th1 or Th2 cytokine predominance environments.
T1781 The Role of TRPA1 in Visceral Hypersensitivity Arising from Neonatal Colon Irritation Julie A. Christianson, Sacha A. Malin, Brian M. Davis Background: During early neonatal development, the sensory nervous system exhibits dramatic plasticity. Inflammation or pain during this period can result in long-term structural and functional alterations of nociceptive pathways, ultimately altering pain perception in the adult. This has been extensively shown to occur in the somatosensory system and we are currently employing a neonatal colon irritation (NCI) model to investigate similar occurrences in the viscerosensory system. Methods: Male C57Bl6 mice were treated with intracolonic mustard oil (MO; 10μL of 2% in saline) on postnatal days 8 and 10. The distal colons were processed 24 hours after each MO application to assess the degree of inflammation by measuring the amount of myeloperoxidase (MPO) in the tissue. Relative mRNA levels of growth factors were also measured in the colon at various timepoints using real time RTPCR. At 8 weeks of age, mice were tested for visceral hypersensitivity by recording the electromyographic activity of the abdominal musculature in response to colorectal distension (CRD), commonly termed the visceromotor response (VMR). Dorsal root ganglion (DRG) neurons from adult NCI and control mice were retrogradely labeled via a subserosal injection of Alexa Fluor 488-conjugated cholera toxin β (CTB) into the distal colon. CTB-positive DRG neurons were evaluated for TRPV1 immunohistochemistry and responses to acute application of capsaicin and MO using calcium imaging. Results: On postnatal day 8, the neonatal colon has significantly higher mRNA expression of NGF, artemin, GDNF, neurturin and NT-3 than the adult colon, which remains elevated for all growth factors tested, with the exception of artemin, until postnatal day 18 in NCI mice. Despite the lack of an inflammatory response following MO instillation, as evidenced by no change in MPO activity, early exposure to intracolonic MO results in long-lasting visceral hypersensitivity, observable as a significantly increased VMR in adult NCI mice. No increase in TRPV1-immunopositive or capsaicin-sensitive colon DRG neurons was observed in adult NCI mice. However, the percentage of MO-sensitive colon afferents was significantly increased in NCI mice. Conclusions: Early exposure to MO results in a long-lasting change in nociceptive processing within the viscerosensory system, possibly driven by an early increase in growth factor expression and carried out by an enhanced population of TRPA1-positive afferents in the adult. This provides an excellent target for potential therapeutic approaches to treating functional bowel disorders.
T1779 Enhanced Ghrelin Secretion and Gastric Excitatory Neural Network in Rats with Gastric Outlet Obstruction Eisuke Iwasaki, Hidekazu Suzuki, Sachiko Suzuki, Yoshimasa Saito, Tatsuhiro Masaoka, Toshihiro Nishizawa, Hitoshi Tsugawa, Juntaro Matsuzaki, Kenro Hirata, Hiroshi Hosoda, Kenji Kangawa, Toshifumi Hibi Background. Ghrelin is a gastrointestinal peptide that exerts distinct effects on the gastrointestinal motility through the vagus nerve and gastric excitatory neural plexus. We previously reported that the plasma ghrelin levels were significantly higher in patients with dysmotilitylike functional dyspepsia (Aliment Pharmacol Ther. 24, S4:104, 2006). The aim of the present study was to investigate the influence of gastric outlet obstruction caused by surgical manipulation on the dynamics of ghrelin and the gastric neural network in rats. Methods. 7-week-old male SD rats were used. After 24 hours' food deprivation, the rats were divided into two groups, as follows; the control group (n=14; sham operation) and the gastric outlet obstruction (GOO) group (n=14; proximal duodenal stricture with an 18Fr.Nelaton catheter). As weight loss is known to increase the plasma levels of ghrelin, a pair-feeding method was applied to equalize the body weights. Rats were sacrificed 2 weeks after the operation after 24 hours' food deprivation. The gastric emptying rate was evaluated by the phenol red method using gavage feeding of a test liquid or a meal with 2.0% methylcellulose. In addition, the catheters were removed one day before sacrifice (n=5), and the gastric motility was evaluated. The plasma and gastric ghrelin were measured by radioimmunoassay. The mRNA expressions in the stomach of preproghrelin, neural choline acetyltransferase (ChAT), c-kit and stem cell factor (scf) were analyzed by quantitative RT-PCR, and immunohistochemistry was performed to determine the gastric c-kit protein expression. Results. The gastric emptying rate in the GOO group was significantly lower as compared with that in the control group (liquid at 15min; 9.8%, 61.0%, p<0.05, meal at 60min; 57.0% 75.0%, p<0.05). The gastric emptying rate after removal of the mechanical duodenal stricture was significantly enhanced as compared with that in the control group (81.2% vs. 75.0%, p<0.05). Rats in the GOO group showed significant increase of the plasma levels of active ghrelin (p<0.01) and decrease of the gastric levels of active ghrelin (p<0.05). The mRNA expressions of preproghrelin, ChAT, c-kit and scf in the stomach were increased in the GOO group. The level of immunostaining for c-kit was enhanced. Conclusion. Elevated of plasma ghrelin levels in rats with gastric outlet obstruction may stimulate gastric motility through activation of cholinergic neurons and interstitial cells of Cajar. These findings suggest that deregulation of gastric emptying might stimulate the ghrelin dynamics and network of gastric cellular communications between nerves and smooth muscle cells.
T1782 Rotenone Recapitulates Parkinson's Disease-Related Gastrointestinal Motility Deficits and Alpha-Synuclein Pathology in the Enteric Nervous System Robert E. Drolet, Jason R. Cannon, Laura M. Montero, J Timothy Greenamyre Gastrointestinal (GI) problems, particularly, severe constipation and delayed gastric emptying are core symptoms of Parkinson's disease (PD) that affect nearly all patients. GI problems can, in fact, dominate the clinical picture for many patients. Despite the importance of these problems for patients, GI symptoms in PD remain poorly characterized. Very little is known about the pathological basis for PD-related GI problems, as few studies have addressed these issues using laboratory models of the disease. The chronic rotenone model of PD is ideal for investigating PD-related GI dysfunction. Following systemic administration, the lipophillic pesticide is able to gain access to all cells of the body, particularly enteric neurons, which line the GI tract and regulate motility. To investigate the consequences of rotenone on GI function and enteric nervous system (ENS) pathology, rats were treated with vehicle or rotenone (2 mg/kg, i.p.) for 6 consecutive weeks. GI function was assessed by measuring total GI transit time of CW800 carboxylate IR-Dye every month for 6 consecutive months after the last injection. Myenteric plexus neuron loss and alpha-synuclein aggregate pathology were assessed either immediately or 6 months after rotenone. Here we show that rotenoneinduced oxidative stress causes a progressive slowing of gastrointestinal motility that begins approximately 5 months after rotenone treatment was stopped. Motility deficits were associated with a progressive increase in alpha-synuclein positive aggregates, and loss of myenteric
T1780 A New Animal Model for the Measurement of Gastric Accommodation Jun Sakurai, Junichi Koseki, Takashi Kondo, Junji Tanaka, Chizuko Hayashi, Toshihiko Tomita, Yongmin Kim, Tadayuki Oshima, Kazutoshi Hori, Takayuki Matsumoto, Hiroto Miwa Background and Aims: Gastric accommodation is defined as the reduction in gastric tone. This phenomenon is attributable to the adaptive relaxation of the stomach, which increases its capacity. Recent studies have demonstrated that functional dyspepsia is associated with visceral hypersensitivity and impairment of gastric adaptive relaxation. Previous studies for gastric accommodation were mainly performed using large animals such as dogs. Since small rodents like rats do not have gastric smooth muscle in their fore-stomachs, accommodation may not be totally and reliably evaluated. Accordingly, in this study, we established a new model for assessing gastric tone using small experimental animal, an awaken guinea pig.
AGA Abstracts
A-578
T1778
We examined effects of various chemical compounds on adaptive relaxation using this model. Methods: A latex balloon was inserted through the gastric distal body and placed at the fundus of the guinea pig. Ten to 14 days after this procedure, water was injected at a speed of 1mL/min (1-10 mL, maximum 10mL) into the balloon, and intragastric pressure was recorded for each volume. Either the nitric oxide synthetase inhibitor L-arginine methyl ester (L-NAME), the muscarinic receptor antagonist atropine, or the serotonin (5-HT) 1B/ D receptor agonist sumatriptan was administered intraperitoneally 15 minutes before starting gastric distention. Results: Stepwise distension increased intragastric pressure, reaching a plateau at 6 to 10 mL. Administration of L-NAME (100 mg/kg) increased intragastric pressure on each volume when compared to control (saline). However atropine (3mg/kg) decreased intragastric pressure, and administration of sumatriptan (0.3 or 1.0 mg/kg) induced a dosedependent relaxation of the gastric fundus (Fig.1, *p < .05). Conclusions: Gastric tone was assessed in a conscious guinea pig, which can be a new animal model for measurement of gastric accommodation.
Reciprocal Modulation of Smooth Muscle Cell Contractility in TH1 and Th2 Dominant Environments Using Murine Model of Early Post Inflammatory Gut Dysfunction Hiroyuki Murao, Hirotada Akiho, Takahiro Mizutani, Mariko Yamada, Noriko Tokunaga, Akira Aso, Haruei Ogino, Kenji Kanayama, Yorinobu Sumida, Yoichiro Iboshi, Soichi Itaba, Kazuhiko Nakamura, Ryoichi Takayanagi, Waliul I. Khan Background & Aim: In the enteric-infection-induced model of inflammation, Th1 and Th2 cytokines have opposing effect on intestinal muscle function and 5-HT signaling (Motomura et al. Gut 2008; Gastroenterology 128;Suppl A 626,2005), but the role of Th1 and Th2 dominant environments for non-infective post-inflammatory (PI) gut dysfunction remains to be determined. Here we investigated the mechanism for gut dysfunction that persists after acute inflammation in Th1 and Th2 dominant environments utilizing mouse model of T cell-induced enteropathy. Methods: BALB/c (Th2 dominant response) and AKR (Th1 dominant response) mice were treated with an anti-CD3 antibody (100 μg), and sacrificed at days 0, 1, 3, 7, and 14 post-treatment to investigate the histological changes, longitudinal smooth muscle cell contraction, various cytokines (Th1, Th2, Th17 cytokines), chemokines, and mediators mRNA /protein expressions and 5-HT-expressing enterochromaffin (EC) cells numbers in the small intestine. Results: In BALB/c mice small intestinal tissue damage was observed from 24 hours after the anti-CD3 antibody injection, but had resolved by day 5. Carbachol-induced smooth muscle cell contractility was significantly increased (p<0.05) from 4 hours after injection, and this muscle hypercontractility was evident (p<0.05) in the early PI phase (at day 7). Th2 cytokines (IL-4, IL-13) were significantly increased (p<0.05) from 4 hours to day 7 in the small intestine. EC cells in the intestine were significantly increased from day 1 to day 7 (p<0.05). On the contrary in AKR mice carbachol-induced smooth muscle cell contractility was significantly decreased from 4 hours after injection (p<0.05), and this muscle hypocontractility was evident until day 14. Th1 cytokines (IFNγ) were increased from 4 hours to day 7 in the small intestine. Conclusions: Intestinal muscle dysfunction in the early PI phase is maintained at the smooth muscle cell level. Th1 and Th2 cytokines have opposing effect on intestinal muscle contraction via 5-HT signaling in the early PI phase in this model. The gut dysfunctions in the early PI phase are influenced by Th1 or Th2 cytokine predominance environments.
T1781 The Role of TRPA1 in Visceral Hypersensitivity Arising from Neonatal Colon Irritation Julie A. Christianson, Sacha A. Malin, Brian M. Davis Background: During early neonatal development, the sensory nervous system exhibits dramatic plasticity. Inflammation or pain during this period can result in long-term structural and functional alterations of nociceptive pathways, ultimately altering pain perception in the adult. This has been extensively shown to occur in the somatosensory system and we are currently employing a neonatal colon irritation (NCI) model to investigate similar occurrences in the viscerosensory system. Methods: Male C57Bl6 mice were treated with intracolonic mustard oil (MO; 10μL of 2% in saline) on postnatal days 8 and 10. The distal colons were processed 24 hours after each MO application to assess the degree of inflammation by measuring the amount of myeloperoxidase (MPO) in the tissue. Relative mRNA levels of growth factors were also measured in the colon at various timepoints using real time RTPCR. At 8 weeks of age, mice were tested for visceral hypersensitivity by recording the electromyographic activity of the abdominal musculature in response to colorectal distension (CRD), commonly termed the visceromotor response (VMR). Dorsal root ganglion (DRG) neurons from adult NCI and control mice were retrogradely labeled via a subserosal injection of Alexa Fluor 488-conjugated cholera toxin β (CTB) into the distal colon. CTB-positive DRG neurons were evaluated for TRPV1 immunohistochemistry and responses to acute application of capsaicin and MO using calcium imaging. Results: On postnatal day 8, the neonatal colon has significantly higher mRNA expression of NGF, artemin, GDNF, neurturin and NT-3 than the adult colon, which remains elevated for all growth factors tested, with the exception of artemin, until postnatal day 18 in NCI mice. Despite the lack of an inflammatory response following MO instillation, as evidenced by no change in MPO activity, early exposure to intracolonic MO results in long-lasting visceral hypersensitivity, observable as a significantly increased VMR in adult NCI mice. No increase in TRPV1-immunopositive or capsaicin-sensitive colon DRG neurons was observed in adult NCI mice. However, the percentage of MO-sensitive colon afferents was significantly increased in NCI mice. Conclusions: Early exposure to MO results in a long-lasting change in nociceptive processing within the viscerosensory system, possibly driven by an early increase in growth factor expression and carried out by an enhanced population of TRPA1-positive afferents in the adult. This provides an excellent target for potential therapeutic approaches to treating functional bowel disorders.
T1779 Enhanced Ghrelin Secretion and Gastric Excitatory Neural Network in Rats with Gastric Outlet Obstruction Eisuke Iwasaki, Hidekazu Suzuki, Sachiko Suzuki, Yoshimasa Saito, Tatsuhiro Masaoka, Toshihiro Nishizawa, Hitoshi Tsugawa, Juntaro Matsuzaki, Kenro Hirata, Hiroshi Hosoda, Kenji Kangawa, Toshifumi Hibi Background. Ghrelin is a gastrointestinal peptide that exerts distinct effects on the gastrointestinal motility through the vagus nerve and gastric excitatory neural plexus. We previously reported that the plasma ghrelin levels were significantly higher in patients with dysmotilitylike functional dyspepsia (Aliment Pharmacol Ther. 24, S4:104, 2006). The aim of the present study was to investigate the influence of gastric outlet obstruction caused by surgical manipulation on the dynamics of ghrelin and the gastric neural network in rats. Methods. 7-week-old male SD rats were used. After 24 hours' food deprivation, the rats were divided into two groups, as follows; the control group (n=14; sham operation) and the gastric outlet obstruction (GOO) group (n=14; proximal duodenal stricture with an 18Fr.Nelaton catheter). As weight loss is known to increase the plasma levels of ghrelin, a pair-feeding method was applied to equalize the body weights. Rats were sacrificed 2 weeks after the operation after 24 hours' food deprivation. The gastric emptying rate was evaluated by the phenol red method using gavage feeding of a test liquid or a meal with 2.0% methylcellulose. In addition, the catheters were removed one day before sacrifice (n=5), and the gastric motility was evaluated. The plasma and gastric ghrelin were measured by radioimmunoassay. The mRNA expressions in the stomach of preproghrelin, neural choline acetyltransferase (ChAT), c-kit and stem cell factor (scf) were analyzed by quantitative RT-PCR, and immunohistochemistry was performed to determine the gastric c-kit protein expression. Results. The gastric emptying rate in the GOO group was significantly lower as compared with that in the control group (liquid at 15min; 9.8%, 61.0%, p<0.05, meal at 60min; 57.0% 75.0%, p<0.05). The gastric emptying rate after removal of the mechanical duodenal stricture was significantly enhanced as compared with that in the control group (81.2% vs. 75.0%, p<0.05). Rats in the GOO group showed significant increase of the plasma levels of active ghrelin (p<0.01) and decrease of the gastric levels of active ghrelin (p<0.05). The mRNA expressions of preproghrelin, ChAT, c-kit and scf in the stomach were increased in the GOO group. The level of immunostaining for c-kit was enhanced. Conclusion. Elevated of plasma ghrelin levels in rats with gastric outlet obstruction may stimulate gastric motility through activation of cholinergic neurons and interstitial cells of Cajar. These findings suggest that deregulation of gastric emptying might stimulate the ghrelin dynamics and network of gastric cellular communications between nerves and smooth muscle cells.
T1782 Rotenone Recapitulates Parkinson's Disease-Related Gastrointestinal Motility Deficits and Alpha-Synuclein Pathology in the Enteric Nervous System Robert E. Drolet, Jason R. Cannon, Laura M. Montero, J Timothy Greenamyre Gastrointestinal (GI) problems, particularly, severe constipation and delayed gastric emptying are core symptoms of Parkinson's disease (PD) that affect nearly all patients. GI problems can, in fact, dominate the clinical picture for many patients. Despite the importance of these problems for patients, GI symptoms in PD remain poorly characterized. Very little is known about the pathological basis for PD-related GI problems, as few studies have addressed these issues using laboratory models of the disease. The chronic rotenone model of PD is ideal for investigating PD-related GI dysfunction. Following systemic administration, the lipophillic pesticide is able to gain access to all cells of the body, particularly enteric neurons, which line the GI tract and regulate motility. To investigate the consequences of rotenone on GI function and enteric nervous system (ENS) pathology, rats were treated with vehicle or rotenone (2 mg/kg, i.p.) for 6 consecutive weeks. GI function was assessed by measuring total GI transit time of CW800 carboxylate IR-Dye every month for 6 consecutive months after the last injection. Myenteric plexus neuron loss and alpha-synuclein aggregate pathology were assessed either immediately or 6 months after rotenone. Here we show that rotenoneinduced oxidative stress causes a progressive slowing of gastrointestinal motility that begins approximately 5 months after rotenone treatment was stopped. Motility deficits were associated with a progressive increase in alpha-synuclein positive aggregates, and loss of myenteric
T1780 A New Animal Model for the Measurement of Gastric Accommodation Jun Sakurai, Junichi Koseki, Takashi Kondo, Junji Tanaka, Chizuko Hayashi, Toshihiko Tomita, Yongmin Kim, Tadayuki Oshima, Kazutoshi Hori, Takayuki Matsumoto, Hiroto Miwa Background and Aims: Gastric accommodation is defined as the reduction in gastric tone. This phenomenon is attributable to the adaptive relaxation of the stomach, which increases its capacity. Recent studies have demonstrated that functional dyspepsia is associated with visceral hypersensitivity and impairment of gastric adaptive relaxation. Previous studies for gastric accommodation were mainly performed using large animals such as dogs. Since small rodents like rats do not have gastric smooth muscle in their fore-stomachs, accommodation may not be totally and reliably evaluated. Accordingly, in this study, we established a new model for assessing gastric tone using small experimental animal, an awaken guinea pig.
AGA Abstracts
A-578