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Sa2061 Peri-Capillary Interstitial Cells of Cajal in the Human Colon Muscle Layers
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
S-372
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
S-372