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S2072 Correlation Between Number of Interstitial Cells of Cajal, Multimodal Electrogastrography and Symptoms in Gastroparesis
AGA Abstracts
in 16D mutants rings was attenuated by 70% compared to control rings; ii) The rate of force generation was ten times slower in 16D mutant rings (0.032 μN/sec) versus control rings (0.374 μN/sec). iii) There was no significant difference in the magnitude or rate of force generation between 16A mutant rings and controls. 3) Upon PdBU stimulation, 16D mutant rings displayed ~33% attenuation in peak maximal force, with no significant change in the rate of force generation. 4) VIP-induced relaxation remained unchanged in magnitude or rate of relaxation between the mutant and control groups. Summary: Phosphomimic HSP20 inhibited direct activation of PKC by PdBU by 30% while it inhibited Ach-induced contraction by 70%. This data confirms that phosphorylated HSP20 inhibits both PKC and non-PKC mediated components of Ach-induced circular smooth muscle contraction. Conclusion: The above results are direct evidence that in CSMC, phosphorylated HSP20 inhibits contraction. The inhibition seems to affect both PKC-mediated and non-PKC mediated contractions. This is the first report of real time force measurements from bioengineered constructs that directly relate inhibition of force generation to the phosphorylation status of HSP20. Supported by NIH/NIDDK RO1DK042876
Neurogastroenterol Motil 2009;21:(A)14 ‡ Lammers et al. Am J Physiol 2009;296(6):G1200-10. Regional Slow Wave Variation
S2072
S2074
Correlation Between Number of Interstitial Cells of Cajal, Multimodal Electrogastrography and Symptoms in Gastroparesis Cheryl E. Bernard, Matthew S. Lurken, Danielle C. Spree, Archana Kedar, Michael Griswold, Thomas L. Abell, Henry P. Parkman, Kenneth L. Koch, William L. Hasler, Pankaj J. Pasricha, William J. Snape, James Tonascia, Aynur Unalp-Arida, Frank A. Hamilton, Gianrico Farrugia
Effects and Mechanisms of Gastric Electrical Stimulation on Visceral Hypersensitivity in Rats With Gastric Ulcers Yan Sun, Chao Qin, Jiande Chen
* p<0.05, **p<0.01 compared to all other gastric regions
Background: Visceral hypersensitivity and/or pain are one of major symptoms in patients with gastric ulcers. Gastric electrical stimulation (GES) has been under investigation as a potential therapy for functional gastric disease. Aims: To investigate the effects and mechanisms of GES on visceral hypersensitivity in a rat model with gastric ulcers. Methods: Under anesthesia, 10 μl of 20% acetic acid was injected into 12-15 sites in the submucosal layer of the stomach wall in 35 Sprague-Dawley (SD) male rats. Each rat was chronically placed with an intragastric balloon and two pairs of electrodes on gastric serosa for GES and at the neck muscles for electromyography (EMG) recordings. The study was composed of 3 experiments (Exp). 1) Exp 1 was designed to select effective GES parameters in reducing EMG response to gastric distention (GD), 20, 40, 60, 80 mmHg in 15 rats 5-7 days after the surgery. GES parameters included: frequency, 10-100Hz; pulse width, 0.25-0.5ms; pulse amplitude, 4-6mA; train duration of 0.1-3s on and 0.4-5s off. 2) Exp 2 was to examine if the opioid pathway was involved in the effects of GES. 10 rats were administrated with opioid receptors antagonist naloxone (1mg/kg, ip). Effects of GES on EMG response to GD were tested 20 minutes after the drug administration. 3) Exp 3 was to assess the spinal afferent mechanisms of GES. Extracellular responses to GD of spinal neurons (T9-T10) were recoded with or without GES in 10 anesthetized rats. Results: 1) GES with train on-time of 0.1s and off-time of 0.4s, 0.25ms, 100Hz, and 6mA significantly reduced GD-induced EMG responses by 41.0±0.9% (P=0.005) to GD at 40mmHg, 40.9±2.6% (P=0.001) at 60mmHg and 48.5±1.8% (P=0.001) at 80mmHg. 2) The inhibitory effects of GES on the GDinduced EMG responses were completely blocked by Naloxone. 3) GES with the parameters effective in reducing the EMG response inhibited 90% high-threshold (HT) spinal neurons total responses to GD of 60mmHg by 49.1% (188.9±59.6 imp/s vs. 384.9±84.0 imp/s, P= 0.0005). But GES with the same parameters only suppressed 36.3% low-threshold (LT) neuronal total response to GD of 60mmHg. Conclusions: GES with appropriate parameters has an ameliorating effect on gastric hypersensitivity; the analgesic effect of GES on visceral pain is mediated via the endogenous opioids pathway and inhibitory mechanism of HT spinal neurons activity with gastric input.
Purpose: Interstitial cells of cajal (ICC) generate signal detected by electrogastrogram (EGG) and contribute to normal gastric function. We studied biopsies from patients with gastroparesis (GP) to assess associations between ICC, EGG values, and symptoms. Patients: Full thickness gastric biopsies were obtained at gastric stimulator (GES) implantation from 16 patients (6M, 10F; mean age 49; 7 idiopathic, 9 diabetic) in an ongoing NIDDK Gastroparesis Clinical Research Consortium registry, and from 16 control patients undergoing obesity surgery, matched for age and sex. Methods: Baseline values for nausea (PN), vomiting (PV), and total symptoms (PTS) were obtained by the Patient Assessment of Upper Gastrointestinal Disorders Symptoms Severity Index (PAGI Sym). At baseline, temporary, and permanent stimulator placement, cutaneous (cut), mucosal (muc), and serosal (ser) EGG frequency (freq) and amplitude (amp) were recorded. EGGs, recorded as cutaneous (cut) at baseline, mucosal (muc) at temporary, and serosal (ser) at permanent device placement, were analyzed by signal averaging, and reported as mean frequency (freq) and amplitude (amp). Circular muscle ICC were counted in 39 high power fields (HPF) and reported as ICC/HPF. Linear regression for all patients was used to determine associations, unadjusted (UnAdj) and adjusted for diabetes (AdjDM). These findings are reported as slope, 95% CI, p-Value. and R2 value. Results: ICC counts were significantly lower for GP patients, at 2.4 (95% CI 1.6, 3.2), than controls, at 5.5 (95% CI 5.0, 6.1)(p<0.0001 by χ-square). ICC values were associated with increased mucosal EGG frequency (UnAdj: slope 0.72; 95% CI -0.06, 1.50; p=0.07, R2=0.17; and AdjDM: slope 0.74; 95% CI 0.04, 1.43; p=0.04, R2=0.35). Increased Serosal EGG amplitude was associated with PV (UnAdj: slope 0.17; 95% CI 0.03, 2.11; p= 0.04; R2=0.20; and AdjDM: slope 1.3; 95% CI 0.28, 2.33; p=0.01, R2=0.30) and with PTS (UnAdj: slope 29; 95% CI 6.7, 51.2; p=0.01; R2=0.30; and AdjDM: slope 29; 95% CI 5.7, 52.4; p=0.01; R2=0.29). Decreased Cutaneous EGG amplitude was associated with PV (UnAdj: slope -15.9; 95% CI -27.5,-4.4; p=0.01; R2 =0.33; and AdjDM: slope -16.03; 95% CI -27.9,-4.2; p=0.01; R=0.33). Conclusion: In this small, but well characterized group, ICC values correspond with mucosal EGG values, and serosal and cutaneous EGG values are associated with symptoms. These data suggest a pathophysiological link between ICC numbers and electrophysiology, and electrophysiology and GP symptoms. Future examination of ICC values, along with mucosal and serosal EGG recordings may help define the relationship of cutaneous EGG to ICC and ICC to symptoms in GP.
S2075 Changes in the Expression of Thin Filament-Associated Proteins in Colonic Smooth Muscle From Mice During TNBS-Induced Colitis Reem M. Alkahtani, Sunila Mahavadi, Olivia Manion, Wimolpak Sriwai, Karnam S. Murthy
S2073
The contractility of smooth muscle in inflammatory bowel disease and experimental colitis is reduced due to inhibition of neurotransmitter release and a decrease in the response of smooth muscle to contractile agonists. Others and we have shown that inflammation induced by TNBS treatment alters the expression and/or activity of signaling molecules involved in the regulation of Ca2+ mobilization, MLC20 phosphorylation and contraction in colonic smooth muscle. Although, thin filament- associated proteins such as calponin, caldesmon, tropomyosin and smoothelin do not directly participate in contraction, they regulate actomyosin interaction and thus, muscle contraction. Calponin, caldesmon and tropomyosin inhibit actomyosin interaction and the inhibition is relieved upon phosphorylation of these proteins. Recent studies have shown that visceral smooth muscle from smoothelin knockout mice exhibited decreased contraction. However, the effect of inflammation on the expression thin filament- associated proteins is not known. Aim. To determine the changes in the expression of calponin, caldesmon, tropomyosin, smoothelin in colonic circular smooth muscle from TNBS-induced colitis mice. Methods. TNBS (2.5% in 50% ethanol) was instilled into the colon of mice. As a control, the same volume of 50% ethanol was instilled. The animals were euthanized on day 3 and a segment of inflamed distal colon was removed. Circular muscle strips were dissected for western blot and real-time RT-PCR analysis; contraction was measured by scanning micrometry in cells isolated from the muscle strips. Results. Contraction in response to acetylcholine in muscle cells isolated from colonic muscle strips derived from mice with TNBS colitis was significantly inhibited compared with the response of cells derived from untreated colon or colon treated with ethanol. Expression of calponin, smoothelin and tropomyosin mRNA in muscle strips from TNBS colitis was significantly increased, whereas expression of caldesmon was unchanged. Similarly, expression of calponin, tropomyosin and smoothelin protein as determined by western blot was significantly increased, whereas caldesmon protein was unchanged. There was no significant change in calponin, tropomyosin, and smoothelin expression in muscle strips derived from colon treated with ethanol compared to control animals. Conclusion. Expression of calponin, tropomyosin and smoothelin is upregulated in colonic circular smooth muscle from TNBS colitis. Increase in the expression of calponin and tropomyosin, which act to inhibit actomyosin interaction, could contribute to decrease in smooth muscle contraction.
High Resolution Mapping of Regional Variations in Porcine Gastric Slow Wave Activity John U. Egbuji, Gregory O'Grady, Peng Du, Leo K. Cheng, Wim Lammers, John A. Windsor, Andrew J. Pullan Background and Aims: The pig is increasingly being used as a model for In-Vivo gastric electrophysiology and electrostimulation studies. However, the normal gastric slow wave (SW) activity of the pig has not been characterized, including normal regional variations in activity. High resolution (HR) electrical mapping has recently enabled an accurate description of gastric SW activity in humans† and canines‡. The aim of this study was to use HR mapping to characterize porcine gastric SW activity and compare it with human and canine data. Methods: Sixteen fasted weaner pigs underwent HR mapping following anesthesia and laparotomy. Flexible printed circuit board arrays were used (160-192 electrodes total; inter-electrode distance 7.62 mm). The stomach was divided into six anatomical regions (see table) and localization of the arrays was performed by measurement against fixed anatomical landmarks. Activation time maps, velocities, amplitudes and frequencies were calculated (mean ± SEM) and ANOVA was used to analyze regional variations (Tukey posttest); significance threshold (p<0.05). Results: From a pacemaker site near the greater curvature of the mid-fundus, the SWs initially propagated radially, then organized into a transverse band propagating in the organo-axial direction towards the pylorus. Multiple slow waves propagated simultaneously, at a frequency of 3.22 ± 0.23 cpm*. Regional variations in amplitude and velocity were defined (see table), and regions adjacent to the lesser curvature and proximal fundus were relatively quiescent. The marked antral transition in SW propagation seen in the human (2x higher amplitude; 2x greater velocity) and canine (2.2x higher amplitude; 3x greater velocity) was not observed in the pig. Conclusions: This study has characterized normal porcine gastric SW activity and will inform future gastric electrophysiology research using this model. The pacemaker region has now been shown to be associated with high-amplitude, high-velocity activity in pig, human† and dog‡. Proximal porcine gastric activity is similar to that shown in the human and dog, however, investigators must allow for substantial differences in distal gastric activity. † O'Grady et al.
AGA Abstracts
S-314
in 16D mutants rings was attenuated by 70% compared to control rings; ii) The rate of force generation was ten times slower in 16D mutant rings (0.032 μN/sec) versus control rings (0.374 μN/sec). iii) There was no significant difference in the magnitude or rate of force generation between 16A mutant rings and controls. 3) Upon PdBU stimulation, 16D mutant rings displayed ~33% attenuation in peak maximal force, with no significant change in the rate of force generation. 4) VIP-induced relaxation remained unchanged in magnitude or rate of relaxation between the mutant and control groups. Summary: Phosphomimic HSP20 inhibited direct activation of PKC by PdBU by 30% while it inhibited Ach-induced contraction by 70%. This data confirms that phosphorylated HSP20 inhibits both PKC and non-PKC mediated components of Ach-induced circular smooth muscle contraction. Conclusion: The above results are direct evidence that in CSMC, phosphorylated HSP20 inhibits contraction. The inhibition seems to affect both PKC-mediated and non-PKC mediated contractions. This is the first report of real time force measurements from bioengineered constructs that directly relate inhibition of force generation to the phosphorylation status of HSP20. Supported by NIH/NIDDK RO1DK042876
Neurogastroenterol Motil 2009;21:(A)14 ‡ Lammers et al. Am J Physiol 2009;296(6):G1200-10. Regional Slow Wave Variation
S2072
S2074
Correlation Between Number of Interstitial Cells of Cajal, Multimodal Electrogastrography and Symptoms in Gastroparesis Cheryl E. Bernard, Matthew S. Lurken, Danielle C. Spree, Archana Kedar, Michael Griswold, Thomas L. Abell, Henry P. Parkman, Kenneth L. Koch, William L. Hasler, Pankaj J. Pasricha, William J. Snape, James Tonascia, Aynur Unalp-Arida, Frank A. Hamilton, Gianrico Farrugia
Effects and Mechanisms of Gastric Electrical Stimulation on Visceral Hypersensitivity in Rats With Gastric Ulcers Yan Sun, Chao Qin, Jiande Chen
* p<0.05, **p<0.01 compared to all other gastric regions
Background: Visceral hypersensitivity and/or pain are one of major symptoms in patients with gastric ulcers. Gastric electrical stimulation (GES) has been under investigation as a potential therapy for functional gastric disease. Aims: To investigate the effects and mechanisms of GES on visceral hypersensitivity in a rat model with gastric ulcers. Methods: Under anesthesia, 10 μl of 20% acetic acid was injected into 12-15 sites in the submucosal layer of the stomach wall in 35 Sprague-Dawley (SD) male rats. Each rat was chronically placed with an intragastric balloon and two pairs of electrodes on gastric serosa for GES and at the neck muscles for electromyography (EMG) recordings. The study was composed of 3 experiments (Exp). 1) Exp 1 was designed to select effective GES parameters in reducing EMG response to gastric distention (GD), 20, 40, 60, 80 mmHg in 15 rats 5-7 days after the surgery. GES parameters included: frequency, 10-100Hz; pulse width, 0.25-0.5ms; pulse amplitude, 4-6mA; train duration of 0.1-3s on and 0.4-5s off. 2) Exp 2 was to examine if the opioid pathway was involved in the effects of GES. 10 rats were administrated with opioid receptors antagonist naloxone (1mg/kg, ip). Effects of GES on EMG response to GD were tested 20 minutes after the drug administration. 3) Exp 3 was to assess the spinal afferent mechanisms of GES. Extracellular responses to GD of spinal neurons (T9-T10) were recoded with or without GES in 10 anesthetized rats. Results: 1) GES with train on-time of 0.1s and off-time of 0.4s, 0.25ms, 100Hz, and 6mA significantly reduced GD-induced EMG responses by 41.0±0.9% (P=0.005) to GD at 40mmHg, 40.9±2.6% (P=0.001) at 60mmHg and 48.5±1.8% (P=0.001) at 80mmHg. 2) The inhibitory effects of GES on the GDinduced EMG responses were completely blocked by Naloxone. 3) GES with the parameters effective in reducing the EMG response inhibited 90% high-threshold (HT) spinal neurons total responses to GD of 60mmHg by 49.1% (188.9±59.6 imp/s vs. 384.9±84.0 imp/s, P= 0.0005). But GES with the same parameters only suppressed 36.3% low-threshold (LT) neuronal total response to GD of 60mmHg. Conclusions: GES with appropriate parameters has an ameliorating effect on gastric hypersensitivity; the analgesic effect of GES on visceral pain is mediated via the endogenous opioids pathway and inhibitory mechanism of HT spinal neurons activity with gastric input.
Purpose: Interstitial cells of cajal (ICC) generate signal detected by electrogastrogram (EGG) and contribute to normal gastric function. We studied biopsies from patients with gastroparesis (GP) to assess associations between ICC, EGG values, and symptoms. Patients: Full thickness gastric biopsies were obtained at gastric stimulator (GES) implantation from 16 patients (6M, 10F; mean age 49; 7 idiopathic, 9 diabetic) in an ongoing NIDDK Gastroparesis Clinical Research Consortium registry, and from 16 control patients undergoing obesity surgery, matched for age and sex. Methods: Baseline values for nausea (PN), vomiting (PV), and total symptoms (PTS) were obtained by the Patient Assessment of Upper Gastrointestinal Disorders Symptoms Severity Index (PAGI Sym). At baseline, temporary, and permanent stimulator placement, cutaneous (cut), mucosal (muc), and serosal (ser) EGG frequency (freq) and amplitude (amp) were recorded. EGGs, recorded as cutaneous (cut) at baseline, mucosal (muc) at temporary, and serosal (ser) at permanent device placement, were analyzed by signal averaging, and reported as mean frequency (freq) and amplitude (amp). Circular muscle ICC were counted in 39 high power fields (HPF) and reported as ICC/HPF. Linear regression for all patients was used to determine associations, unadjusted (UnAdj) and adjusted for diabetes (AdjDM). These findings are reported as slope, 95% CI, p-Value. and R2 value. Results: ICC counts were significantly lower for GP patients, at 2.4 (95% CI 1.6, 3.2), than controls, at 5.5 (95% CI 5.0, 6.1)(p<0.0001 by χ-square). ICC values were associated with increased mucosal EGG frequency (UnAdj: slope 0.72; 95% CI -0.06, 1.50; p=0.07, R2=0.17; and AdjDM: slope 0.74; 95% CI 0.04, 1.43; p=0.04, R2=0.35). Increased Serosal EGG amplitude was associated with PV (UnAdj: slope 0.17; 95% CI 0.03, 2.11; p= 0.04; R2=0.20; and AdjDM: slope 1.3; 95% CI 0.28, 2.33; p=0.01, R2=0.30) and with PTS (UnAdj: slope 29; 95% CI 6.7, 51.2; p=0.01; R2=0.30; and AdjDM: slope 29; 95% CI 5.7, 52.4; p=0.01; R2=0.29). Decreased Cutaneous EGG amplitude was associated with PV (UnAdj: slope -15.9; 95% CI -27.5,-4.4; p=0.01; R2 =0.33; and AdjDM: slope -16.03; 95% CI -27.9,-4.2; p=0.01; R=0.33). Conclusion: In this small, but well characterized group, ICC values correspond with mucosal EGG values, and serosal and cutaneous EGG values are associated with symptoms. These data suggest a pathophysiological link between ICC numbers and electrophysiology, and electrophysiology and GP symptoms. Future examination of ICC values, along with mucosal and serosal EGG recordings may help define the relationship of cutaneous EGG to ICC and ICC to symptoms in GP.
S2075 Changes in the Expression of Thin Filament-Associated Proteins in Colonic Smooth Muscle From Mice During TNBS-Induced Colitis Reem M. Alkahtani, Sunila Mahavadi, Olivia Manion, Wimolpak Sriwai, Karnam S. Murthy
S2073
The contractility of smooth muscle in inflammatory bowel disease and experimental colitis is reduced due to inhibition of neurotransmitter release and a decrease in the response of smooth muscle to contractile agonists. Others and we have shown that inflammation induced by TNBS treatment alters the expression and/or activity of signaling molecules involved in the regulation of Ca2+ mobilization, MLC20 phosphorylation and contraction in colonic smooth muscle. Although, thin filament- associated proteins such as calponin, caldesmon, tropomyosin and smoothelin do not directly participate in contraction, they regulate actomyosin interaction and thus, muscle contraction. Calponin, caldesmon and tropomyosin inhibit actomyosin interaction and the inhibition is relieved upon phosphorylation of these proteins. Recent studies have shown that visceral smooth muscle from smoothelin knockout mice exhibited decreased contraction. However, the effect of inflammation on the expression thin filament- associated proteins is not known. Aim. To determine the changes in the expression of calponin, caldesmon, tropomyosin, smoothelin in colonic circular smooth muscle from TNBS-induced colitis mice. Methods. TNBS (2.5% in 50% ethanol) was instilled into the colon of mice. As a control, the same volume of 50% ethanol was instilled. The animals were euthanized on day 3 and a segment of inflamed distal colon was removed. Circular muscle strips were dissected for western blot and real-time RT-PCR analysis; contraction was measured by scanning micrometry in cells isolated from the muscle strips. Results. Contraction in response to acetylcholine in muscle cells isolated from colonic muscle strips derived from mice with TNBS colitis was significantly inhibited compared with the response of cells derived from untreated colon or colon treated with ethanol. Expression of calponin, smoothelin and tropomyosin mRNA in muscle strips from TNBS colitis was significantly increased, whereas expression of caldesmon was unchanged. Similarly, expression of calponin, tropomyosin and smoothelin protein as determined by western blot was significantly increased, whereas caldesmon protein was unchanged. There was no significant change in calponin, tropomyosin, and smoothelin expression in muscle strips derived from colon treated with ethanol compared to control animals. Conclusion. Expression of calponin, tropomyosin and smoothelin is upregulated in colonic circular smooth muscle from TNBS colitis. Increase in the expression of calponin and tropomyosin, which act to inhibit actomyosin interaction, could contribute to decrease in smooth muscle contraction.
High Resolution Mapping of Regional Variations in Porcine Gastric Slow Wave Activity John U. Egbuji, Gregory O'Grady, Peng Du, Leo K. Cheng, Wim Lammers, John A. Windsor, Andrew J. Pullan Background and Aims: The pig is increasingly being used as a model for In-Vivo gastric electrophysiology and electrostimulation studies. However, the normal gastric slow wave (SW) activity of the pig has not been characterized, including normal regional variations in activity. High resolution (HR) electrical mapping has recently enabled an accurate description of gastric SW activity in humans† and canines‡. The aim of this study was to use HR mapping to characterize porcine gastric SW activity and compare it with human and canine data. Methods: Sixteen fasted weaner pigs underwent HR mapping following anesthesia and laparotomy. Flexible printed circuit board arrays were used (160-192 electrodes total; inter-electrode distance 7.62 mm). The stomach was divided into six anatomical regions (see table) and localization of the arrays was performed by measurement against fixed anatomical landmarks. Activation time maps, velocities, amplitudes and frequencies were calculated (mean ± SEM) and ANOVA was used to analyze regional variations (Tukey posttest); significance threshold (p<0.05). Results: From a pacemaker site near the greater curvature of the mid-fundus, the SWs initially propagated radially, then organized into a transverse band propagating in the organo-axial direction towards the pylorus. Multiple slow waves propagated simultaneously, at a frequency of 3.22 ± 0.23 cpm*. Regional variations in amplitude and velocity were defined (see table), and regions adjacent to the lesser curvature and proximal fundus were relatively quiescent. The marked antral transition in SW propagation seen in the human (2x higher amplitude; 2x greater velocity) and canine (2.2x higher amplitude; 3x greater velocity) was not observed in the pig. Conclusions: This study has characterized normal porcine gastric SW activity and will inform future gastric electrophysiology research using this model. The pacemaker region has now been shown to be associated with high-amplitude, high-velocity activity in pig, human† and dog‡. Proximal porcine gastric activity is similar to that shown in the human and dog, however, investigators must allow for substantial differences in distal gastric activity. † O'Grady et al.
AGA Abstracts
S-314