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66 Novel Non-Invasive Method to Assess Colonic Motility in Conscious Mice: Influence of Various Stressors
AGA Abstracts
are attenuated by pretreatment with 5-HT2A/2C stimulation. Methods: The neonatal Wistar rat pups received a subcutaneous injection of either saline or 5-HT2A/2C agonist (±)-1(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI)(0.5mg/kg) every day, and the injection was given 15 min before the maternal separation (MS) manipulation for 180 min daily during 2-14 postnatal days. After growing, rats were exposed to the elevated plus maze (EPM) to measure anxiety-like behavior and colonic motor function. The visceromotor response was evaluated by electromyogram of the abdominal muscle in response to phasic colorectal distention (CRD) (20, 40, 60, and 80 mmHg) for three times. On the next day, tonic CRD (60 mmHg; 15min duration) was loaded to measure plasma adrenocorticoticotropic hormone and serum corticosterone. Results: MS rats spent significantly more time (60.6±6.1%) in the closed arms than controls (43.4±5.3%, p<0.05). MS rats showed significantly more fecal pellet output (3.3±0.8) during the EPM than controls (1.0±0.6, p<0.05). MS rats expressed significantly more visceromotor response to phasic CRD than controls (p=0.001). Administration of DOI significantly reduced anxiety (40.9±2.7%, p<0.05) and fecal pellet output (1.2±0.5, p<0.05) in DOI+MS rats compared with MS rats. The significant difference in visceromotor response between controls and MS rats was also abolished by DOI. Conclusions: Our results support the hypotheses that rats after the maternal separation show anxiety, colonic hypermotility and visceral hypersensitivity, and that administration 5-HT2A/2C receptor agonist reverses these changes. The results suggest a salient role of 5HT2A/2C pathway in the development of pathophysiology mimicking IBS.
and tropical chronic pancreatitis. Loss-of-function alterations in CTRC predispose to pancreatitis by diminishing the protective trypsin degrading activity of chymotrypsin C. 68 Mortality Rate in Patients with Hereditary Pancreatitis (HP), Compared with the French General Population Vinciane Rebours, Marie-Christine Boutron-Ruault, Valérie Jooste, Anne-Marie Bouvier, Apch Apch, Pascal Hammel, Philippe B. Ruszniewski, Philippe Levy HP is an inherited disease due to cationic trypsinogen mutations. The risk of pancreatic adenocarcinoma has been shown to be very high in these patients (pts). Aims: to compare the survival rate of HP pts with that of the general French population, according to several risk factors. Patients and Methods: A national French exhaustive cohort of pts with HP was established (1). Diagnostic criteria were the presence of PRSS1 mutation or chronic pancreatitis in at least 2 first degree relatives or 3 second-degree relatives without another cause. Pts younger than 20 years were excluded. Excess mortality as compared with the general French population was calculated over 2 periods, ie 20-50 and 50-70 years. Esteve model (2) was used to estimate the risk; that was also calculated according to gender, tobacco use, diabetes mellitus and presence of R122H mutation on PRSS1 gene. Results: the cohort comprised 200 pts (181 alive, males: 53 %, smokers: 34 %). PRSS1 mutations were found in 68 %. 140 pts were older than 20 years. Among the 19 deaths, occurring at the median age of 60, 10 were attributable to HP. Global median survival was 74 years (CI 71-79) for the whole cohort. Gender, tobacco use in pts over 18 years old and diabetes mellitus were not associated with differences in survival. Excess mortality risk in HP pts older than 20 years as compared to the general population was 0.02 % between 20 and 50 years, and 0.61 % between 50 and 70 years (NS). According to gender, corresponding figures were 0.004 % and 1 % in males (NS), and 0.09 % in females aged 20-50 (NS). Presence of R122H mutation, diabetes mellitus or tobacco use was not associated with increased death rate in any of the 2 periods. Conclusion: Even if the risk of pancreatic adenocarcinoma is higher in HP patients, patients with HP do not have an excess mortality risk as compared with the general population, irrespective of gender, tobacco use or diabetes mellitus. These data should be brought to the knowledge of both patients and insurance companies. (1) Risk of pancreatic adenocarcinoma in patients with hereditary pancreatitis. A national exhaustive series. Am J Gastroenterol 2007; in press. (2) Relative survival and the estimation of net survival :elements for further discussion. Stat Med, 9,529-538 (1990).
66 Novel Non-Invasive Method to Assess Colonic Motility in Conscious Mice: Influence of Various Stressors Guillaume Gourcerol, Lixin Wang, David W. Adelson, Muriel H. Larauche, Yvette Tache, Mulugeta Million BACKGROUND: Colonic motility plays an important role in colonic physiology and diseases (Dig.Dis.Sci. 36:827-62, 1991; J Am.Geriatr.Soc. 48:1142-50, 2000). Despite the advent of genetically modified mice, there is a glaring lack of knowledge on colonic motility of conscious mice, primarily due to lack of adequate recording methods. AIM: To characterise distal colonic motility and determine the neurochemical pathway involved in the conscious mice colonic responses to stress. METHODS: Adult male C57/BL6 and a genetic model of chronic stress, CRF-overexpressing (CRF-OE) mice were used. Under brief (2 min) isoflorane anesthesia (UBIA), miniaturised-pressure transducers were inserted 2 and 4 cm past the anus and colonic manometry (CM) monitored for 60-120 min in conscious mice placed in acute partial restraint stress (APRS) cage. Colonic pressure changes in response to a 60 min APRS and the influence of cholinergic, nitrergic and CRF pathways was determined. Colonic myenteric neurones activation was assessed by Fos-immunohistochemistry. Separate groups of C57/Bl6 mice underwent, UBIA, sham or abdominal surgery and CM monitored for 60 min before and after surgery. For chronic stress effects, CM was monitored in CRF-OE mice and in C57/Bl6 mice submitted to 60 min APRS for 15 consecutive days. CM data were acquired via a Micro1401 A/D interface and recorded using Spike 2 software. RESULTS: Conscious mice exposed to APRS displays transient (first 20 min) phasic colonic contractions mainly composed of frequent (15-25/h) giant contractions (GCs, >25 mmHg) of which >50% were propagative. Intraperitoneal astressin B (100 mg/kg), a CRF1/2 antagonist, or atropine (0.5 mg/kg) blocked the responses to APRS. Distal colonic contraction was positively correlated with defecation and colonic myenteric activation. Chronic stress in C57/Bl6 or in CRF-OE mice, blunted the APRS-induced defecation and the transient colonic stimulation by shortening the duration of the contractile response to10 min and reducing selectively the frequency of low amplitude contractions. Abdominal surgery, an interoceptive stress, decreased the frequency and duration of both low and high amplitude (GC) contractions compared to the sham surgery group. CONCLUSIONS: We validate a novel non-invasive colonic motility measurement method in conscious mice and established the presence of highly frequent phasic giant contraction in response to acute restraint that involves cholinergic and CRF pathways. Data show also that chronic exposure to homotypic stressors leads to an adaptive colonic motility response. Supported by NIH grants: DK 068155; 33061; 57238
69 Intestinal Current Measurement As a Diagnostic Procedure in Cystic Fibrosis in Infants and Young Children Michael Wilschanski, Yasmin Yaakov, Gidon Stern, David Shoseyov, Joseph Rivlin, Lea Bentur, Shmuel Goldberg, Edna Shachar, Eitan Kerem Aim: Assessing functional CFTR activity using nasal potential difference measurement is becoming an integral component of CF diagnosis in non-classical presentations. However, the need for patient cooperation limits the application of this test to older children. We present our experience of an alternative method of assessing functional CFTR activity, which involves intestinal current measurement (ICM) of rectal biopsies, thereby removing the agelimitation from this useful diagnostic aid. Methods: Rectal biopsies were taken from 3 sets of patients: known CF, controls, and suspected CF. This last group included patients with a mean age 3.2 ± 2.7 years, who were undergoing investigation for a variety of symptoms including recurrent pneumonia, unexplained bronchiectasis, chronic diarrhea and failure to thrive. ICMs were then performed using standard protocols by mounting the rectal biopsy in an Ussing chamber and sequentially adding secretagogues while recording current changes. Results: Comparing the ICMs of the known CF group and the controls revealed a significant difference on addition of the chloride channel activators: carbachol (p<0.0001), histamine (p<0.0001) and cAMP (p=0.003). This enabled the undiagnosed group to be differentiated into those whose values resembled the CF group (13/38) and those who resembled the control group (25/38) (Table 1). Significantly, there was no difference in the sweat tests of these two groups. Conclusion: In this preliminary study we have shown that, like nasal PD measurements, ICM tests may be useful to differentiate between CF and non-CF patients, in particular in those young patients with atypical presentations, in whom performing nasal PD is impractical. Larger studies are needed to confirm these results. Table 1
67 Genetic Defects in the Trypsin Degrading Enzyme Chymotrypsin C (CTRC) Are Associated with Chronic Pancreatitis. Jonas Rosendahl, Heiko Witt, Richard Szmola, Niels Teich, Miklós Sahin-Tóth Background and Aims: The digestive enzyme trypsin plays a critical role in the development of chronic pancreatitis. We recently demonstrated that chymotrypsin C (CTRC) degrades all human trypsin isoforms with high specificity (Szmola and Sahin-Tóth (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 11227-11232). We proposed that CTRC dependent trypsin degradation serves as a protective mechanism against trypsin mediated cellular injury and CTRC might be a candidate for a pancreatitis-associated gene. The aim of the present study was to analyze the CTRC gene in subjects with chronic pancreatitis and to characterize the newly identified CTRC variants functionally. Methods: Genetic analysis included 1320 unrelated individuals with the diagnosis of alcoholic (n=348), hereditary (n=143), idiopathic (n=758) or tropical (n=71) chronic pancreatitis and 3320 control subjects. Wild-type and mutant CTRC were expressed in HEK 293T and AR42J cells via transient transfection and the expression was characterized by SDS-PAGE, activity assays and Western blots. Results: We identified several alterations in the CTRC gene of pancreatitis patients with an overall frequency of 4.7%. The two most frequent alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the idiopathic and hereditary pancreatitis group (3.3%) relative to healthy controls (0.7%; OR=4.6; CI=2.6-8.0; P=1.3x10-7). A replication study on subjects with alcohol-related diseases identified these two variants in 2.9% of individuals with alcoholic chronic pancreatitis but only in 0.7% of subjects with alcoholic liver disease (OR=4.2; CI= 1.2-15.5; P=0.02). Finally, CTRC variants were also found in 14.1% of Indian subjects with tropical chronic pancreatitis but only in 1.2% of healthy controls (OR=13.6; CI=1.7-109.2; P=0.0028). Functional analysis of CTRC variants showed reduced activity and/or secretion, indicating that the mutations result in a loss of CTRC function. Conclusions: In summary, our results identify CTRC as a novel susceptibility gene for alcoholic, hereditary, idiopathic
AGA Abstracts
70 Lessons from the Secretin Stimulation Test: S-MRCP May Not Be the Best Test to Diagnose Small Duct Chronic Pancreatitis John G. Lieb, Cynthia W. Garvan, Cheryl W. Curington, Phillip P. Toskes Background: Secretin stimulated MRCP (S-MRCP) attempts to combine physiologic pancreatic function testing with imaging as a novel way to diagnose chronic pancreatitis (CP). This technology is based on the enhancement of pancreatic duct flow and volume production by secretin. However, it is unclear if volume of pancreatic fluid is really an appropriate endpoint in pancreatic function testing. We retrospectively reviewed 132 consecutive Secretin
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are attenuated by pretreatment with 5-HT2A/2C stimulation. Methods: The neonatal Wistar rat pups received a subcutaneous injection of either saline or 5-HT2A/2C agonist (±)-1(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI)(0.5mg/kg) every day, and the injection was given 15 min before the maternal separation (MS) manipulation for 180 min daily during 2-14 postnatal days. After growing, rats were exposed to the elevated plus maze (EPM) to measure anxiety-like behavior and colonic motor function. The visceromotor response was evaluated by electromyogram of the abdominal muscle in response to phasic colorectal distention (CRD) (20, 40, 60, and 80 mmHg) for three times. On the next day, tonic CRD (60 mmHg; 15min duration) was loaded to measure plasma adrenocorticoticotropic hormone and serum corticosterone. Results: MS rats spent significantly more time (60.6±6.1%) in the closed arms than controls (43.4±5.3%, p<0.05). MS rats showed significantly more fecal pellet output (3.3±0.8) during the EPM than controls (1.0±0.6, p<0.05). MS rats expressed significantly more visceromotor response to phasic CRD than controls (p=0.001). Administration of DOI significantly reduced anxiety (40.9±2.7%, p<0.05) and fecal pellet output (1.2±0.5, p<0.05) in DOI+MS rats compared with MS rats. The significant difference in visceromotor response between controls and MS rats was also abolished by DOI. Conclusions: Our results support the hypotheses that rats after the maternal separation show anxiety, colonic hypermotility and visceral hypersensitivity, and that administration 5-HT2A/2C receptor agonist reverses these changes. The results suggest a salient role of 5HT2A/2C pathway in the development of pathophysiology mimicking IBS.
and tropical chronic pancreatitis. Loss-of-function alterations in CTRC predispose to pancreatitis by diminishing the protective trypsin degrading activity of chymotrypsin C. 68 Mortality Rate in Patients with Hereditary Pancreatitis (HP), Compared with the French General Population Vinciane Rebours, Marie-Christine Boutron-Ruault, Valérie Jooste, Anne-Marie Bouvier, Apch Apch, Pascal Hammel, Philippe B. Ruszniewski, Philippe Levy HP is an inherited disease due to cationic trypsinogen mutations. The risk of pancreatic adenocarcinoma has been shown to be very high in these patients (pts). Aims: to compare the survival rate of HP pts with that of the general French population, according to several risk factors. Patients and Methods: A national French exhaustive cohort of pts with HP was established (1). Diagnostic criteria were the presence of PRSS1 mutation or chronic pancreatitis in at least 2 first degree relatives or 3 second-degree relatives without another cause. Pts younger than 20 years were excluded. Excess mortality as compared with the general French population was calculated over 2 periods, ie 20-50 and 50-70 years. Esteve model (2) was used to estimate the risk; that was also calculated according to gender, tobacco use, diabetes mellitus and presence of R122H mutation on PRSS1 gene. Results: the cohort comprised 200 pts (181 alive, males: 53 %, smokers: 34 %). PRSS1 mutations were found in 68 %. 140 pts were older than 20 years. Among the 19 deaths, occurring at the median age of 60, 10 were attributable to HP. Global median survival was 74 years (CI 71-79) for the whole cohort. Gender, tobacco use in pts over 18 years old and diabetes mellitus were not associated with differences in survival. Excess mortality risk in HP pts older than 20 years as compared to the general population was 0.02 % between 20 and 50 years, and 0.61 % between 50 and 70 years (NS). According to gender, corresponding figures were 0.004 % and 1 % in males (NS), and 0.09 % in females aged 20-50 (NS). Presence of R122H mutation, diabetes mellitus or tobacco use was not associated with increased death rate in any of the 2 periods. Conclusion: Even if the risk of pancreatic adenocarcinoma is higher in HP patients, patients with HP do not have an excess mortality risk as compared with the general population, irrespective of gender, tobacco use or diabetes mellitus. These data should be brought to the knowledge of both patients and insurance companies. (1) Risk of pancreatic adenocarcinoma in patients with hereditary pancreatitis. A national exhaustive series. Am J Gastroenterol 2007; in press. (2) Relative survival and the estimation of net survival :elements for further discussion. Stat Med, 9,529-538 (1990).
66 Novel Non-Invasive Method to Assess Colonic Motility in Conscious Mice: Influence of Various Stressors Guillaume Gourcerol, Lixin Wang, David W. Adelson, Muriel H. Larauche, Yvette Tache, Mulugeta Million BACKGROUND: Colonic motility plays an important role in colonic physiology and diseases (Dig.Dis.Sci. 36:827-62, 1991; J Am.Geriatr.Soc. 48:1142-50, 2000). Despite the advent of genetically modified mice, there is a glaring lack of knowledge on colonic motility of conscious mice, primarily due to lack of adequate recording methods. AIM: To characterise distal colonic motility and determine the neurochemical pathway involved in the conscious mice colonic responses to stress. METHODS: Adult male C57/BL6 and a genetic model of chronic stress, CRF-overexpressing (CRF-OE) mice were used. Under brief (2 min) isoflorane anesthesia (UBIA), miniaturised-pressure transducers were inserted 2 and 4 cm past the anus and colonic manometry (CM) monitored for 60-120 min in conscious mice placed in acute partial restraint stress (APRS) cage. Colonic pressure changes in response to a 60 min APRS and the influence of cholinergic, nitrergic and CRF pathways was determined. Colonic myenteric neurones activation was assessed by Fos-immunohistochemistry. Separate groups of C57/Bl6 mice underwent, UBIA, sham or abdominal surgery and CM monitored for 60 min before and after surgery. For chronic stress effects, CM was monitored in CRF-OE mice and in C57/Bl6 mice submitted to 60 min APRS for 15 consecutive days. CM data were acquired via a Micro1401 A/D interface and recorded using Spike 2 software. RESULTS: Conscious mice exposed to APRS displays transient (first 20 min) phasic colonic contractions mainly composed of frequent (15-25/h) giant contractions (GCs, >25 mmHg) of which >50% were propagative. Intraperitoneal astressin B (100 mg/kg), a CRF1/2 antagonist, or atropine (0.5 mg/kg) blocked the responses to APRS. Distal colonic contraction was positively correlated with defecation and colonic myenteric activation. Chronic stress in C57/Bl6 or in CRF-OE mice, blunted the APRS-induced defecation and the transient colonic stimulation by shortening the duration of the contractile response to10 min and reducing selectively the frequency of low amplitude contractions. Abdominal surgery, an interoceptive stress, decreased the frequency and duration of both low and high amplitude (GC) contractions compared to the sham surgery group. CONCLUSIONS: We validate a novel non-invasive colonic motility measurement method in conscious mice and established the presence of highly frequent phasic giant contraction in response to acute restraint that involves cholinergic and CRF pathways. Data show also that chronic exposure to homotypic stressors leads to an adaptive colonic motility response. Supported by NIH grants: DK 068155; 33061; 57238
69 Intestinal Current Measurement As a Diagnostic Procedure in Cystic Fibrosis in Infants and Young Children Michael Wilschanski, Yasmin Yaakov, Gidon Stern, David Shoseyov, Joseph Rivlin, Lea Bentur, Shmuel Goldberg, Edna Shachar, Eitan Kerem Aim: Assessing functional CFTR activity using nasal potential difference measurement is becoming an integral component of CF diagnosis in non-classical presentations. However, the need for patient cooperation limits the application of this test to older children. We present our experience of an alternative method of assessing functional CFTR activity, which involves intestinal current measurement (ICM) of rectal biopsies, thereby removing the agelimitation from this useful diagnostic aid. Methods: Rectal biopsies were taken from 3 sets of patients: known CF, controls, and suspected CF. This last group included patients with a mean age 3.2 ± 2.7 years, who were undergoing investigation for a variety of symptoms including recurrent pneumonia, unexplained bronchiectasis, chronic diarrhea and failure to thrive. ICMs were then performed using standard protocols by mounting the rectal biopsy in an Ussing chamber and sequentially adding secretagogues while recording current changes. Results: Comparing the ICMs of the known CF group and the controls revealed a significant difference on addition of the chloride channel activators: carbachol (p<0.0001), histamine (p<0.0001) and cAMP (p=0.003). This enabled the undiagnosed group to be differentiated into those whose values resembled the CF group (13/38) and those who resembled the control group (25/38) (Table 1). Significantly, there was no difference in the sweat tests of these two groups. Conclusion: In this preliminary study we have shown that, like nasal PD measurements, ICM tests may be useful to differentiate between CF and non-CF patients, in particular in those young patients with atypical presentations, in whom performing nasal PD is impractical. Larger studies are needed to confirm these results. Table 1
67 Genetic Defects in the Trypsin Degrading Enzyme Chymotrypsin C (CTRC) Are Associated with Chronic Pancreatitis. Jonas Rosendahl, Heiko Witt, Richard Szmola, Niels Teich, Miklós Sahin-Tóth Background and Aims: The digestive enzyme trypsin plays a critical role in the development of chronic pancreatitis. We recently demonstrated that chymotrypsin C (CTRC) degrades all human trypsin isoforms with high specificity (Szmola and Sahin-Tóth (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 11227-11232). We proposed that CTRC dependent trypsin degradation serves as a protective mechanism against trypsin mediated cellular injury and CTRC might be a candidate for a pancreatitis-associated gene. The aim of the present study was to analyze the CTRC gene in subjects with chronic pancreatitis and to characterize the newly identified CTRC variants functionally. Methods: Genetic analysis included 1320 unrelated individuals with the diagnosis of alcoholic (n=348), hereditary (n=143), idiopathic (n=758) or tropical (n=71) chronic pancreatitis and 3320 control subjects. Wild-type and mutant CTRC were expressed in HEK 293T and AR42J cells via transient transfection and the expression was characterized by SDS-PAGE, activity assays and Western blots. Results: We identified several alterations in the CTRC gene of pancreatitis patients with an overall frequency of 4.7%. The two most frequent alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the idiopathic and hereditary pancreatitis group (3.3%) relative to healthy controls (0.7%; OR=4.6; CI=2.6-8.0; P=1.3x10-7). A replication study on subjects with alcohol-related diseases identified these two variants in 2.9% of individuals with alcoholic chronic pancreatitis but only in 0.7% of subjects with alcoholic liver disease (OR=4.2; CI= 1.2-15.5; P=0.02). Finally, CTRC variants were also found in 14.1% of Indian subjects with tropical chronic pancreatitis but only in 1.2% of healthy controls (OR=13.6; CI=1.7-109.2; P=0.0028). Functional analysis of CTRC variants showed reduced activity and/or secretion, indicating that the mutations result in a loss of CTRC function. Conclusions: In summary, our results identify CTRC as a novel susceptibility gene for alcoholic, hereditary, idiopathic
AGA Abstracts
70 Lessons from the Secretin Stimulation Test: S-MRCP May Not Be the Best Test to Diagnose Small Duct Chronic Pancreatitis John G. Lieb, Cynthia W. Garvan, Cheryl W. Curington, Phillip P. Toskes Background: Secretin stimulated MRCP (S-MRCP) attempts to combine physiologic pancreatic function testing with imaging as a novel way to diagnose chronic pancreatitis (CP). This technology is based on the enhancement of pancreatic duct flow and volume production by secretin. However, it is unclear if volume of pancreatic fluid is really an appropriate endpoint in pancreatic function testing. We retrospectively reviewed 132 consecutive Secretin
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