- Email: [email protected]
Su2053 Gastrointestinal Effects Induced by Microinjection of 6-OHDA in the Substantia Nigra of Rats
Su2053 Su2055 Gastrointestinal Effects Induced by Microinjection of 6-OHDA in the Substantia Nigra of Rats Luca Toti, R. Alberto Travagli
Gender Dependent Effects of Cholera Toxin on Colonic Motility - A Role for Sex Steroids Gayathri K. Balasuriya, Joel C. Bornstein
Idiopathic Parkinson's disease (PD) is a late-onset, chronic and progressive motor dysfunction due to loss of nigrostriatal dopamine (DA) neurons. Various experimental models of PD have been used to study the motor-associated dysfunctions of PD; these models principally involve the destruction of DA neurons with neurotoxins such as 6-OHDA. Since almost all patients with PD show gastrointestinal (GI) dysfunctions, such as gastroparesis, we aimed to evaluate whether gastric emptying correlated with biochemical changes in brainstem vagal nuclei controlling gastrointestinal functions in a rat model of PD. PD was induced in rats (N=18) by unilateral administration of 6-OHDA into the substantia nigra pars compacta (SN); control animals (N=10) were injected with saline. The non-invasive [13C]-octanoic acid breath test was performed weekly to evaluate the rate of gastric emptying. After four weeks, rats were perfused-fixed and the SN, brainstem, esophagus, stomach and duodenum were collected and processed for light microscopy for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). Four weeks after 6OHDA microinjection in the SN, rats that were later confirmed to have sustained a .90% decrease in TH-immunoreactivity (IR) in the SN significantly delayed gastric emptying, with increase of time to peak [13C], 85±2.2 and 62±3.6min, and half emptying time, i.e. 113±3.9 and 95±8.9min in 6-OHDA, N=6, and control rats, N=5, respectively; P ,0.05. In control rats, microinjection of the indirect sympathomimetic, tyramine (40nmoles/60nl) in the dorsal vagal complex decreased gastric tone by ~150mg (N=2), the effect was prevented by 4th ventricular application of yohimbine (500pmoles/60nl) and prazosin (100pmoles/60nl). Conversely, in 6-OHDA treated rats (N=2), microinjection of tyramine had no effect upon gastric tone. The IHC results (expressed as neurons/slice) are summarized in the table below. There was also a qualitative decrease in the expression of nNOS-IR neurons within the myenteric plexus of the esophagus, stomach and duodenum while the distribution of ChATIR neurons and TH-IR fibers in the same areas was not modified. Our data suggest that the delayed gastric emptying in a 6-OHDA rat model of PD with a selective loss of dopaminergic neurons in SN may be caused by changes in expression of neurotransmitters in both brainstem and myenteric neurons. Number of Neurons per Slice
Purpose: Diarrhea is a combination of hypersecretion and increased gut motility. Although the effect of Cholera Toxin (CT) on hypersecretion is highly characterised, its effect on motility remains largely unexplored. We evaluated the effect of CT on colonic migrating motor complexes (CMMCs) in female and male C57Bl/6 mice and further characterised this effect in female mice in estrus and proestrus. The distribution of the estrogen receptor alpha (ERα) in mouse colon was also characterised immunohistochemically. Method: Video imaging was used to construct high resolution spatiotemporal maps of colonic motor patterns in vitro. The entire colon was cannulated at each end and mounted horizontally in an organ bath containing physiological saline warmed to 37oC. The proximal end was connected to a reservoir of physiological saline, the distal end to an outflow tube. CT (0.125 μg/ml, 1.25 μg/ml, 12.5 μg/ml) was introduced to the lumen of 3 groups of female C57Bl/6 mice without considering their stage in the estrus cycle. CT (1.25 μg/ml) was introduced to the male colon and to females in the proestrus (highest estrogen concentration) and estrus (lowest estrogen concentration) stages (determined by screening vaginal smears). ER α was localized immunohistochemically in whole mounts of myenteric and submucosal plexus of colon using PA1-308 antiserum (AffinityBioreagents), along with a pan neuronal marker (Hu) and nitric oxide synthase (NOS). Results: In randomly selected female colon, all three concentrations of CT significantly and reversibly reduced the number of CMMCs (p , 0.001, 2 way ANOVA, n = 5 in each case). This occurred within 15 min of exposure to CT and was sustained for 1 h, but reversed when CT was flushed from the colonic lumen. The effect was blocked by granisetron (1 μM, 5-HT3 receptor antagonist) with the number of CMMCs in the presence of both CT and granisetron being indistinguishable from control (p . 0.05). CT (1.25 μg/ml) produced a significant (p . 0.001, n = 5) reduction in CMMC frequency in females during estrus but had no significant effect during proestrus or in male colon. ERα were expressed in myenteric and submucosal neurons. They co-localised with NOS in some neurons, but ER α expressing neurons that did not express NOS were also seen. Conclusion: The colon from female mice in estrus behaves significantly differently in response to luminal CT compared to both male colon and colon from mice in proestrus. This may be due to the lower level of sex steroidal hormones in estrus, which is consistent with our finding that ERα is expressed in colonic myenteric and submucosal ganglia. Su2056 New Vista Into Delayed Emesis Assessment: Validation of the Emetic Response in the Ferret Monitored by Telemetry Using Computerized Detection Sonia Goineau, Philippe Guillaume, Sonia Rompion, Toni Wolinsky, David J. Virley Nausea and vomiting are common side effects of cancer chemotherapy. While early emesis can be easily investigated in ferrets by direct observation, alternative methods are required to quantify delayed emesis which is time-consuming. This study was therefore designed to validate the computerized detection of changes in abdominal pressure related to retches or vomits induced by a cytotoxic substance in the telemetered ferret. Five ferrets were implanted with telemetry devices (Data Sciences International). They were then intra-peritoneally challenged with cisplatin (8 mg/kg) and abdominal pressure was recorded in unrestrained animals for 72 hours. The pressure signals were analyzed both manually and automatically using an adapted version of ecgAUTO software (Emka Technologies). Over the first 3 hours, the emetic response was also quantified via direct observation of the animals. The data produced by the 3 methods of detection were compared using a Spearman's rank correlation coefficient. Visual, manual and computerized detections of early emetic events over the first 3-hour recording period were well-correlated when compared per 30-, 15- or 5-minute epoch: correlation coefficients ranging from 0.8640 to 0.9289, p , 0.0001 for all comparisons. Manual and computerized detections of early and delayed emetic events over the 72hour recording period were also well-correlated when compared per 3-hour epoch: correlation coefficient = 0.9190, p , 0.0001. These findings demonstrate that computerized detection of abdominal pressure changes with adapted software is a reliable method for measuring emetic events in the ferret. This approach opens a new vista into emesis assessment and research to permit rapid, comprehensive and objective analysis of delayed emesis and to facilitate the development of novel anti-emetic therapies.
Su2054 Nitric Oxide Mediated Colonic Motility Is Altered in the Neuroligin-3 R451c Mouse Model of Autism Mathusi Swaminathan, Jaime Pei Pei Foong, Melina Ellis, Joel C. Bornstein, Elisa L. HillYardin Purpose: Gastrointestinal function is compromised in up to 90% of patients with Autism spectrum disorder (ASD) and the underlying causes are unknown. NL3R451C mice express a missense mutation in the Nlgn3 gene coding for the postsynaptic adhesion protein, neuroligin-3 (NL3) found in ASD patients. We have previously shown that the excitatory component of organized colonic motility (GABAA- and serotonergic- mediated) is perturbed in NL3R451C mice compared to wild type (WT) controls. Neuronal release of nitric oxide (NO) is responsible for tonic inhibition between colonic migrating motor complexes (CMMCs). Here, we examined whether the NO-mediated inhibitory component of spontaneous CMMCs is altered in NL3R451C mice. Methods: Full length colons were removed from NL3R451C, WT littermates (control) and C57Bl/6 mice (comparison strain). Colonic motility was examined using video imaging techniques. After a 1 h control recording, the NO synthase (NOS) inhibitor L-Nitro-arginine (NOLA, 100μm) was applied into the superfusing solution and activity was recorded for 1 h. The NOLA was then washed out for 1 h while a further recording was made. The proportion of all myenteric neurons (marked by the pan neuronal marker, Hu) that contained GABA and/or NOS was investigated using immunohistochemistry. Myenteric neuronal density and connectivity between GABA+ and NOS+ neurons were examined. Results: NOLA significantly increased CMMC frequency in NL3R451C (n = 9; p = 0.031), but not in WT littermates (n = 9; p = 0.831). NOLA also increased CMMC frequency in C57/Bl6 colons (n = 9; p = 0.008). No differences in neuronal density were observed between proximal (adjacent to cecum) and mid colon regions of WT and NL3R451C mice
S-543
AGA Abstracts
AGA Abstracts
(n = 3 each; p . 0.05). Furthermore, there was no difference in the mean number of neurons per ganglion in the distal colon of WT and NL3R451C mice (n = 3 each; p = 0.216). In each colonic region, the proportions of neurons that were NOS+ and/or GABA+ were similar in WT and NL3R451C mice (GABA+; p . 0.05; NOS+; p . 0.05; GABA+/NOS+; p . 0.05; n=3 each). No GABA+ appositions onto NOS+ neurons were observed, and GABA+ neurons rarely receive NOS+ appositions in NL3R451C mice. Conclusions: NL3R451C mice show altered NOS-mediated colonic motility compared with WT controls. However, our findings also suggest a strain specific effect of NOLA on CMMC frequency. The changes in motility in NL3R451C colon are unlikely to be due to altered neuronal density or proportions of GABA or NOS neurons in NL3R451C mice suggesting that altered transmission at the level of the synapse is a likely explanation. These results provide the first evidence that a synaptic mutation associated with ASD can affect inhibitory elements of gastrointestinal motility.
(5.16x106) (P=0.03) or controls (2.14x106) (P=0.02). While as previously reported, C. jejuniinfected rats exhibited significant changes in mucosal defense mediators compared to controls, including increases in TNFalpha (P,0.05), IL-8 (P,0.01), beta-defensin 2 (P,0.05), betadefensin 6 (P,0.05) and TLR-4 (P,0.001), these changes were not linked to SIBO or the level of Aeromonas spp. Conclusions: In this translational study, rats with SIBO had increased Aeromonas spp in the duodenum, similar to humans with IBS. This supports the similarity between our translational animal model and human IBS. We previously concluded that the cytokine elevations in our rat model were dependent only on C. jejuni infection and not SIBO. Given that Aeromonas is mildly pathogenic, we wanted to verify that our previous conclusions remain sound. It appears that Aeromonas is not responsible for the cytokine changes in this rat model of IBS.
Gender Dependent Effects of Cholera Toxin on Colonic Motility - A Role for Sex Steroids Gayathri K. Balasuriya, Joel C. Bornstein
Idiopathic Parkinson's disease (PD) is a late-onset, chronic and progressive motor dysfunction due to loss of nigrostriatal dopamine (DA) neurons. Various experimental models of PD have been used to study the motor-associated dysfunctions of PD; these models principally involve the destruction of DA neurons with neurotoxins such as 6-OHDA. Since almost all patients with PD show gastrointestinal (GI) dysfunctions, such as gastroparesis, we aimed to evaluate whether gastric emptying correlated with biochemical changes in brainstem vagal nuclei controlling gastrointestinal functions in a rat model of PD. PD was induced in rats (N=18) by unilateral administration of 6-OHDA into the substantia nigra pars compacta (SN); control animals (N=10) were injected with saline. The non-invasive [13C]-octanoic acid breath test was performed weekly to evaluate the rate of gastric emptying. After four weeks, rats were perfused-fixed and the SN, brainstem, esophagus, stomach and duodenum were collected and processed for light microscopy for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). Four weeks after 6OHDA microinjection in the SN, rats that were later confirmed to have sustained a .90% decrease in TH-immunoreactivity (IR) in the SN significantly delayed gastric emptying, with increase of time to peak [13C], 85±2.2 and 62±3.6min, and half emptying time, i.e. 113±3.9 and 95±8.9min in 6-OHDA, N=6, and control rats, N=5, respectively; P ,0.05. In control rats, microinjection of the indirect sympathomimetic, tyramine (40nmoles/60nl) in the dorsal vagal complex decreased gastric tone by ~150mg (N=2), the effect was prevented by 4th ventricular application of yohimbine (500pmoles/60nl) and prazosin (100pmoles/60nl). Conversely, in 6-OHDA treated rats (N=2), microinjection of tyramine had no effect upon gastric tone. The IHC results (expressed as neurons/slice) are summarized in the table below. There was also a qualitative decrease in the expression of nNOS-IR neurons within the myenteric plexus of the esophagus, stomach and duodenum while the distribution of ChATIR neurons and TH-IR fibers in the same areas was not modified. Our data suggest that the delayed gastric emptying in a 6-OHDA rat model of PD with a selective loss of dopaminergic neurons in SN may be caused by changes in expression of neurotransmitters in both brainstem and myenteric neurons. Number of Neurons per Slice
Purpose: Diarrhea is a combination of hypersecretion and increased gut motility. Although the effect of Cholera Toxin (CT) on hypersecretion is highly characterised, its effect on motility remains largely unexplored. We evaluated the effect of CT on colonic migrating motor complexes (CMMCs) in female and male C57Bl/6 mice and further characterised this effect in female mice in estrus and proestrus. The distribution of the estrogen receptor alpha (ERα) in mouse colon was also characterised immunohistochemically. Method: Video imaging was used to construct high resolution spatiotemporal maps of colonic motor patterns in vitro. The entire colon was cannulated at each end and mounted horizontally in an organ bath containing physiological saline warmed to 37oC. The proximal end was connected to a reservoir of physiological saline, the distal end to an outflow tube. CT (0.125 μg/ml, 1.25 μg/ml, 12.5 μg/ml) was introduced to the lumen of 3 groups of female C57Bl/6 mice without considering their stage in the estrus cycle. CT (1.25 μg/ml) was introduced to the male colon and to females in the proestrus (highest estrogen concentration) and estrus (lowest estrogen concentration) stages (determined by screening vaginal smears). ER α was localized immunohistochemically in whole mounts of myenteric and submucosal plexus of colon using PA1-308 antiserum (AffinityBioreagents), along with a pan neuronal marker (Hu) and nitric oxide synthase (NOS). Results: In randomly selected female colon, all three concentrations of CT significantly and reversibly reduced the number of CMMCs (p , 0.001, 2 way ANOVA, n = 5 in each case). This occurred within 15 min of exposure to CT and was sustained for 1 h, but reversed when CT was flushed from the colonic lumen. The effect was blocked by granisetron (1 μM, 5-HT3 receptor antagonist) with the number of CMMCs in the presence of both CT and granisetron being indistinguishable from control (p . 0.05). CT (1.25 μg/ml) produced a significant (p . 0.001, n = 5) reduction in CMMC frequency in females during estrus but had no significant effect during proestrus or in male colon. ERα were expressed in myenteric and submucosal neurons. They co-localised with NOS in some neurons, but ER α expressing neurons that did not express NOS were also seen. Conclusion: The colon from female mice in estrus behaves significantly differently in response to luminal CT compared to both male colon and colon from mice in proestrus. This may be due to the lower level of sex steroidal hormones in estrus, which is consistent with our finding that ERα is expressed in colonic myenteric and submucosal ganglia. Su2056 New Vista Into Delayed Emesis Assessment: Validation of the Emetic Response in the Ferret Monitored by Telemetry Using Computerized Detection Sonia Goineau, Philippe Guillaume, Sonia Rompion, Toni Wolinsky, David J. Virley Nausea and vomiting are common side effects of cancer chemotherapy. While early emesis can be easily investigated in ferrets by direct observation, alternative methods are required to quantify delayed emesis which is time-consuming. This study was therefore designed to validate the computerized detection of changes in abdominal pressure related to retches or vomits induced by a cytotoxic substance in the telemetered ferret. Five ferrets were implanted with telemetry devices (Data Sciences International). They were then intra-peritoneally challenged with cisplatin (8 mg/kg) and abdominal pressure was recorded in unrestrained animals for 72 hours. The pressure signals were analyzed both manually and automatically using an adapted version of ecgAUTO software (Emka Technologies). Over the first 3 hours, the emetic response was also quantified via direct observation of the animals. The data produced by the 3 methods of detection were compared using a Spearman's rank correlation coefficient. Visual, manual and computerized detections of early emetic events over the first 3-hour recording period were well-correlated when compared per 30-, 15- or 5-minute epoch: correlation coefficients ranging from 0.8640 to 0.9289, p , 0.0001 for all comparisons. Manual and computerized detections of early and delayed emetic events over the 72hour recording period were also well-correlated when compared per 3-hour epoch: correlation coefficient = 0.9190, p , 0.0001. These findings demonstrate that computerized detection of abdominal pressure changes with adapted software is a reliable method for measuring emetic events in the ferret. This approach opens a new vista into emesis assessment and research to permit rapid, comprehensive and objective analysis of delayed emesis and to facilitate the development of novel anti-emetic therapies.
Su2054 Nitric Oxide Mediated Colonic Motility Is Altered in the Neuroligin-3 R451c Mouse Model of Autism Mathusi Swaminathan, Jaime Pei Pei Foong, Melina Ellis, Joel C. Bornstein, Elisa L. HillYardin Purpose: Gastrointestinal function is compromised in up to 90% of patients with Autism spectrum disorder (ASD) and the underlying causes are unknown. NL3R451C mice express a missense mutation in the Nlgn3 gene coding for the postsynaptic adhesion protein, neuroligin-3 (NL3) found in ASD patients. We have previously shown that the excitatory component of organized colonic motility (GABAA- and serotonergic- mediated) is perturbed in NL3R451C mice compared to wild type (WT) controls. Neuronal release of nitric oxide (NO) is responsible for tonic inhibition between colonic migrating motor complexes (CMMCs). Here, we examined whether the NO-mediated inhibitory component of spontaneous CMMCs is altered in NL3R451C mice. Methods: Full length colons were removed from NL3R451C, WT littermates (control) and C57Bl/6 mice (comparison strain). Colonic motility was examined using video imaging techniques. After a 1 h control recording, the NO synthase (NOS) inhibitor L-Nitro-arginine (NOLA, 100μm) was applied into the superfusing solution and activity was recorded for 1 h. The NOLA was then washed out for 1 h while a further recording was made. The proportion of all myenteric neurons (marked by the pan neuronal marker, Hu) that contained GABA and/or NOS was investigated using immunohistochemistry. Myenteric neuronal density and connectivity between GABA+ and NOS+ neurons were examined. Results: NOLA significantly increased CMMC frequency in NL3R451C (n = 9; p = 0.031), but not in WT littermates (n = 9; p = 0.831). NOLA also increased CMMC frequency in C57/Bl6 colons (n = 9; p = 0.008). No differences in neuronal density were observed between proximal (adjacent to cecum) and mid colon regions of WT and NL3R451C mice
S-543
AGA Abstracts
AGA Abstracts
(n = 3 each; p . 0.05). Furthermore, there was no difference in the mean number of neurons per ganglion in the distal colon of WT and NL3R451C mice (n = 3 each; p = 0.216). In each colonic region, the proportions of neurons that were NOS+ and/or GABA+ were similar in WT and NL3R451C mice (GABA+; p . 0.05; NOS+; p . 0.05; GABA+/NOS+; p . 0.05; n=3 each). No GABA+ appositions onto NOS+ neurons were observed, and GABA+ neurons rarely receive NOS+ appositions in NL3R451C mice. Conclusions: NL3R451C mice show altered NOS-mediated colonic motility compared with WT controls. However, our findings also suggest a strain specific effect of NOLA on CMMC frequency. The changes in motility in NL3R451C colon are unlikely to be due to altered neuronal density or proportions of GABA or NOS neurons in NL3R451C mice suggesting that altered transmission at the level of the synapse is a likely explanation. These results provide the first evidence that a synaptic mutation associated with ASD can affect inhibitory elements of gastrointestinal motility.
(5.16x106) (P=0.03) or controls (2.14x106) (P=0.02). While as previously reported, C. jejuniinfected rats exhibited significant changes in mucosal defense mediators compared to controls, including increases in TNFalpha (P,0.05), IL-8 (P,0.01), beta-defensin 2 (P,0.05), betadefensin 6 (P,0.05) and TLR-4 (P,0.001), these changes were not linked to SIBO or the level of Aeromonas spp. Conclusions: In this translational study, rats with SIBO had increased Aeromonas spp in the duodenum, similar to humans with IBS. This supports the similarity between our translational animal model and human IBS. We previously concluded that the cytokine elevations in our rat model were dependent only on C. jejuni infection and not SIBO. Given that Aeromonas is mildly pathogenic, we wanted to verify that our previous conclusions remain sound. It appears that Aeromonas is not responsible for the cytokine changes in this rat model of IBS.