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ABSTRACT: Objective: Neural cross-talk between visceral organs may play a role in mediating inflammation and pain remote from the site of the insult. We hypothesized such a cross-talk exists between the duodenum and pancreas and further it induces pancreatitis in response to intraduodenal toxins. Design: A dichotomous spinal innervation serving both the duodenum and pancreas was examined and splanchnic nerve responses to mechanical stimulation of these organs were detected. This pathway was then excited on the duodenal side by exposure to ethanol followed by luminal mustard oil to activate TRPA1. Ninety minutes later, pancreatic inflammation was examined. Ablation of duodenal afferents by resiniferatoxin (RTX) or blocking TRPA1 by CHEM5861528 was used to further investigate the duodeno-pancreatic neural reflex via TRPA 1. Results: About 40% of dorsal root ganglia (DRG) from the spinal cord originated from both duodenum and pancreas via dichotomous peripheral branches; about 50% splanchnic nerve single units responded to mechanical stimulation of both organs. Ethanol sensitized TRPA1 currents in cultured DRG neurons. Pancreatic edema and myeloperoxidase (MPO) activity significantly increased after intraduodenal ethanol followed by mustard oil (but not capsaicin), but remarkably decreased after ablation of duodenal afferents by using RTX or blocking TRPA1 by CHEM5861528. Conclusion: We found the existence of a neural cross-talk between the duodenum and pancreas that can promote acute pancreatitis in response to intraduodenal chemicals. It also proves a previously unexamined mechanism by which alcohol can induce pancreatitis, which is novel both in terms of the site (duodenum), process (neurogenic) and receptor (TRPA1).
AJP Gastrointestinal and Liver Physiology 01/2013; · 3.43 Impact Factor
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ABSTRACT: A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU(+) cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.
PLoS ONE 01/2013; 8(3):e59452. · 4.09 Impact Factor
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ABSTRACT: Due to the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and 3-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical-clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with μm-level resolution. Four examples are given: (1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intra-ganglionic at myenteric plexus, (2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface, (3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts, and (4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.
AJP Gastrointestinal and Liver Physiology 10/2012; · 3.43 Impact Factor
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ABSTRACT: Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Pluripotent embryonic stem (ES) cells represent an attractive cell source; however, little or no information is currently available on how ES cells will respond to the gut environment. In this study, we investigated the ability of ES cells to respond to environmental cues derived from the ENS and related tissues, both in vitro and in vivo.
Neurospheres were generated from mouse ES cells (ES-NS) and co-cultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LM-MP).
LM-MP co-culture led to a significant increase in the expression of pan-neuronal markers (βIII-tubulin, PGP 9.5) as well as more specialized markers (peripherin, nNOS) in ES-NS, both at the transcriptional and protein level. The increased expression was not associated with increased proliferation, thus confirming a true neurogenic effect. LM-MP preparations exerted also a myogenic effect on ES-NS, although to a lesser extent. After transplantation in vivo into the mouse pylorus, grafted ES-NS failed to acquire a distinct phenotype al least 1 week following transplantation.
This is the first study reporting that the gut explants can induce neuronal differentiation of ES cells in vitro and induce the expression of nNOS, a key molecule in gastrointestinal motility regulation. The inability of ES-NS to adopt a neuronal phenotype after transplantation in the gastrointestinal tract is suggestive of the presence of local inhibitory influences that prevent ES-NS differentiation in vivo.
BMC Gastroenterology 06/2012; 12:81. · 2.42 Impact Factor
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ABSTRACT: Morphological changes of interstitial cells of Cajal (ICC) have been proposed to characterize motility disorders. However, a global view of the network orientations of ICC subgroups has not been established to illustrate their three-dimensional (3-D) architectures in the human colon. In this research, we integrate c-kit immunostaining, 3-D microscopy with optical clearing, and image rendering to present the location-dependent network orientations with high definition. Full-depth colonic tissues were obtained from colectomies performed for nonobstructing carcinoma. Specimens of colon wall were prepared away from the tumor site. C-kit and nuclear fluorescent staining were used to identify the ICC processes and cell body. Optical clearing was used to generate transparent colon specimens, which led to panoramic visualization of the fluorescence-labeled ICC networks at the myenteric plexus (ICC-MY), longitudinal (ICC-LM) and circular (ICC-CM) muscles, and submucosal boundary (ICC-SM) up to 300 μm in depth via confocal microscopy with subcellular level resolution. We observed four distinct network patterns: 1) periganglionic ICC-MY that connect with ICC-LM and ICC-CM, 2) plexuses of ICC-LM within the longitudinal muscle and extending toward the serosa, 3) repetitive and organized ICC-CM layers running parallel to the circular muscle axis and extending toward the submucosa, and 4) a condensed ICC-SM layer lining the submucosal border. Among the four patterns, the orderly aligned ICC-CM layers provide an appropriate target for quantitation. Our results demonstrate the location-dependent network orientations of ICC subgroups and suggest a practical approach for in-depth imaging and quantitative analysis of ICC in the human colon specimen.
AJP Gastrointestinal and Liver Physiology 03/2012; 302(10):G1099-110. · 3.43 Impact Factor
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ABSTRACT: Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.
AJP Gastrointestinal and Liver Physiology 02/2012; 302(9):G958-65. · 3.43 Impact Factor
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ABSTRACT: We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.
AJP Gastrointestinal and Liver Physiology 01/2012; 302(1):G176-81. · 3.43 Impact Factor
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ABSTRACT: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract for which there are few safe and effective therapeutic options for long-term treatment and disease maintenance. Here, we applied a computational approach to discover new drug therapies for IBD in silico, using publicly available molecular data reporting gene expression in IBD samples and 164 small-molecule drug compounds. Among the top compounds predicted to be therapeutic for IBD by our approach were prednisolone, a corticosteroid used to treat IBD, and topiramate, an anticonvulsant drug not previously described to have efficacy for IBD or any related disorders of inflammation or the gastrointestinal tract. Using a trinitrobenzenesulfonic acid (TNBS)-induced rodent model of IBD, we experimentally validated our topiramate prediction in vivo. Oral administration of topiramate significantly reduced gross pathological signs and microscopic damage in primary affected colon tissue in the TNBS-induced rodent model of IBD. These findings suggest that topiramate might serve as a therapeutic option for IBD in humans and support the use of public molecular data and computational approaches to discover new therapeutic options for disease.
Science translational medicine 08/2011; 3(96):96ra76. · 7.80 Impact Factor
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ABSTRACT: Current advances in our understanding of stem and precursor cell biology and in the protocols of stem cell isolation and transplantation have opened up the possibility of transplanting neural stem cells for the treatment of gastrointestinal motility disorders. This review summarises the current status of research in this field, identifies the major gaps in our knowledge and discusses the potential opportunities and hurdles for clinical application.
Gut 08/2011; 61(4):613-21. · 10.11 Impact Factor
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ABSTRACT: Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.
AJP Gastrointestinal and Liver Physiology 08/2011; 301(4):G644-55. · 3.43 Impact Factor
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ABSTRACT: The pathogenesis of pain in chronic pancreatitis (CP) is poorly understood and treatment remains difficult. We hypothesized that nerve growth factor (NGF) plays a key role in this process via its effects on the transient receptor potential vanilloid 1, TRPV1.
CP was induced by intraductal injection of trinitrobenzene sulfonic acid in rats. After 3 weeks, anti-NGF antibody or control serum was administered daily for 1 week. Pancreatic hyperalgesia was assessed by nocifensive behavioral response to electrical stimulation of the pancreas as well as by referred somatic pain assessed by von Frey filament testing. TRPV1 currents in pancreatic sensory neurons were examined by patch-clamp. The expression and function of TRPV1 in pancreas-specific nociceptors was examined by immunostaining and quantification of messenger RNA levels.
Blockade of NGF significantly attenuated pancreatic hyperalgesia and referred somatic pain compared with controls. It also decreased TRPV1 current density and open probability and reduced the proportion of pancreatic sensory neurons that expressed TRPV1 as well as levels of TRPV1 in these neurons.
These findings emphasize a key role for NGF in pancreatic pain and highlight the role it plays in the modulation of TRPV1 expression and activity in CP.
Gastroenterology 04/2011; 141(1):370-7. · 11.68 Impact Factor
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Pankaj Jay Pasricha
Gastroenterology 02/2011; 140(4):1126-1128.e1. · 11.68 Impact Factor
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ABSTRACT: Calcitonin gene-related peptide (CGRP), substance P and nerve growth factor play an important role in inflammatory pain in various somatic pain models but their role in chronic pancreatitis has not been well studied.
The aim of this study was to investigate the effects of intrathecal administration of calcitonin gene-related peptide antagonist and substance P receptor antagonist on pain behavior in a rat model of chronic pancreatitis and to determine whether nerve growth factor drives the up-regulation of expression of these neuropeptides in sensory neurons.
Pancreatitis was induced by retrograde infusion of trinitobenzene sulfonic acid into the pancreatic duct of adult rats. Three weeks post infusion continuous intrathecal infusion of the calcitonin gene-related peptide antagonist alpha CGRP8-37 or neurokinin-1 receptor antagonist CP-96345 or its inactive enantiomer CP-96344 was administered for seven days. The effects of treatment on pancreatic hyperalgesia were assessed by sensitivity of the abdominal wall to von Frey filament probing as well as by the nocifensive response to electrical stimulation of the pancreas. In a separate experiment chronic pancreatitis was induced and pancreas specific dorsal root ganglion neurons labeled with DiI were assessed for calcitonin gene-related peptide and substance P immunoreactivity.
Intrathecal infusion of calcitonin gene-related peptide and neurokinin-1 receptor antagonists significantly attenuated behavioral pain responses in rats with chronic pancreatitis. Further, treatment of chronic pancreatitis rats with nerve growth factor antibody significantly reduced pancreas specific neurons expressing calcitonin gene-related peptide and substance P in thoracic dorsal root ganglion.
Calcitonin gene-related peptide and substance P mediate pancreatic hyperalgesia in chronic pancreatitis and nerve growth factor in turn sustains the up-regulation of these neuropeptides in pancreatic sensory neurons.
JOP: Journal of the pancreas 01/2011; 12(4):389-94.
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ABSTRACT: In this inaugural year of a historic presidency, gastroenterologists and gastrointestinal surgeons may well want to turn their attention to more immediate transformative events that have the potential to revolutionize their own practice in the near future. The most visible and, perhaps, controversial of these is natural orifice transluminal endoscopic surgery (NOTES), but other equally important changes are emerging as investigators around the globe vie with one another in the demonstration of increasingly audacious procedures. As is to be expected, we are also already seeing a backlash from more conservative scholars attempting to temper what they believe to be the surgical equivalent of irrational exuberance. However, by far the most common attitude among gastroenterologists toward these changes is one of indifference. In this piece, we discuss the circumstances that led to the development of NOTES and other innovative procedures, the peril that lies in ignoring them, and the true promise that they hold for our specialties.
The American Journal of Gastroenterology 10/2009; 104(10):2384-6. · 7.28 Impact Factor
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Gut 08/2009; 58(7):897-8. · 10.11 Impact Factor
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ABSTRACT: Cryotherapy or the application of exceptionally cold temperatures for tissue destruction has long been used as a mainstay
therapy in a wide variety of medical fields such as dermatology, gynecology, and otolaryngology. However, not until relatively
recently over the past decade has the use of cryotherapy in therapeutic endoscopy been seriously investigated. Beginning with
seminal work at Johns Hopkins in the early 1990s, this treatment modality is now increasingly being evaluated for human use
in the ablation of Barrett’s esophagus (BE), vascular abnormalities, and the treatment and palliation of gastric and esophageal
malignancy. This chapter will focus on the application of this therapy for Barrett’s esophagus.
Key WordsCryoablation–Barrett’s esophagus–Esophageal dysplasia–Esophageal cancer
01/2009: pages 155-164;
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ABSTRACT: Background Enteric neuronal dopamine (DA) inhibits acetylcholine release and gastric motility; this has been thought to be mediated
via neuronal dopamine-2 receptor (D2R). The aim of this study was to investigate the modulation of gastric motility by the
dopamine-3 receptor (D3R). Methods Adult Sprague–Dawley rats were used. Pyloric relaxation in response to electrical field stimulation (EFS) was assessed in
an organ bath in the presence of varying concentrations of a selective D3R agonist, PD128907. Gastric emptying was assessed
by the phenol red method after rats were treated with varying doses of PD128907 or DA with and without a selective D3R antagonist,
l-nafadotride. Results Immunoblotting and immunohistochemistry confirmed the presence of D3R in the myenteric neurons in the rat pylorus. D3R activation
reduced EFS-induced relaxation of pyloric strips in a dose-dependent manner and significantly delayed gastric emptying compared
with vehicle. The D3R antagonist partially reversed the effect of DA on gastric emptying. Conclusions Our data suggest a novel role for D3R in regulation of gastric motility. D3R activation delays gastric emptying, an effect
that may be due to impairment of pyloric relaxation. D3R antagonists therefore hold promise as useful agents for treatment
of gastric motility disorders.
Digestive Diseases and Sciences 12/2008; 54(1):57-62. · 2.12 Impact Factor
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ABSTRACT: Gastric electrical stimulation has been used for the treatment of drug refractory GI motility disorders and for the treatment of obesity. Both these indications have involved surgical placement of gastric electrodes, which adds to the complexity and cost of the procedure. Endoscopic placement is therefore an attractive alternative approach for this therapy.
Our purpose was to investigate the feasibility, safety, and efficacy of percutaneous endoscopic electrodes for gastric electrical stimulation.
Experimental animal study in hound dogs.
Percutaneous endoscopic transgastric electrode (PETE) placement was carried out by using a pair of gastric pacing wires attached to a percutaneous endoscopic gastrostomy tube. In addition, 4 pairs of gastric serosal electrodes were implanted surgically for comparison. The efficacy of the percutaneous endoscopic electrodes was defined by their ability to entrain gastric slow waves and the induction of dysrhythmia.
(1) The PETE recorded gastric slow waves comparable to the serosal electrodes. (2) Gastric electrical stimulation with long pulses delivered by the PETE, at a frequency of 10% higher than the intrinsic gastric slow wave frequency, entrained gastric slow waves. (3) Gastric electrical stimulation delivered by the PETE, at a tachygastric frequency, induced gastric dysrhythmia.
This was an animal study; however, its results are expected to be reproducible in humans, with PETE kept in place for even a longer duration than 6 to 8 weeks.
PETE placement is both feasible and safe. PETEs are effective, having a potential for use in treatment of both gastroparesis and obesity.
Gastrointestinal endoscopy 09/2008; 68(4):754-9. · 6.71 Impact Factor
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ABSTRACT: The 20-year technology slump in endoscopic innovation is finally giving way to a flurry of technologies, of which many are directed specifically at improving or even replacing traditional colonoscopy. These technologies include "smart" overtubes, electronically mapped and driven instruments, and completely self-propelled devices. In addition to nonendoscopic technologies such as CT, these innovations may dramatically alter the practice of colorectal cancer screening, the "bread and butter" of gastroenterologists in this country. There are multiple and complex forces driving these changes, including a mismatch between the supply and demand in colonoscopy, patient convenience and comfort, costs, and more recently, a growing concern about the miss rate of conventional colonoscopy.
Gastrointestinal Endoscopy Clinics of North America 08/2008; 18(3):607-17, xi.
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ABSTRACT: Although several pathophysiologic abnormalities have been noted in functional dyspepsia (FD), their pathogenesis is poorly understood. We hypothesized that chronic gastric hypersensitivity and gastric motor dysfunction seen in FD patients can be modeled in rats by transient gastric irritation during the neonatal period, a time of known neuronal vulnerability to long-term plasticity.
Ten-day-old male rats received 0.2 mL 0.1% iodoacetamide (IA) in 2% sucrose daily by oral gavages for 6 days; controls received 2% sucrose. Rats in both groups were then followed to adulthood (8-10 weeks) at which point behavioral, visceromotor, and great splanchnic nerve responses to graded gastric balloon distention (GD; 20-80 mm Hg) and gastric motor function were tested.
IA-treated rats exhibited hypersensitivity to GD in a dose-dependent manner, as compared with the control group. The threshold of afferent nerve activation was lower and nerve responses to GD were significantly increased in IA-treated rats. Although IA-treated rats ingested food at a lower rate, gastric emptying was not significantly different between IA and control groups. However, gastric accommodation was significantly reduced in the IA group. No significant gastric pathology was seen in hypersensitive adult rats compared with controls.
These studies demonstrate that gastric irritation in the neonatal period can result in chronic gastric hypersensitivity and gastric motor dysfunction in adults even in the absence of significant detectable gastric pathology. Our results offer insight into the pathogenesis of chronic functional dyspepsia and provide a potential model for further study to this important clinical problem.
Gastroenterology 07/2008; 134(7):2070-9. · 11.68 Impact Factor
