A Rat Model of Chronic Gastric Sensorimotor Dysfunction Resulting From Transient Neonatal Gastric Irritation

Background & Aims: 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. Methods: 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. Results: 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. Conclusions: 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. Background & Aims: 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. Methods: 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. Results: 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. Conclusions: 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. Functional dyspepsia (FD) is a common clinical gastrointestinal disorder, defined as persistent or recurrent pain or discomfort centered in the upper abdomen without evidence of organic structural abnormalities that can explain the symptoms. The US household survey suggests that approximately 25% of the normal population in the United States suffers from FD.1Drossman D.A. Li Z. Andruzzi E. et al.US householder survey of functional gastrointestinal disorders Prevalence, sociodemography, and health impact.Dig Dis Sci. 1993; 38: 1569-1580Crossref PubMed Scopus (1973) Google Scholar A number of pathophysiologic mechanisms have been proposed to explain the clinical symptoms in this condition including hypersensitivity to gastric distention, impaired gastric accommodation to a meal, delayed gastric emptying, altered duodenal sensitivity to lipids or acid, abnormal duodenojejunal motility, and central nervous system dysfunction, evidence for which has been found to varying degrees in patients.2Tack J. Bisschops R. Sarnelli G. Pathophysiology and treatment of functional dyspepsia.Gastroenterology. 2004; 127: 1239-1255Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar However, the etiopathogenesis of these abnormalities and their molecular basis remains unknown. In part, this is due to a lack of suitable animal models. A previous attempt to find a "spontaneous" model using the "Flinders Sensitive Line" rat (a strain that has been bred for hypersensitivity to cholinergic stimuli) revealed some similarities to human FD; however, unlike patients, these rats demonstrated an increase in gastric accommodation.3Mattsson H. Arani Z. Astin M. et al.Altered neuroendocrine response and gastric dysmotility in the Flinders Sensitive Line rat.Neurogastroenterol Motil. 2005; 17: 166-174Crossref PubMed Scopus (16) Google Scholar According to one hypothesis, the development of FD is related to a preceding infectious event.2Tack J. Bisschops R. Sarnelli G. Pathophysiology and treatment of functional dyspepsia.Gastroenterology. 2004; 127: 1239-1255Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 4Spiller R.C. Inflammation as a basis for functional GI disorders.Best Pract Res Clin Gastroenterol. 2004; 18: 641-661Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar This is suggested by studies that demonstrate an abrupt onset of symptoms in a subset of patients5Tack J. Demedts I. Dehondt G. et al.Clinical and pathophysiological characteristics of acute-onset functional dyspepsia.Gastroenterology. 2002; 122: 1738-1747Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar as well as by observations that gastric discomfort and pain thresholds are lower in patients with Helicobacter gastritis.6Monnikes H. van der Voort I.R. Wollenberg B. et al.Gastric perception thresholds are low and sensory neuropeptide levels high in Helicobacter pylori-positive functional dyspepsia.Digestion. 2005; 71: 111-123Crossref PubMed Scopus (32) Google Scholar Experimentally, acute gastric inflammation can induce hypersensitivity to gastric distention in adult rats. However, hypersensitivity resolves over time as the tissue recovers.7Lamb K. Kang Y.M. Gebhart G.F. et al.Gastric inflammation triggers hypersensitivity to acid in awake rats.Gastroenterology. 2003; 125: 1410-1418Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8Ozaki N. Bielefeldt K. Sengupta J.N. et al.Models of gastric hyperalgesia in the rat.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G666-G676Crossref PubMed Scopus (87) Google Scholar By contrast, abnormal events in the gastrointestinal tract during the immediate postnatal period can have a long-lasting, perhaps permanent, impact on the neural processing of sensory information. Thus, we have previously shown that the neonatal period is a particularly vulnerable window in time for the development of long-term changes in colonic function and nociception in response to mild irritation.9Winston J. Shenoy M. Medley D. et al.The vanilloid receptor initiates and maintains colonic hypersensitivity induced by neonatal colon irritation in rats.Gastroenterology. 2007; 132: 615-627Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Al-Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar These pups stay sensitized to noxious colonic stimulation during adulthood in the absence of overt histologic changes, mimicking the situation in humans with irritable bowel syndrome. These findings are consistent with reports in somatic and other visceral models as well.11Ruda M.A. Ling Q.D. Hohmann A.G. et al.Altered nociceptive neuronal circuits after neonatal peripheral inflammation.Science. 2000; 289: 628-631Crossref PubMed Scopus (360) Google Scholar, 12Randich A. Uzzell T. DeBerry J.J. et al.Neonatal urinary bladder inflammation produces adult bladder hypersensitivity.J Pain. 2006; 7: 469-479Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In this study, we therefore set out to create a model for FD based on the general principle of neonatal vulnerability combined with acute mild gastric irritation in the neonatal period. Our hypothesis was that gastric irritation early in life will result in chronic gastric hypersensitivity and disturbances in gastric motor function that persist into adulthood. Male Sprague-Dawley rats were used in all the experiments (Harlan, Indianapolis, IN). The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch. Ten-day-old rat pups received 0.2 mL of 0.1% iodoacetamide (IA) in 2% sucrose daily for 6 days by oral gavages. The control group received 0.2 mL of 2% sucrose. After overnight fasting, 8-week-old rats were anesthetized with intraperitoneal ketamine (60 mg/kg) and xylazine (7 mg/kg). Balloons (2.5 cm long) made from latex condoms were attached to a long catheter (PE-240). A left lateral epigastric incision was made, and the balloon was placed in the stomach through an incision at the tip of the fundus. The pylorus is not obstructed. The catheter was exteriorized at the back of the neck. For electromyographic (EMG) recordings, a pair of stainless steel wires (Cooner Wire, Chatsworth, CA) was implanted into the acromiotrapezius (a superficial neck muscle) and externalized at the back of the neck. The behavioral and EMG studies were done 1 week after surgery. Balloon implantation for gastric accommodation was similar to the one described above, except the balloon had a spherical shape with a wall thickness of 15 μm, a nondistensible diameter of 2.0–2.5 cm, and a maximal volume of 7 mL. Gastric accommodation testing was performed 1 week after surgery. The behavioral response to gastric balloon distention (GD) was studied on day 6 after surgery. The rats were placed in individual plastic rat restrainers in a quiet environment and allowed to adapt for an hour. The catheter from the gastric balloon was connected to a sphygmomanometer. Graded intragastric pressure (20, 40, 60, and 80 mm Hg) was achieved by inflating the balloon for 20 seconds with 5 minutes rest. Behavioral responses (based on the abdominal withdrawal reflex [AWR]) were graded as previously published on visceral pain10Al-Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 13Stam R. van Laar T.J. Weigant V.M. Physiological and behavioral responses to duodenal pain in freely moving rats.Physiol Behav. 2004; 81: 163-169Crossref PubMed Scopus (17) Google Scholar: 0, no behavioral response to GD; 1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of abdomen; 4, body arching, lifting of pelvic structures, and stretching of body. Behaviors were videotaped and analyzed by a blinded observer. Visceromotor responses (VMRs) to GD were recorded by quantifying EMG activity simultaneously with grading behavior. The EMG recordings (baseline and during constant pressure distention of the stomach) were amplified with a low-noise AC differential amplifier (Iso-DAM8A Bio-amplifier; WPI, Sarasota, FL), digitized, and integrated using the CED 1401/ SPIKE2 program (Cambridge Electronic Design, Cambridge, UK). The raw EMG data were rectified and quantified by calculating the area under the curve. Responses to GD were expressed as percent increase over baseline during GD. Eight-week-old rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and maintained with a constant infusion of 50 mg sodium pentobarbital in 9 mL 0.9% NaCl at 1.0 mL/h through the jugular vein. Tracheotomy was performed for artificial ventilation with room air, and rats were paralyzed with gallamine triethiodide. Body temperature was monitored at 37°C by a rectal probe feedback-controlled electric heating blanket (Harvard Apparatus). Balloons were implanted as before, and a flank incision was made to expose the left greater splanchnic nerve (GSN). The skin and abdominal muscles were retracted using silk sutures under a microscope, and the preganglionic GSN was carefully dissected and cut just below the diaphragm with the distal segment placed in a pool of warm mineral oil on a black base plate. The nerve was then teased into fine bundles that were draped over one arm of a bipolar silver hook electrode while an equally fine bundle of connective tissue was placed on the other arm as reference. Action potentials were amplified, processed, and recorded through a window discriminator using the CED 1401/ SPIKE2 data acquisition program. GSN fibers responsive to GD were identified by an increase in activity over baseline in response to a short test stimulus (60 mm Hg). Single unit recordings were differentiated and compiled into rate histograms (1-second bin width) using wave-mark template in SPIKE 2. Graded intragastric pressure (20, 40, 60, and 80 mm Hg) was produced by inflating the balloon for 20 seconds with 2-minute intervals via a sphygmomanometer. A responsive fiber was defined as one that increased its activity (spikes/second) by ≥30% from baseline. Response thresholds were defined as low-threshold (LT) (=20 mm Hg) or high-threshold (HT) (≥40 mm Hg). In a separate group of rats, gastric emptying of a solid meal was measured with a modification as described.14Martinez V. Barquist E. Rivier J. et al.Central CRF inhibits gastric emptying of a nutrient solid meal in rats: the role of CRF2 receptors.Am J Physiol. 1998; 274: G965-G970PubMed Google Scholar In brief, overnight fasting rats were allowed free access to water and preweighed normal chow for 3 hours. Food and water were then removed, and gastric emptying of the ingested meal was assessed 3 hours later. Animals were killed, and the stomach was removed and emptied thoroughly. The amount (grams) of food contained in the stomach was the difference between the total weight of the stomach before and after stomach emptying. The rate of gastric emptying for the entire 6-hour period was calculated by the following: gastric emptying (%) = 100-(gastric content/food intake) × 100. In a separate group of fasted rats, an electronic barostat (G & J Electronics Inc, Willowdale, Ontario, Canada) was applied to assess gastric accommodation.3Mattsson H. Arani Z. Astin M. et al.Altered neuroendocrine response and gastric dysmotility in the Flinders Sensitive Line rat.Neurogastroenterol Motil. 2005; 17: 166-174Crossref PubMed Scopus (16) Google Scholar Starting with a minimum distention pressure of 1 mm Hg (P1) maintained for 10 minutes (baseline), pressure was increased by 1 mm Hg per minute up to a maximum of 10 mm Hg (P10) during the ramp phase. The pressure was then maintained at 10 mm Hg for 10 minutes (tonic phase). After the tonic phase, the pressure was dropped to the minimum distention pressure and maintained for another 10 minutes. The gastric volume produced at minimum distention pressure during the first 10 minutes is referred to as the baseline. Rats were perfused through the heart with cold Tyrode's solution (20 mL for neonates and 200 mL for adults). Stomachs were removed, cut opened along the greater curvature, and immersed in 10% formalin for 72 hours at 4°C. Four-micrometer sections from paraffin-embedded tissue specimens were processed for H&E staining for histologic evaluation. Myeloperoxidase (MPO) activity was measured 6 days following exposure to IA or sucrose and 8-week-old rats as described15Bhatia M. Saluja A.K. Hofbauer B. et al.Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.Proc Natl Acad Sci U S A. 1998; 95: 4760-4765Crossref PubMed Scopus (269) Google Scholar and expressed as activity per unit of dry weight. All values were presented as means ± SE. Student t test or 2-way repeated measures ANOVA were used for comparison. Post hoc comparisons were made using the Student-Newman-Keuls test. Statistical analysis was performed using Sigma Stat (Systat Software Inc, San Jose, CA); P < .05 was considered statistically significant. Body weight was determined on days 2, 3, 4, 5, and 6 during 0.1% IA in 2% sucrose or 2% sucrose treatment and on day 18 post-treatment. Weight gain was significantly lower in the IA-treated rats during days 3–6 of the treatment period (*P < .05, Figure 1). However, by day 18, body weights in both groups were similar. Gastric histology in neonatal rats 6 days following exposure to IA or sucrose (n = 8 in each group) revealed only superficial sloughing of the mucosa in the IA group without any evidence of deeper injury or inflammation (Figure 2A). This was confirmed by MPO activity in gastric tissues that was similar in both groups (Figure 2B). In another set of experiments, rats with neonatal IA or sucrose exposure were followed to adulthood, killed, and their stomachs examined (n = 8 per group). There was neither change in gastric histology (Figure 2C) nor MPO activity between the groups (Figure 2D). At 8 weeks, neonatal treated (IA or sucrose) rats (n = 8 per group) were tested with graded GD. Behavioral scores in response to GD in IA-treated rats were significantly increased compared with controls (neonatal treatment: P = .011; distention pressure: P < .001, Figure 3A). Post hoc analysis found significant differences in AWR scores at 40 (P = .045), 60 (P = .0017), and 80 mm Hg (P = .045). These results were corroborated using EMG of the acromiotrapezius to quantify the VMR to GD (Figure 3B). Rats treated with IA as neonates exhibited significantly greater EMG responses compared with controls (2-way repeated measures ANOVA, P = .003). Significant increases in the EMG activity in IA-treated rats were observed at GD pressures of 60 (196.1% vs 147.5%, respectively, P = .004) and 80 mm Hg (271.2% vs 205.5%, respectively, P < .001, Figure 3C). A trend toward an increase was also seen at 40 mm Hg (141.9% vs 112.1%, respectively, P = .07). A total of 82 and 90 afferent fibers in the GSN were identified in IA-treated and control rats, respectively (n = 9 in each group). All fibers recorded in both groups displayed ongoing baseline activity (Figure 4A) ranging between 0.3 and 9.3 (IA-treated rats) and 0.2 and 8.9 spikes/seconds (control rats). The average spontaneous activity in IA-treated rats (2.4 ± 0.18 spikes/second, n = 82) was significantly higher compared with controls (1.8 ± 0.17 spikes/second, n = 90; P = .01, Figure 4B). In the IA-treated rats, 47 (57.3%), 61 (74.4%), 79(96.3%), and 80 (97.6%) fibers were responsive to graded GD of 20, 40, 60, and 80 mm Hg, respectively. Similarly, 35 (38.9%), 57 (63.3%), 80 (88.9%), and 87 (96.7%) fibers in control group were responsive to 20, 40, 60, and 80 mm Hg of GD, respectively (Table 1). The proportion of fibers responding at 20 mm Hg was significantly higher in IA-treated rats compared with controls (57% vs 39%, respectively; P = .03).Table 1Number of GSN afferent fibers responsive to GD20406080 mmHg2% Sucrose38.9% (35/90)63.3% (57/90)88.9% (80/90)96.7% (87/90)0.1% IA57.3% (47/82)74.4% (61/82)96.3% (79/82)97.6% (80/82)P.034.465.147.641The numerators indicate the number of fibers activated by GD at the level within that group. Open table in a new tab The numerators indicate the number of fibers activated by GD at the level within that group. When distention-induced increases in GSN activity were expressed as a percent increase over the baseline, these responses were significantly greater in the IA-treated rats compared with controls (treatment: P = .002; pressure: P < .0001). Post hoc analysis found significant differences in distention-evoked GSN activity at 60 and 80 mm Hg (P < .05) but not at lower distention pressures (Figure 4C). Furthermore, the responses of LT fibers (defined by response to all 4 distention pressures) to graded GD in IA- and sucrose-treated rats were compared by 2-way ANOVA: There was significant effect of IA-treated treatment as well as distention pressure between the 2 groups (treatment: P < .0001; pressure: P < .0001). Post hoc analysis found significant differences in distention-evoked GSN activity at 60 and 80 mm Hg, P < .05 (Figure 4D). Among HT fibers, there was significant effect of pressure (P < .0001) but no significant treatment effect (P = .098). The post hoc analysis did not find significant difference (Figure 4E). As shown in Figure 5A, after an overnight (18 hours) fast, IA-treated rats consumed less solid food than controls over a 3-hour period (6.3 ± 0.3 g vs 8.0 ± 0.4 g, respectively; P = .01; n = 6 in each group). However, the rate of gastric emptying of the food ingested was not significantly different between the 2 groups (75.6 ± 5.4% in IA-treated rats and 69.5 ± 4.7% in sucrose-treated rats, P = .4, Figure 5B). We measured gastric volume as a function of distention pressure in a separate group of IA-treated and sucrose-treated rats (n = 10 in each group). During the ramp phase of gastric distention, gastric volume increased gradually with no distinct deflection point in either group. Compared with controls, the mean volumes of the IA-treated group were significantly lower (F1160 = 19.136, P < .0001, Figure 6) throughout this phase (note that significant differences were detected in post hoc tests at P6–P10 but not at P1–P5). In the tonic phase of gastric distention, the gastric volume showed no difference between the 2 groups (P = .260). In the current study, a sulfhydryl acetylating agent, IA, was used to induce a transient mild gastric inflammation in neonatal rats. This is based on reports that 0.1% IA for 5–7 days in adult rats or mice induces mild gastritis.16Barnett K. Bell C.J. McKnight W. et al.Role of cyclooxygenase-2 in modulating gastric acid secretion in the normal and inflamed rat stomach.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1292-G1297PubMed Google Scholar, 17Nishio H. Hayashi Y. Terashima S. et al.Role of endogenous nitric oxide in mucosal defense of inflamed rat stomach following iodoacetamide treatment.Life Sci. 2006; 79: 1523-1530Crossref PubMed Scopus (19) Google Scholar, 18Piqueras L. Corpa J.M. Martinez J. et al.Gastric hypersecretion associated to iodoacetamide-induced mild gastritis in mice.Naunyn Schmiedebergs Arch Pharmacol. 2003; 367: 140-150Crossref PubMed Scopus (18) Google Scholar, 19Takeeda M. Hayashi Y. Yamato M. et al.Roles of endogenous prostaglandins and cyclooxygenase izoenzymes in mucosal defense of inflamed rat stomach.J Physiol Pharmacol. 2004; 55: 193-205PubMed Google Scholar Furthermore, IA has been shown to result in acute changes of gastric sensory and motor function.8Ozaki N. Bielefeldt K. Sengupta J.N. et al.Models of gastric hyperalgesia in the rat.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G666-G676Crossref PubMed Scopus (87) Google Scholar, 20Bielefeldt K. Ozaki N. Gebhart G.F. Mild gastritis alters voltage-sensitive sodium currents in gastric sensory neurons in rats.Gastroenterology. 2002; 122: 752-761Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Our study showed that treatment with 0.1% IA orally once daily for 6 days in neonates induced mild but transient damage to the surface epithelium of gastric mucosa without evidence of deeper injury. Furthermore, after a few days, rats seem to recover completely and maintain their body weight in the same range as controls. At 8 weeks, no evidence of significant morphologic and histologic changes was seen in the stomach, attesting to the transient nature of the insult. Despite a normal appearance of their stomachs, IA-treated rats had significant disturbances in gastric sensory and motor function. Together, these features are characteristic of what has been described as functional dyspepsia in humans. FD is a common clinical syndrome that is often chronic and relatively difficult to treat satisfactorily. This is in large part because of our inability to identify putative molecular targets, a situation that in turn has arisen from the lack of appropriate animal models. Although the precise definition of FD continues to evolve, pain, presumably of upper gastrointestinal origin, remains a prominent feature in a significant subset of patients. This is also important from an experimental perspective; modeling "pain" in animals, although not simple, is conceptually more straightforward than simulating other symptoms that make up the FD complex (eg, early satiation or postprandial fullness). The primary aim of this study was therefore to create a model that displayed gastric hypersensitivity to mechanical distention in the absence of overt morphologic or histologic changes in the stomach, both features that are characteristic, if not pathognomic, of FD. Subsequently, we also investigated the robustness of this model by identifying other pathophysiologic derangements that have been associated with FD. Visceral hypersensitivity, particularly to mechanical distention, is regarded as a hallmark of many functional bowel disorders including IBS and FD and, depending on the methodology used, is found in approximately 35%–65% of patients.21Mertz H. Fullerton S. Naliboff B. et al.Symptoms and visceral perception in severe functional and organic dyspepsia.Gut. 1998; 42: 814-822Crossref PubMed Scopus (257) Google Scholar, 22Tack J. Caenepeel P. Fischler B. et al.Symptoms associated with hypersensitivity to gastric distention in functional dyspepsia.Gastroenterology. 2001; 121: 526-535Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 23Vandenberghe J. Vos R. Persoons P. et al.Dyspeptic patients with visceral hypersensitivity: sensitisation of pain specific or multimodal pathways?.Gut. 2005; 54: 914-919Crossref PubMed Scopus (73) Google Scholar In patients with FD, the presence of visceral hypersensitivity to graded GD appears to correlate with postprandial pain22Tack J. Caenepeel P. Fischler B. et al.Symptoms associated with hypersensitivity to gastric distention in functional dyspepsia.Gastroenterology. 2001; 121: 526-535Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar; furthermore, in hypersensitive patients, other nonpainful symptoms such as nausea, satiety, and fullness are also triggered at lower distention pressures suggesting sensitization of multimodal pathways.23Vandenberghe J. Vos R. Persoons P. et al.Dyspeptic patients with visceral hypersensitivity: sensitisation of pain specific or multimodal pathways?.Gut. 2005; 54: 914-919Crossref PubMed Scopus (73) Google Scholar We reproduced this finding in our experimental model. First, we graded the behavioral response to graded GD at pressures ranging from 20 to 80 mm Hg of pressure, following an experimental paradigm that has been validated by others.8Ozaki N. Bielefeldt K. Sengupta J.N. et al.Models of gastric hyperalgesia in the rat.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G666-G676Crossref PubMed Scopus (87) Google Scholar Neonatal IA-treated rats displayed significantly higher behavioral scores in response to GD at 40, 60, and 80 mm Hg. Allodynia is a phenomenon in which stimuli that are normally innocuous elicit pain. This has been invoked to explain symptoms in functional bowel disease on the basis that physiologic events such as contractions or mild distentions result in pain. Experimentally, the demonstration of allodynia requires an understanding of the threshold intensity at which a stimulus is perceived as noxious. Based on a combination of electrophysiologic, behavioral, and visceromotor reflex activity, Ozaki et al have suggested that GD pressure of 30 mm Hg or more be considered to be in the noxious range.8Ozaki N. Bielefeldt K. Sengupta J.N. et al.Models of gastric hyperalgesia in the rat.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G666-G676Crossref PubMed Scopus (87) Google Scholar In our study, extrapolation of the linear portion of the stimulus response curves for both behavioral and VMR activity in control rats (Figures 3A and C) suggests a noxious threshold closer to 40 mm Hg, which is similar to that reported for the colorectal distention in rats.24Ness T.J. Gebhart G.F. Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat.Brain Res. 1988; 450: 153-169Crossref PubMed Scopus (591) Google Scholar At this threshold, the average response of the IA-treated rats was greater than that of controls. Spontaneous GSN activity was also greater in sensitized animals (Figure 4B). Finally, the number of GSN fibers responsive to subnoxious stimulation (ie, 20 mm Hg) was greater in the IA-treated rats as compared with controls (Table 1). Taken together, these findings suggest an allodynic component to the observed sensitization in IA-treated animals. In our model, the responses to stimuli considerably above the noxious threshold (ie, 60 and 80 mm Hg) were also greater, suggesting the equivalent of hyperalgesia, an important component of visceral hypersensitivity. This was corroborated by the VMR assay, with higher acro