Study On The Molecular Mechanisms Of The Nervous Dysfunction In Irritable Bowel Syndrome

Part I Role of enteric nervous dysfunction in the abdominal pain in patients with irritable bowel syndrome Ph.D student Yu Yan-Bo Supervisor Prof. Li Yan-Qing Major Gastroenterology ABSTRACTBackgroundIrritable bowel syndrome (IBS) is a common intestinal disorder characterized by recurrent abdominal pain or discomfort associated with disturbance in bowel habits. Abdominal pain/discomfort is the most likely complaints to result in medical consultation for IBS and is considered a clinical hallmark of the disease. Although the pathogenesis of the increased pain sensitivity in IBS is not fully understood, several mechanisms have been proposed, including (1) the enhanced perception of the intestinal signal in the brain, (2) hypersensitivity of dorsal horn neurons in the central limb of the visceral afferent system, and (3) hyperexcitability of primary visceral afferent fibers at the end-organ level.The molecular mechanisms of the end-organ hypersensitivity in IBS have been increasingly studied. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, originally known for its effects on the development and regeneration of nervous system, has aroused attention because of its critical roles in chronic pain conditions. In humans, BDNF is overexpressed in chronic pancreatitis and the expression is associated with pain in these patients. Injections of complete Freund's adjuvant into the hind paw increased BDNF mRNA levels in the ipsilateral dorsal horn, supporting an important role for these nociceptive mediators in the amplification of ascending pain signaling. BDNF is expressed in the enteric nervous system (ENS) and gut mucosa of various species, including humans. These studies raise the possibility that endogenous BDNF may have a pathophysiologic role in the altered gut sensation in IBS.Substance P (SP) and calcitonin gene-related peptide (CGRP) are markers of sensory neurons mainly involved with pain perception. SP is a potent splanchnic vasodilator and appears to mediate visceral pain. Mucosal mast cell interaction with nerve fibers including SP-positive fibers was confirmed to be an important trigger for the onset of IBS symptoms. CGRP, found in intrinsic and extrinsic nerves throughout the gastrointestinal tract, was involved in TNBS induced referred colonic hypersensitivity. Notably, SP and CGRP were found to be co-expressed with BDNF in sensory neurons of both the peripheral and central nervous system. Whether co-release of both types of molecules occurs in IBS patients is unknown and needs further investigation.In humans, the ENS is composed of neurons and glial cells organized in ganglionated and aganglionated plexuses that control the physiologic functions of the bowel. The plexuses are present in the muscle, in the submucosa and in the lamina propria of the mucosa. In the colon, mucosal nerve fibers form a plexus that is generally denser near the crypts in the deeper part of the lamina propria. The autonomous function of the intestine largely depends on appropriate regulation by the ENS. A number of observations have supported the structural alterations of the ENS in inflammatory bowel disease (IBD), which likely lead to the altered sensory perception and bowel motility even in the quiescent phase of the disease. Further, high concentrations of BDNF are detected in the adult human colon, which implies that the factor may be essential for the maintenance and plasticity of the adult ENS. However, few studies have specifically addressed any involvement of BDNF-induced ENS plasticity in colonic biopsies of IBS patients.Objectives1. To investigate the possible alterations in expression of BDNF in the plasma and colonic mucosa of IBS patients.2. To evaluated the association of mucosal BDNF expression and abdominal pain in IBS and parameters known to be related to the altered sensation, such as nociceptive neuropeptides secretion (SP and CGRP), mucosal nerve fiber density and ultrastructural alterations.Methods1. Subjects enrollment and specimens collectionA total of 40 IBS patients and 21 control subjects participated in the study. This study was approved by the Clinical Ethical Committee of Qilu Hospital of Shandong University and all participants gave their written informed consent before participation. The diagnosis of IBS was based on the RomeⅡcriteria, and patients were further subclassified as having IBS that was diarrhea-predominant (IBS-D) or constipation-predominant (IBS-C). Controls were selected from patients undergoing colonoscopy for polyps and cancer surveillance. Blood samples were collected before the colonoscopy. To standardize the site of sampling, all specimens were taken from the rectosigmoid junction. In all cases,2 biopsies were used for routine hematoxylin and eosin histology and immunohistochemistry. Another 2 biopsies were obtained for BDNF, SP and CGRP release assays. And 2 additional specimens were taken for electron microscopy.2. Abdominal pain/discomfort questionnaire, hospital anxiety and depression questionnairePatients were asked to score the frequency and severity of their abdominal symptoms over the last 2 weeks before interview by using a validated questionnaire. The severity of abdominal pain/discomfort was graded 0～4 according to the impact on patients'daily activities:0, absent; 1, mild (not influencing activities); 2, relevant (diverting from, but not urging modification of activities); 3, severe (influencing activities markedly enough to urge modifications); 4, extremely severe (precluding daily activities). The frequency of abdominal pain/discomfort was graded 0～4 according to the following scale:0, absent; 1, up to 1 day/week; 2,2 or 3 days/week; 3,4-6 days/week; 4, daily.The Hospital Anxiety and Depression Scale (HADS) was given to each subject to complete. It is a 14-item self-report questionnaire (7 items for anxiety, and 7 items for depression), with a 4-point intensity scale for each item to measure anxiety and depression severity in a medical context. Participants were asked to complete the HADS based on how they had been feeling over the past 2 weeks. A score of 8 or above on anxiety/depression subscale signifies a degree of anxiety/depression.3. Histology and immunohistochemistryBiopsy specimens were fixed in 10% neutral buffered formalin. Histopathological and immunohistological studies were performed on paraffin-embedded 4μm thick sections. PGP9.5-immunoreactive areas per mm2 of mucosa were quantified by use of the Image Pro-Plus 5.0 under a 40 x objective. For BDNF analysis, the immunostaining was measured by the integrated optical density (IOD) with the above software under a 20×objective, and the results were expressed as IOD/sum stained area to indicate the mean intensity of staining. All sections were inspected independently by 2 blinded observers, and the mean values of readings were used for final analysis.4. Transmission electron microscopyBiopsy specimens for transmission electron microscopy were immediately fixed in cacodylate-buffered 2.5% glutaraldehyde solution, postfixed with osmium tetroxide, and routinely processed and embedded in araldite. Toluidine blue-stained semithin sections were screened under an optical microscope to observe the enteric nerves in mucosa. Following this, ultrathin sections were double stained with uranyl acetate and lead citrate, and observed under a Hitachi H-600 electron microscope.5. Enzyme-linked immunosorbent assay (ELISA)Mucosal samples were homogenized in the homogenization buffer and were centrifuged at 4℃for 15 min at 15000 g. The supernatants were collected and stored at-80℃. After quantification by BCA assay, the levels of BDNF, SP and CGRP were measured by commercially available specific ELISA kits according to the manufacturer's protocols. Each sample was analyzed in duplicate.Venous blood samples were collected in EDTA-treated tubes and then centrifuged at 4000rmp,4℃for 15 min. The plasma was then collected and transferred to new tubes and frozen at -80℃. The concentration of BDNF was evaluated by ELISA kits according to the manufacturer's protocols. Each sample was analyzed in duplicate.Results1. Participants characteristicsThe participants in IBS group and control group were not statistically different for age or sex. Among the 40 IBS patients included,52.5%(n=21) and 47.5%(n=19) were considered IBS-D and IBS-C, respectively. The mean duration of symptoms was similar between IBS subgroups. The mean pain intensity was moderate for IBS patients and significantly higher than that for controls. The anxiety subscale scores and depression subscale scores in the IBS group and control group were not statistically different. And the colonic mucosa was macroscopically normal in all subjects.2. ImmunohistochemistryPGP9.5-immunoreactive nerve fibers were seen scattered throughout the mucosa and muscularis mucosa in all the specimens. The median area occupied by PGP9.5-immunoreactive fibers was greater in IBS patients than in control subjects (P<0.001 in mucosa and P<0.01 in muscularis mucosa). Furthermore, there was no statistically significant difference in the median density of colonic mucosal nerve fibers between the IBS subgroups (P=0.55 in mucosa and P=0.42 in muscularis mucosa).BDNF-like immunoreactivity was abundant in mucosal epithelial cells. Quantification revealed that the expression of BDNF in the colonic mucosa was significantly elevated in the IBS patients than in controls (P=0.001). With further analysis of IBS subgroups on BDNF expression, no significant difference was found between the IBS-D and IBS-C (P=0.46).3. Transmission electron microscopy of nerve bundlesNerve bundles were identified by electron microscopy in the mucosal lamina propria region in all colonic biopsies investigated. Injury was demonstrated in both the nerve bundles and the perinuclear cytoplasm in the biopsies from IBS patients. Nerve axons appeared swollen, lucent, and sometimes with membrane-bound vacuoles. As well, the arrangement of microtubules and microfilaments inside the axon chambers was obviously disrupted. The basement membrane surrounding the nerve bundles was thickened, with a fuzzy border and sometimes even disrupted. The cytoplasm of Schwann cells exhibited significantly decreased electron density, with dilated rough endothelial reticulum fragments and swollen mitochondria.4. Measurement of BDNF, SP and CGRPCompared with the normal controls, the plasma concentration of BDNF was lower but not significantly in IBS patients (P=0.34). And there was no significant difference in terms of plasma BDNF concentrations between IBS-D and IBS-C (P=0.85). However, IBS patients showed a significant upregulation of BDNF in the intestinal mucosa as compared with controls (P=0.003). The IBS subtypes did not differ in BDNF levels in intestinal mucosa (P=0.051). Furthermore, the level of BDNF was not significantly higher in IBS-C patients than in controls (P=0.1).Mucosal concentration of SP was significantly elevated in IBS patients (P=0.013). However, the increase in mucosal CGRP levels did not reach a statistically significant difference between IBS patients and controls (P=0.55). And there were no differences between IBS subtypes in mucosal SP and CGRP production (P=0.87 for SP and P=0.31 for CGRP).5. Correlation analysisCorrelations between the expression of BDNF in colonic biopsies and severity and frequency of abdominal pain/discomfort are analyzed. In the IBS patients, a significant correlation was found between BDNF levels and both severity and frequency of abdominal pain/discomfort (r=0.57, P<0.001 and r=0.46, P=0.003, respectively), but not in the control group (r=0.01, P=0.98 and r=0.06, P=0.81, respectively). For all patients and control subjects, the BDNF levels also correlated statistically with the severity and frequency of abdominal pain/discomfort (r=0.53, P<0.001 and r=0.54, P<0.001, respectively). Conclusions1. Structural damage of colonic mucosal nerve fibers and elevated mucosal BDNF expression may contribute to abdominal pain/discomfort in IBS patients.2. The ultrastructural damage of mucosal nerve fibers in IBS patients may induce the increased colonic expression of BDNF, and the elevated BDNF levels consequently induce increased mucosal nerve fibers in IBS patients.3. BDNF may play an indirect role to cause abdominal pain/discomfort through promoting the mucosal release of Substance P in IBS patients. Part IIEnteric nervous dysfunction participates in the visceral sensitivity in BDNF gene knock-out mice ABSTRACTBackgroundAs a member of the nerve growth factor family, brain-derived neurotrophic factor (BDNF), widely distributed in the central and peripheral nervous system, plays fundamental roles in the differentiation, survival and maintenance of neurons. Many studies about the role of BDNF in modulating the sensation in CNS have proven that BDNF is an important factor in chronic pain and visceral sensitivity. In the Tanure MT et al.'s study, BDNF serum levels were significantly higher during migraine attack than in pain-free period, which reinforced the view that BDNF might be implicated in the physiopathology of migraine. Another study showed that BDNF was involved in the process of long-term potentiation (LTP). LTP was one of the fundamental mechanisms of learning and memory and played important roles in the development and maintenance of neuropathic pain. Vanja Duric et al. found that injections of complete Freund's adjuvant into the hind paw increased BDNF mRNA levels in the ipsilateral dorsal horn, supporting an important role for this mediator in the amplification of ascending pain signaling. BDNF was increased in rat models of bladder inflammation and nerve injury, while neutralizing the upregulated BDNF by anti-BDNF antibody, was effective in relieving mechanical allodynia after nerve injury.In the colon, BDNF may also play a crucial role in the regulation of enteric nervous system. BDNF mRNA expression was detected in epithelia and internal circular muscle layer throughout the intestinal tract of the mice. Delafoy et al. reported that intraperitoneal injection of anti-BDNF antibody could alleviate TNBS induced referred colonic hypersensitivity in rats, which demonstrated involvement of this neurotrophin in the pathophysiology of visceral hyperalgesia following colitis. Both BDNF mRNA and protein were elevated in lumbosacral dorsal root ganglia following colitis. While in lumbosacral spinal cord only increases in BDNF protein were observed. These results that BDNF was not transcribed in the spinal cord, but rather that BDNF was likely synthesized in primary sensory neurons and transported to the spinal dorsal horn. In the Part I, we found that elevated mucosal BDNF expression induced increased mucosal nerve fibers and contributed to abdominal pain in IBS patients. However, whether colonic sensitivity could be down-regulated by inhibiting the expression of BDNF in the colonic mucosal has not been known. In the present study, BDNF+/- mice were applied for the first time to further investigate the role of BDNF in the colonic hyperalgesia.Objectives1. To investigate the colonic mucosal BDNF expression and mucosal nerve fiber density in BDNF+- mice and in BDNF+/+ mice, and make clear the role of BDNF in colonic sensitivity.2. To investigate the effects of different doses of exogenous BDNF on the colonic sensitivity of BDNF+/+ mice.Methods1. AnimalsHeterozygous BDNF+/- mice (C57B1/6 background) and wild-type BDNF+ littermates were generous gifts from Neurobiology Laboratory of Shandong University. Animals were genotyped by PCR on DNA isolated from tail biopsy samples and housed under conditions of controlled temperature (20±1℃), hygrometry (50±5%), and lighting. All experiments were performed with adult male mice at 4 months of age. Animal care and treatment were in accordance with the guidelines of the International Association for the Study of Pain. Great efforts were made to minimize animal suffering.2. Experimental protocolTwo series of experiments were conducted. In the first series of experiments, fecal water content and fecal pellet output were measured for all mice. To determine the involvement of BDNF in the visceral sensitivity, the study was conducted on heterozygous BDNF+- mice and wild-type BDNF+/+ littermates. Visceral sensitivity was evaluated by measuring behavioral responses of abdominal withdrawal reflex (AWR) to colorectal distension (CRD) in each animal group.In the second series of experiments, the effect of exogenous BDNF on the colonic sensitivity of wild-type BDNF+/+ mice was investigated. Four groups of mice received BDNF (Peprotech, Rocky Hill, NJ, USA) in 0.1% BSA at doses of 0.1,1,10ng/mice, or its vehicle (0.1% BSA), intraperitoneally (0.3ml/mice),30 minutes before CRD. And the threshold intensity of CRD was recorded in all animal groups.3. Colorectal distension and AWR scoringBriefly, mice were lightly sedated with halothane while a flexible latex balloon was inserted intra-anally into the descending colon 1cm proximal to the anus. And the balloon was secured in place by taping the attached tubing to the tail. The animals were allowed to recover 30min fully from the halothane anesthesia. For measuring the threshold intensity of CRD, the colorectal balloon was progressively inflated with an increment of 5 mm Hg until the pain behavior displayed. For measuring the AWR, the balloon was rapidly inflated to constant pressure (15,30,45,60 mmHg). The AWR scores were graded on a scale of 0-4:0, no behavioral response to CRD; 1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of abdomen; 4, body arching and lifting of pelvic structures. During the measurement, mice were given CRD with 30-second duration and then 4-minute interval. All the measurements were observed by two blinded observers and performed in triplicate.4. ImmunofluorescenceColon tissues from heterozygous BDNF+/- mice and wild-type BDNF+/+ littermates were processed for PGP9.5 immunofluorescence using widely applied methods. Sections were examined with a fluorescence microscope equipped with separate filters. PGP 9.5-immunoreactive areas per mm of mucosa were quantified using the Image Pro-Plus 5.0. Analysis were done on five fields of eight BDNF+/- mice and eight BDNF+/+ mice.5. ImmunohistochemistryColonic tissues from heterozygous BDNF+/- mice and wild-type BDNF+/+ littermates were fixed in 10% neutral buffered formalin. Immunohistological studies were performed on paraffin-embedded 4μm thick sections. For BDNF analysis, the immunostaining was measured by the integrated optical density (IOD) with the above software under a 20 x objective, and the results were expressed as IOD/sum stained area to indicate the mean intensity of staining. All sections were inspected independently by 2 blinded observers, and the mean values of readings were used for final analysis.6. Enzyme-linked immunosorbent assay (ELISA)Colonic mucosal tissues from heterozygous BDNF+- mice and wild-type BDNF+ littermates were homogenized in the homogenization buffer and were centrifuged at 4℃for 15 min at 15000 g. The supernatants were collected and stored at -80℃. After quantification by BCA assay, the levels of BDNF were measured by commercially available specific ELISA kits according to the manufacturer's protocols. Each sample was analyzed in duplicate.Results1. Fecal water content and fecal pellet output Fecal pellets expelled by each mouse in 24h were collected and desiccated.And percent fecal water content was calculated.The fecal water content was significantly lowered in BDNF+- mice than in BDNF+/+ mice(39.7±4.9% vs.50.7±3.8%,P<0.05). Meanwhile,fecal pellet output was obviously reduced in BDNF+- mice than in BDNF+/ mice(5.3±2.4 pellets/h vs.7.8±2.2 pellets/h,P<0.05).2.Behavior study of miceVisceral pain was determined by measuring the AWR scores in response to CRD in BDNF+- mice and BDNF+/+ littermates.BDNF+- and BDNF+/+ mice did not differ in visceral response to CRD at distension pressures≤30 mm Hg.However,BDNF+- mice showed significant lower AWR scores than BDNF+/+ mice at 45 and 60 mm Hg distension pressures(*P=0.03,**P<0.001).The threshold pressure to elicit a distinctive abdominal muscle contraction in control mice,treated with vehicle only(0.1％BSA),was 21.8±2.2 mm Hg.Intraperitoneal injection of BDNF in 0.1％BSA(0.1-10 ng/mice),30 minutes before distension,induced a significant dose dependent decrease in threshold pressure compared with control mice. With a dose of 0.1 ng/mice,the decrease was not statistically significant(19.8±2.4 mm Hg vs.21.8±2.2 mm Hg,P=0.23).For BDNF at 1 and 10 ng/mice,the threshold pressure was 15.4±3.4 mm Hg and 12.6±1.9 mm Hg respectively(**P<0.01,***P<0.001).3.ImmunofluorescenceColonic nerve fibers were identified by PGP 9.5 immunostaining and fiber density was assessed by the surface(um2)occupied by PGP 9.5 immunoreactivity per mm2 of the mucosa.The mucosal surface of PGP 9.5 immunoreactivity was significant lower in BDNF +- mice than in BDNF++ mice.4.ImmunohistochemistryBDNF-like immunoreactivity was abundant in colonic mucosal epithelial cells of the mice.Quantification revealed that the expression of BDNF in the colonic mucosa was significantly lowered in BDNF+/- mice than in BDNF+/+ mice.5.ELISAThe mucosal concentration of BDNF in BDNF+/- mice was obviously lowered, approximately half that in BDNF+/+ mice.Conclusions1.Compared with BDNF+/+ mice,BDNF+/+ mice showed lower BDNF levels and decreased nerve fiber density in the colonic mucosa,and the colonic sensitivity in BDNF+/- mice was also reduced.2. Intraperitoneal injection of BDNF induced a significant dose-dependent increase in colonic sensitivity in BDNF+/+ mice. PartⅢTransient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) in dorsal root ganglia contribute to visceral hyperalgesia in irritable bowel syndrome ABSTRACTBackgroundIrritable bowel syndrome (IBS) is a common functional gastrointestinal disorder which is characterized by recurrent abdominal pain or discomfort associated with altered bowel habits in the absence of structural and biochemical abnormalities. Altered visceral sensitivity with increased perception of colorectal balloon distention appears to consistently present and is recognized as a clinical hallmark of IBS. Therefore visceral hypersensitivity is proposed to be a significant component in the pathophysiology of IBS. The mechanisms underlying visceral hypersensitivity are multifactorial and complex. Nevertheless, accumulating clinical evidence suggests that psychological stress is an important determinant in the initiation, exacerbation and perpetuation of the symptoms of IBS patients. Early life stressors associated with childhood neglect, parental influence, physical or social abuse and life threatening situations have been shown to increase the risk of IBS development. Furthermore, IBS patients tend to be more vulnerable to the stressful events in daily life. However, the mechanism through which the stress contributes to visceral hyperalgesia in IBS patients remains elusive. The repetitive water avoidance stress (WAS) model, which simulates the pattern of daily stress experienced by humans, is a well-accepted animal model to study the possible mechanisms involved in the altered gut sensation.Transient Receptor Potential (TRP) channels have emerged as a family of evolutionarily conserved ligand-gated ion channels which are proposed to be primary transducers of thermal, mechanical and chemical stimuli. The TRP Vanilloid (TRPV) is probably the best known sub-family of TRP channels. TRPV1 is recognized as a polymodal channel expressed on primary sensory afferents which could be stimulated by mechanical stimuli, high temperature, low pH and capsaicin. Growing evidence suggests that TRPV1 is involved in the development and maintenance of visceral hypersensitivity induced by neonatal irritation or experimental colitis. The TRP ankyrin (TRPA) family has only one member-TRPA1, which is expressed on polymodal nociceptor endings and contributes to the detection of mechanical stimuli, cold temperature and chemical irritants such as allyl isothiocyanates (AITC). Activation of TRPA1 is proposed to contribute to the somatic hyperalgesia triggered by tissue damage and inflammation. Furthermore, TRPA1 is known to co-express with TRPV1 in primary sensory neurons. Therefore the role of TRPA1 in the field of gut sensation arouses recent interest of researchers. It is reported that TRPA1 plays as a mechanosensor and TRPA1 deletion markedly reduced colitis-induced mechanical hyperalgesia in the mice. However, whether TRPV1 and TRPA1 channels are involved in the altered visceral sensation in the chronic psychological stress is not yet evident.Sensory information from the colorectum is transmitted to the central nervous system via colonic sensory afferents whose cell bodies exist in dorsal root ganglia (DRG). Two anatomical divisions of these extrinsic sensory neurons could be distinguished. Thoracolumbar spinal neurons (TL, existing in T10-L1 DRG) innervate the colon through the lumbar splanchnic nerves (LSN) while lumbosacral neurons (LS, existing in L6-S1 DRG) via the pelvic nerves (PN).Objectives1. Exposed rats to repetitive water avoidance stress and studied the outcome with respect to visceral sensitivity.2. To investigate the protein expression of TRPV1 and TRPA1 in the corresponding DRGs of the lumbar splanchnic nerves and pelvic nerves in the stress-induced visceral hyperalgesia rat model.3. To examine the effects of direct intrathecal administration of an antagonist for each TRP channel on the visceral hyperalgesia, and determine whether behavioral responses were specifically mediated by the TRPV1 and TRPA1 channels in the WAS rat model.Methods1. AnimalsAdult male Wistar rats (250-300g) were used for all experiments. The animals were housed under standardized conditions of temperature (20±1℃), humidity (50±5%) and lighting (12-hr day/12-hr night). All experimental procedures were performed at the same time of the day between 9:30 am and 12:00 am to avoid the effect of diurnal variations. This study was approved by Chinese Institutional Animal Care Committee and adhered to the ethical guidelines of the International Association for the Study of Pain.2. Water avoidance stressRats were exposed to water avoidance stress (WAS) or sham stress for 1 hour each day for 10 consecutive days. Briefly, WAS rats were placed individually on a small platform (10×8×8cm), which was fixed in the center of a plexiglass tank filled with fresh room temperature water (25℃) to 1 cm below the height of the platform. Control littermates were placed on the same platform in a tank without water.3. Colorectal distension and AWR scoringBriefly, rats were lightly sedated with halothane while a flexible latex balloon (4 cm) made of a surgical glove finger attached to a Tygon tubing with thread was inserted intra-anally into the descending colon and rectum until the thread end was 1cm proximal to the anal sphincter. And the balloon was secured in place by taping the tubing to the tail. Rats were then placed in small Lucite cubicles (20×8×8 cm) and allowed 30 min for recovery from sedation before testing. For measuring the threshold intensity of CRD, the colorectal balloon was progressively inflated with an increment of 5 mm Hg until the pain behavior displayed or until a cutoff pressure of 80 mm Hg was reached in order to avoid invincible damage to the rat. For measuring the AWR, the balloon was rapidly inflated to constant pressure (10,20,40,60,80 mm Hg). The AWR scores were graded on a scale of 0 to 4:0, no behavioral response to CRD;1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of abdomen; 4, body arching and lifting of pelvic structures. For both measurements (threshold and AWR), the animals were given CRD with 30-second duration and then 4-minute interval. All the measurements were observed by two blinded observers and performed in triplicate.4. Western-blotting analysisBilateral TL (T10-L1) and LS (L6-S1) DRGs were dissected. Proteins were extracted with RIPA buffer and quantified. Equal amounts of protein extracts were loaded and separated by electrophoresis on the premade 12% sodium dodecyl sulfate/polyacrylamide gel. The separated proteins were then electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% fat-free milk for 2 hour at room temperature and then incubated with rabbit anti-TRPV1 or goat anti- TRPA1 primary antibody at 4℃overnight. After washing, the membranes were administered the horseradish peroxidase-conjugated anti-rabbit or anti-goat secondary antibody and incubated for 2 hours. The bands were detected by an enhanced chemiluminescence technique on Kodak biomax light film and the densitometric quantification was performed by the Alpha Ease FC Imaging software 4.0. Data were expressed as the ratios of TRPV1 (or TRPA1) toβ-actin band intensity.5. Effects of the selective TRPV1 and TRPA1 antagonist on the visceral hyperalgesia induced by WAS The involvements of TRPV1 and TRPA1 in the visceral hyperalgesia induced by WAS were evaluated using the selective antagonist for TRPV1 (capsazepine) and antagonist for TRPA1 (HC030031). The WAS rats were treated with capsazepine (1μg. 10μg, 100μg) or with HC030031 (1μg,10μg,100μg) or its vehicle (0.01mM PBS) by intrathecal route,2 hours before CRD. And the threshold intensity of CRD was recorded in all animal groups.Based on the above initial results obtained in the visceral hyperalgesia, the dose of 100μg capsazepine/HC030031 was chosen for further studies on the AWR scores of the WAS rats in response to phasic CRD. For this purpose, the WAS rats were treated with 100μg capsazepine/HC030031 or 0.01 mM PBS intrathecally. Two hours later, measurements of the abdominal withdrawal response in response to colorectal distention were carried out by two independent observers blinded to the randominzation.Results1. Behavioral study1.1 The threshold pressure of CRDThe threshold in the WAS group was significantly lower than that in the control group.1.2 AWR scoresAt the distension pressure<40 mmHg, the mean AWR scores in the WAS group were numerically higher than those in the control group, but not statistically. At the distension pressure≥40 mmHg, rats in the WAS group exhibited stronger responses than those in the control group as reflected by significantly higher AWR scores.2. Western blotting2.1 Water avoidance stress increased TRPV1 expression in DRGDensitometric analysis of the bands revealed that in both TL and LS DRG, the expression of TRPV1 from the WAS-treated rats was significantly greater than that from the sham stress treated rats.2.2 Water avoidance stress increased TRPA1 Expression in DRGIn parallel with the increased TRPV1 levels, immunoblotting analysis and quantification of the bands revealed a significant higher level of TRPA1 protein expression in the WAS-treated rats compared with the sham stress-treated controls. And this trend could be detected in both TL (T10-L1) and LS (L6-S1) DRGs.3. Effects of the selective TRPV1 and TRPA1 antagonist on the visceral hyperalgesia induced by WAS3.1 Effects of capsazepine on the visceral hyperalgesia induced by WAS Intrathecal injection of capsazepine (1-100μg),2 hours before distension, induced a significant dose dependent increase in threshold pressure compared with PBS-treated rats. With a dose of 1μg/rat, the increase was not statistically significant (P=0.16). For capsazepine at 10 and 100μg/rat, the threshold pressure was 24.80mmHg (20.98～27.78mmHg) and 31.05mmHg (27.65-34.20mmHg) respectively (*P=0.001, **P<0.001).Intrathecal injection of capsazepine 100μg could induce a significant decrease in the visceral hyperalgesia induced by water avoidance stress. WAS rats treated with capsazepine 100μg showed statistically weaker responses with distension pressures >40mm Hg (*P=0.04 at 40mmHg,**P=0.001 at 60mmHg,***P<0.001 at 80mmHg).3.2 Effects of HC030031 on the visceral hyperalgesia induced by WASIntrathecal injection of HC030031 (1-100μg),2...