The Experimental Study On The Molecular Dysregulation Of Post-inflammatory Visceral Hypersensitivity

BackgroundAbdominal pain is a prevalent symptom in inflammatory bowel?disease (IBD) and irritable bowel syndrome (IBS) patients which affects life quality seriously. Current studies present that visceral hypersensitivity plays an important role in abdominal pain. Visceral sensitivity is mediated by spinal sensory afferent nerves, with their cell bodies in dorsal root ganglia (DRG). The mechanisms of visceral hypersensitivity include peripheral and central sensitization. Approximately 1/3 IBS patients are reported to be post-infectious IBS (PI-IBS). Studies proposed that colonic samples of PI-IBS patients showed increased epithelial permeability, more inflammatory cells and mediators gathered. Inflammation could sensitize the peripheral sensory neurons. Post-inflammatory sensitization of enteric afferent nerves might play an essential role in the mechanism of visceral hypersensitivity in IBS patients.Brain-derived neurotrophic factor (BDNF) is one member of the neurotrophin family. Previous studies suggest that BDNF could modulate the modality of synapses and maintain the environment of nervous system. Besides, BDNF is involved in the studying and memory functions.Recent studies raise that BDNF is also involved in the pain mediation. BDNF is up-regulated in a number of inflammatory somatic hyperalgesic models. The application of anti-BDNF antibody could reverse the somatic hyperalegsia.BDNF not only participates in the nervous system, but also expresses in many peripheral organs such as the epithelium of heart, lung, bladder and colon. But the receptors of BDNF-TrkB and P75 are not expressed on the visceral epithelium while selectively expressed in nervous system. Therefore BDNF is predicted to mediate the visceral sensitivity through the nervous system innervating the viscera,The present study used trinitro benzene sulfonic acid (TNBS) to induce colitis.7 days after induction, the expression level of BDNF in DRG and colonic sensitivity were tested. Meanwhile we combined the BDNF heterozygous knock-down (BDNF+/-) mice in this study and evaluated the alterations of visceral sensitivity after the suppression of BDNF expression to investigate the role of BDNF in the colonic hypersensitivity following colitis.ObjectiveTo investigate the role of BDNF in the colonic hypersensitivity following colitis.Methods1. Collectively, four groups (BDNF+/+/vehicle, BDNF+/+/TNBS, BDNF+/-/vehicle and BDNF+/-/TNBS) were used in this study which were divided by their genotypes and treatments.2. Colitis was induced by TNBS (1.75mg in 50% ethanol, total 0.1 mL) through rectum. Control mice were administered the same volume of saline.3. Histopathology:seven days after colitis induction, mice were euthanized by an overdose of pentobarbital (200mg/kg i.p.) and the distal colon was removed. The sample was 3cm proximal to the anus. Half of the sample was fixed in formalin, embedded in paraffin, stained with hematoxylin-eosin and graded on a published semi-quantitative scale.4. Myeloperoxidase (MPO) activity test:Part of the distal colon was weighed, homogenized and processed with a mouse MPO kit.5. Determination of BDNF protein by enzyme-linked immunosorbent assay (ELISA)Tissue samples separated from bilateral thoracolumbar (TL, T10-L1), lumbosacral (LS, L6-S1) DRG and part of distal colon were homogenized and extracted for protein determination and ELISA assay. Total protein content was measured by the detergent-compatible BCA protein assay. Commercial ELISA kits were used to measure the level of BDNF protein according to the manufacturer's instructions. Concentrations of BDNF were calculated as nanograms BDNF per gram total protein.6. Visceral response to colorectal distensionWith mice briefly sedated with diethyl ether, a small balloon catheter (2-cm polyethylene plastic balloon secured to polytetrafluroethylene-24 tubing) was inserted into the distal colon,0.5cm proximal to the anus.(1) While animals were fully awake and recovered for 30min, the abdominal withdrawal response (AWR) in response to phasic colorectal distension (CRD) (at 0, 15,30,45,60,80 mmHg,20-s inflation and then 4 min intervals of deflation) was measured. All the measurements were performed in triplicate, and the average of the results was obtained. The AWR was graded on a scale of 0 to 4:0, no behavioral response to CRD; 1, immobility of the body or occasionally clinches of the head; 2, contraction of abdominal muscles; 3, lifting of abdomen; 4, body arching and lifting of pelvic structures.(2) The catheter with balloon was connected to a pressure gauge. Initial perception threshold (the pressure of immobility of the body or occasionally clinches of the head) and pain threshold (the pressure of contraction of abdominal muscles) were recorded.Results 1. Seven days after treatment, TNBS-induction of colitis induced ulcer formation, mucin depletion and inflammatory infiltrations with areas of transmural inflammation as compared with vehicle treatment.2. BDNF+/- and BDNF+/+ mice did not differ in histological damage scores with colitis induction.3. BDNF+/- and BDNF+/+ mice did not differ in MPO activity with colitis induction.4. Expression of BDNF protein(1) In vehicle-treated groups, the protein level of BDNF in BDNF+/- mice was approximately half of that in BDNF+/+ mice in both DRG and colon.(2) After colitis induction, BDNF was significantly up-regulated in both BDNF+/+ and BDNF+/- genotypes at levels of DRG and colon.(3) The protein level of BDNF in BDNF+/- mice with colitis was significantly lower than that in BDNF+/+ mice with colitis.(4) The protein level of BDNF in BDNF+/- mice with colitis was relatively lower than that in BDNF+/+ mice without colitis.5. Visceral response to colorectal distension(1) Comparisons of AWR scores at graded pressures.BDNF+/- and BDNF+/+ mice did not differ in visceral responses to colorectal distension at< 60mmHg. However, BDNF+/- mice showed significantly weaker responses to the distension of pressure?60 mmHg than did BDNF+/+ mice.With colitis, TNBS triggered a significant increase in wild-type BDNF+/+ mice as compared with vehicle treatment with distension pressures exceeding 30mmHg. BDNF+/- mice showed statistically stronger responses to distension following colitis than with vehicle treatment but only with pressure?45 mmHg.The response in BDNF+/- mice with colitis was significantly weaker than that in BDNF+/+ mice with colitis, but still stronger than that in normal wild-type mice.(2) Comparisons of initial perception thresholds and pain thresholdsBDNF+/- and BDNF+/+ mice did not differ in initial perception threshold and pain thresholds in non-inflammatory state.With colitis, TNBS triggered significant decreases in initial perception thresholds and pain thresholds in both BDNF+/+ mice and BDNF+/- mice as compared with their own vehicle controls.The initial perception threshold and pain threshold in BDNF+/- mice with colitis were significantly higher than those in BDNF+/+ mice with colitis, but still lower than those in normal wild-type mice.Conclusion1. In non-inflammatory state, compared with BDNF+/+ mice, BDNF+/- mice showed lower BDNF levels at both colon and DRG levels, but the colonic sensitivity in BDNF+/- mice was not significantly different at the distension pressures<60mmHg. At pressures?60mmHg, BDNF+/- mice showed reduced colonic sensitivity.2. We set up TNBS colitis model and found that after colitis the expression level of BDNF was significantly increased at the levels of DRG and colon while the colonic sensitivity was enhanced. The post-inflammatory hypersensitivity was partially reduced in heterozygous BDNF+/- mice. The findings identified the role of BDNF in the mediation of post-inflammatory colonic hypersensitivity BackgroundPatients with inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) often complain some urinary complications such as frequent urination, urgency and urinary pain which aggravate the incontinence. The underlying mechanism remains elusive. Current studies suggest that the overlapping of colonic and bladder dysfunctions comes from neural convergence and/or cross-talk at the levels of dorsal root ganglia or spinal cord.The DRGs innervating colon locate at T10-L1 and L6-S1 while the DRGs innervating bladder locate at T13-L1 and L6-S1. Studies present that the cell bodies of colonic DRG and urinary DRG are overlapped to some extent. The nerve fibers from overlapped neurons could be dichotomized into two different organs and supply a "convergent" model of afferent fibres. One organ is sensitized by peripheral inflammation and the noceptive signal is tranferred to DRG. Activated DRG could amplify the normal visceral signal from the other organ and transfer to central nervous system which appears as the sensitization of the other organ as well.The view of neural cross-talk focuses on the cross-activation of neighbouring neurons innervating different organs through the release of neurotransmitters or cytokines which has been proved by animal researches. Colitis could sensitize DRG neurons innervating bladder which shows the bladder hypersensitivity to the distension of saline infusion.Either neural convergence or cross-talk, to investigate the molecules involved in the signal amplification or cross-sensitization is significant to identify the underlying mechanisms of referred bladder hypersensitivity following colitis and provide useful insights into potential pharmalogical treatments. Study proposes that Calcitonin gene-related peptide (CGRP) is up-regulated in colitic model, sensitizes the DRG neurons innervating bladder and is involved in the referred bladder hyperalgeisa. Brain-derived neurotrophic factor (BDNF) could be released by DRG neurons and mediate the transduction of nociception. Therefore we predict that BDNF is also involved in the mechanism of referred bladder hypersensitivity following colitis.ObjectiveTo investigate the role of BDNF in the referred bladder hypersensitivity following colitis.Methods1. Collectively, four groups (BDNF+/+/vehicle, BDNF+/+/TNBS, BDNF+/-/vehicle and BDNF+/-/TNBS) were used in this study which were divided by their genotypes and treatments. Because the evaluations of bladder sensitivity require in-vitro exposed examinations while the DRGs innervating bladder and colon are not the same, this part of study is totally separate from the first part.2. Colitis was induced by TNBS (1.75mg in 50% ethanol, total 0.1 mL) through rectum. Control mice were administered the same volume of saline.3. Histopathology:seven days after colitis induction, mice were euthanized by an overdose of pentobarbital (200mg/kg i.p.) and the bladder was removed. Half of the sample was fixed in formalin, embedded in paraffin, stained with hematoxylin-eosin and graded on a semi-quantitative scale.4. Myeloperoxidase (MPO) activity test:Part of the bladder was weighed, homogenized and processed with a mouse MPO kit.5. Determination of BDNF protein by enzyme-linked immunosorbent assay (ELISA)Tissue samples separated from bilateral thoracolumbar (TL, T13-L1) and lumbosacral (LS, L6-S1) DRGs and part of bladder were homogenized and extracted for protein determination and ELISA assay. Total protein content was measured by the detergent-compatible BCA protein assay. Commercial ELISA kits were used to measure the level of BDNF protein according to the manufacturer's instructions. Concentrations of BDNF were calculated as nanograms BDNF per gram total protein.6. Bladder sensitivity tests(1) Short-term voluntary urination test:seven days after induction of colitis, free moving mouse was left on the filter paper in a big breaker and the small diameter urination spots (<0.2 cm2) were recorded by UVP imaging system one hour later.(2) Bladder capacity:Mouse was anesthetized and PE-10 catheter was gently inserted into the bladder through urethra. Pre-warmed saline was infused at the speed of 1mL/hour and inflated until voiding. The bladder capacity was termed as the volume to induce the voiding response.(3) CytometrographyMouse was anesthetized and bladder was exposed. PE-50 catheter was inserted into the dome of bladder through a small incision with the other end connected to infusion pump and pressure transducer.2 minutes later, pre-warmed saline was infused consistently at the speed of 20ul/min for 2 hours.Interval time:the time between three voidings was recorded.Initial voiding pressure:the pressure was recorded at which the voiding started.Results1. TNBS could induce severe colitis but no inflammation existed in bladder shown by histopathology and MPO activity evaluation.2. The protein level of BDNF in DRG innervating bladder(1) In non-inflammatory state, the levels of BDNF in TL and LS DRG of BDNF+/- mice were approximately half of those of BDNF+/+ mice.(2) TNBS could induce significant increases of BDNF expression of TL and LS DRG in both genotypes. (3) The level of BDNF following colitis in BDNF+/- mice was significantly lower than that of BDNF+/+ mice following colitis.(4) The levels of BDNF in BDNF+/- mice with colitis and BDNF+/- mice without colitis had no significant difference.3. The protein level of BDNF in bladder(1) In non-inflammatory state, the levels of BDNF of BDNF+/- mice were approximately half of those of BDNF+/+ mice.(2) TNBS could induce significant increases of BDNF expression in both genotypes.(3) The level of BDNF following colitis in BDNF+/- mice was significantly lower than that of BDNF+/+ mice following colitis.(4) The level of BDNF in BDNF+/- mice with colitis was lower than that of BDNF+/- mice without colitis.4. Bladder sensitivity evaluations(1) In non-inflammatory state, the sensitivities of BDNF+/- and BDNF+/+ mice had no significant differences.(2) TNBS induced significant hypersensitivity in both BDNF+/- and BDNF+ mice compared with vehicle-treated mice. In colitic mice, the short-term voluntary urination was increased, bladder capacity was reduced while initial urination pressure and interval time were reduced.(3) BDNF+/- mice with colitis showed lower sensitivity compared with BDNF+/ mice with colitis, but the sensitivity of BDNF+/- mice with colitis was still higher than normal wild-type mice.ConclusionWe used TNBS to induce colitis and found no inflammation existed in bladder. Following colitis, the levels of BDNF in bladder and DRG innervating bladder were increased while the bladder sensitivity was enhanced. Heterozygous BDNF knock-out mice showed partially reduced bladder sensitivity compared with wild-type mice following colitis which indicated the involvement of BDNF in referred bladder hypersensitivity. BackgroundVisceral hypersensitivity is suggested as the major mechanism of inflammatory bowel disease (IBD) and post-infectious irritable bowel syndrome (PI-IBS). Studies propose that post-inflammatory peripheral neural sensitization in colon play an important role in the etiology of visceral hypersensitivity in IBD and PI-IBS patients.Visceral sensitivity is mediated by the spinal afferents with the cell bodies in dorsal root ganglia (DRG), therefore the molecular changes of DRG following inflammation are the key targets to investigate the mechanism of visceral hypersensitivity.Transient Receptor Potential (TRP) channels have emerged as a family of evolutionarily conserved ligand-gated ion channels that contribute to the detection of mechanical, thermal and chemical stimuli. Increasing amount of evidence suggests that members of TRPs family, such as transient receptor potential vanilloid-1(TRPV1) and TRPV4, are widely involved in visceral hyperalgesia.A novel member of TRP channels, the transient receptor potential ankyrin-1(TRPA1) channel has been cloned recently and found to be selectively expressed by the peptidergic subset of sensory fibers that also expressed TRPV1. Initially characterized as a sensor of noxious cold and mechanical stimuli, this channel was further suggested to be activated by a number of exogenous pungent compounds, proalgesic bradykinin and multiple products of oxidative stress. Pharmacological blockade of TRPA1 in peripheral sensory neurons reversed somatic nociceptive responses caused by tissue injury and inflammation. Previous studies were mainly concerned with the role of TRPA1 in somatic sensation and few referred to the visceral hyperalgesia. Here we hypothesized that TRPA1 receptor might participate in the visceral hyperalgesia following colonic inflammation. The present study applied TNBS (trinitro benzene sulfonic acid) to induce colitis and examine the protein expression levels of TRPV1 and TRPA1 in L6-S2 DRG (dorsal root ganglia) while the visceral mechanical and chemical sensitivity after colitis. Then with antisense oligodeoxynucleotide directed to TRPA1, we tried to knock down TRPA1 to examine whether the protein expression and colitis-induced visceral hyperalgesia could be altered.ObjectiveTo investigate the role of TRPA1 in the mediation of visceral hypersensitivity after colitisMethods1. Collectively, rats involved in our study were divided into six groups. Control and TNBS groups were each further sub-divided into vehicle group, TRPA1 antisense oligodeoxynucleotides (AS-ODN) group and mismatch oligodeoxynucleotides (MM-ODN) group.2.20mg TNBS in 50% ethanol (total volume 0.4ml) was instilled into the lumen of the colon using a 7 cm long oral gavage needle that was inserted into the descending colon. Control rats were treated with a similar volume of vehicle (saline) only. To avoid leakage of instilled solutions, the rats were kept in a vertical position for 3 min.3. Visceral sensitivity tests3.1 Adult male Wistar rats (250-300g body weight) were anesthetized by sodium pentobarbital (50mg/kg i. p.) with Teflon-coated stainless steel electrodes sewn into the external oblique abdominal musculature and tunneled subcutaneously to a small incision made on the nape of the neck.3.2 For visceral sensitivity to colonic distension, briefly sedated with halothane, the rats (n=8/group) were placed in a small cubicle (20 cm×8 cm×8 cm) on a platform while a flexible latex balloon(5cm in length) was inserted into the descending colon and held in place by taping the attached tubing to the tail. The rats were allowed 30 min to acclimatize before testing. The balloon was inflated to various pressures (20,40,60,80mmHg) using a sphygmomanometer. Distension pressures were held constant during the 30s stimulation with 4-min inter-stimulus interval. The EMG signal was amplified, filtered, and recorded on the computer with BL-420E Biological function system for off-line analysis. The area under the curve for EMG recording was measured for further analysis.3.3 For visceral sensitivity to intracolonic chemical irritation, the animal was placed in the small cubicle with PE-10 tubing inserted into the distal colon. The tube was kept in place by taping to the tail with the opposite end attached to a 1.0ml syringe. Solutions of allyl isothiocyanate (AITC,0.25%vol/vol) and capsaicin (0.03%vol/vol) were handled as separated cases with the volumes both restricted to 100ul and the duration over 10s to avoid mechanical stimulation (n=6/case/group). The raw EMG data were recorded during a 45 s period following the stimulus and the area under the curve was calculated4. Antisense oligodeoxynucleotide (5'-TCTATGCGGTTATGTTGG-3') or mismatch oligodeoxynucleotide (5'-ACTACTACACTAGACTAC-3') directed to TRPA1 which were designed as described previously. Nuclease-free saline was applied as the vehicle control. An intrathecal catheter (PE-10) was implanted at the level of the L6-S1 spinal cord in the second day after TNBS or vehicle enema. The distal end of the catheter was fire sealed and placed subcutaneously.4 d after the catheter implantation, the rats were injected intrathecally (i. t.) with sustained mini-osmotic pumps operating at a rate of 1 u 1/hr for a period of 3 days.5. For western-blotting, bilateral lumbosacral (L6-S2) DRG tissues were immediately dissected and pooled to extract protein. The levels of TRPAland TRPV1 protein expression in different groups were assessed using western-blotting.6. For histopathology, distal colon tissues from different groups were removed, fixed in formalin, embedded in paraffin, cut in 5 u m sections, and stained with hematoxylin-eosin.ResultsThe TRPA1 and TRPV1 protein levels were both significantly up-regulated following colitis. The TRPA1 antisense ODN, but not the mismatch ODN, significantly reduced the TRPA1 expression in control and TNBS rats. In contrast, the antisense ODN had no significant effect on the TRPV1 expression.TNBS resulted in significantly higher response to colonic distension (at?40mmHg). At above 60mmHg, The TRPA1 antisense ODN reduced visceromotor response to colonic distension significantly in TNBS group while the mismatch ODN and vehicle treatments had no effect. The visceral mechanosensitivity of control rats was not changed following intrathecal antisense or mismatch ODN.Meanwhile, TNBS resulted in significantly higher response to intracolonic chemical irritations. The antisense ODN, but not the mismatch ODN efficiently suppressed responses to AITC in both TNBS and control rats. In contrast, the antisense ODN had no influence on the response to capsaicin, a potent agonist for TRPV1. No significant difference existed between the mismatch ODN and vehicle treatments for protein expression or visceral sensitivity.ConclusionTRPA1 is involved in the mediation of visceral hyperalgesia following colitis.