The Effect Of Oxytocin On Visceral Sensitivity In Rats

Part Ⅰ:The Impact of Oxytocin on Mesenteric Afferent Sensitivity and Visceral pain in Normal RatAimsOxytocin (OT) is known to be a nonapeptide synthesized in the paraventricular nucleus of the hypothalamus and released from the pituitary. As a classical hormone, its physiological function is related to uterine contraction during parturition and milk ejection reflex through by OT receptor (OTR). OT and OTRare synthesized not only in the central nerve system, but also in many peripheral organs including the gut. It is involved in regulating gastric, duodenal and colonic motility. Mounting evidence suggests that OT plays an important role in pain sensation and antinociception in the central nervous system, but little is known about possible roles of peripheral OT on antinociception. So we explore the effect of OT on mesenteric afferent sensitivity in and visceral pain in rats.MethodsTissue preparation and mesenteric afferent nerve recordingsRats were anaesthetized with pentobarbital sodium (60mg kg-1i.p.). A midline laparotomy was performed and a2cm-long segment of jejunum with its attached mesenteric arcade quickly excised and placed into ice-cold Krebs buffer with continuously oxygenated (95%O2and5%CO2). After flushing the gut lumen, thetissue was transferred to the perfusion chamber of organ bath where the segment was continuously superfused with Krebs buffer. Both ends of the segment were cannulated and the lumen perfused with Krebs solution. To record changes of intraluminal pressure, the oral end was connected to a pressure transducer (CED single channel1902preamplifier/filter; Cambridge Electronic Design, Cambridge, UK) and the other end left open. To make afferent nerve recordings the mesenteric arcade was drawn through an aperture leading into a separate recording chamber. Under a stereo microscope, one of the two perivascular nerve bundles was isolated and attached to one arm of a bipolar platinum recording electrode, while one strip of connective tissue, the size of which was similar to the nerve, was attached to the other electrode. The two electrodes were connected to a CED single channel1902preamplifier/filter (CED, as above), by which the signal was amplified (10,000×) and filtered (bandwidth of100-1,000Hz). Signals from the amplifiers were passed into Micro1401interface system (CED) and viewed on a PC running Spike2software (version5.01; CED).Visceral Pain Behavioral AssessmentEach animal was lightly anesthe tized by inhalation of isoflurane. A4-6cm-long air sac was tied to a4mm diameter silicone catheter, which was connected to a viscera dilator. And the end of air sac was carefully inserted into the colorectum with a depth of1-2cm. Then the catheter was taped to the base of the tail to prevent displacement. In order to make a rat model of visceral hyperalgesia, CRD stimulation was carried out by rapidly inflating the air sac to80mmHg for30s duration which was followed by a30s interval. This inflation and deflation cycle was repeated for60times. The behavioral responses to acute pain were tested4h later. The graded CRD was conducted by manually infusing different volume of water in to the sac. For every rat, the distension was repeated four times with different volumes of water (0.4ml,0.8ml,1.2ml and1.6ml). For every CRD, the duration of the distension was20s, and there is a5min interval before the successive distension. Oxytocin (1mg kg-1) or the inhibitors were applied (i.p.)0.5h or1h before graded CRD.All measurements were made by at least two observers blinded to the experimental procedures.Data analysis The baseline of the mesenteric afferent discharge (impulse s-1) was determined as the average frequency multiunit or single unit spikes during a2min recording period prior to administration of test stimuli. Responses to stimulation with drugs were measured as the peak impulse frequency above baseline at30s intervals up to120s after the onset of administration. Intestinal motility was measured at baseline as the mean intraluminal pressure during a30s interval before test stimuli were administered. After administration of oxytocin or bradykinin (BK), intestinal pressure was measured as the mean pressure over2min following the stimulus. Responses to distension were determined by quantifying the firing frequency over a3s period at10cmH2O increments in intraluminal pressure. The baseline firing prior to distension was subtracted in order to provide values for the increase in discharge in response to distension. In some experiments single unit analysis was performed using the template matching algorithm in SPIKE2(CED) as described previously.Descriptive statistics were presented as mean±SEM and evaluated using one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s multiple comparison test or Students’ t-test using Sigmaplot statistical software (Sigma, USA). For all cases, n denotes the number of animals and P<0.05was considered to be statistically significant.ResultsEffect of OT on mesenteric afferent dischargeOxytocin (10-7M), when added to the Krebs perfusing the serosal side of the gut segment for2min, failed to elicit significant changes in afferent multiunit firing rates (n=6).Effect of OT on afferent response to BKPretreatment with OT dose-dependently decreased the response to BK. The peak increase of the discharge to BK was decreased by OT (10-7M or10-6M). Lower doses of OT did not appear to influence the response to BK. Pretreatment of the tissue with the OTR antagonist atosiban (10-6M) reversed the inhibitory effect of OT (10-7M) on the response to BK.Effect of OT and BK on average intraluminal pressureOT and BK both decreased intraluminal the average intraluminal pressure, and that therefore BK evoked increases in firing rate cannot be attributed to an increase in intraluminal pressure.Effect of nitro oxide synthase (NOS) inhibitors on OT induced down regulation of the response to BKThe inhibitory effect of OT on the BK-evoked excitation was significantly reduced by adding the non-specific NOS inhibitor L-NAME (10-4M) to the serosal superfusate for10min.Effect of SNP on mesenteric afferent dischargeTo investigate the effect of NO on the mesenteric afferent response to bradykinin, the intestinal segment was pretreated for5min with the NO donor SNP (4mM) before bradykinin applied. Similar to oxytocin, SNP decreased the maximal bradykinin-induced multiunit discharge rate.Effect of OT on distension-evoked responsesThe effect of OT on mechanosensitivity was investigated by comparing the response to ramp distension up to60cm H2O before and after treatment with OT. Forty-one distension sensitive single units were identified from multiunit recordings for which OT was tested. OT reduced the response to distension only for high threshold units; the low threshold and dynamic range afferentswere unaffected.Effect of OT on acute visceral pain caused by CRD stimulationOT significantly decreased the AWR score with1.2ml and1.6ml distention. With the pretreatment of atosiban (0.5mg kg-1, i.p.), the AWR score was significantly higher than that of the OT group. Pretreatment of L-NAME (20mg kg-1, i.p.)also attenuated the effect of OT on pain sensation.ConclusionsThe sensitivity of the jejunal afferent nerve to BK, the high threshould afferent to mechanical stimulation and the acute visceral pain were significantly decreased by OT, and NO was involved in the inhibitory effect of OT. Part II:Involvement of nNOS-NO-KATP Channel Pathway in the Inhibitory Effect of Oxytocin on the Sensitivity of Mesenteric AfferentsAimsOxytocin (OT) and OT receptor (OTR) were expressed in the longitudinal muscle my enteric plexus (LMMP). OT was found to increased the intracellular concentration of Ca2+in calbindin containing neurons within the rat small intestine myenteric plexus Nitric oxide (NO) is an important neurotransmitter in enteric nervous system (ENS) and involved in the peripheral antinociception. NO synthesis appears to be dependent on the influx of Ca2+in enteric neurons. It is reporeted that ATP-activated potassium (KATP) channel is assotiated with the peripheral antinociceptive effect of NO. So we hypothesized that OT in gut might play an inhibitory effect on the sensitivity of spinal mesenteric afferents through by the nNOS-NO-KATP channel pathway.MethodsTissue preparation and mesenteric afferent nerve recordingsRats were anaesthetized with pentobarbital sodium (60mg kg-1i.p.).A midline laparotomy was performed and a2cm-long segment of jejunum with its attached mesenteric arcade quickly excised and placed into ice-cold Krebs buffer bubbled with95%O2and5%CO2. After flushing the gut lumen, thetissue was transferred to the perfusion chamber of organ bath where the segment was continuously superfused with Krebs buffer. Both ends of the segment were cannulated and the lumen perfused with Krebs solution. To record changes of intraluminal pressure, the oral end was connected to a pressure transducer (CED single channel1902preamplifier/filter; Cambridge Electronic Design, Cambridge, UK) and the other end left open. To make afferent nerve recordings the mesenteric arcade was drawn through an aperture leading into a separate recording chamber. Under a stereo microscope, one of the two perivascular nerve bundles was isolated and attached to one arm of a bipolar platinum recording electrode, while one strip of connective tissue, the size of which was similar to thenerve, was attached to the other electrode. The two electrodes were connected to a CED single channel1902preamplifier/filter (CED, as above), by which the signal was amplified (10,000×) and filtered (band width of100-1,000Hz). Signals from the amplifiers were passed into Micro1401interface system (CED) and viewed on a PC running Spike2software (version5.01;CED).Measurement of NOLMMP preparations of jejunum were made by carefully removing the mucosa, submucosa and circular muscle layers by microdissection and leaving the myenteric plexus with attached longitudinal muscle. LMMP preparations were divided into five groups, and treated with the following agents respectively:vehicle, vehicle plus OT (10"7M), atosiban (10-6M, Ferring AB, Malmoe, Sweden) plus OT (10-7M), N (ω)-Propyl-L-arginine hydrochloride (NPLA,10’6M) plus OT (10-7M). The concentration of NO was assayed using a commercial NO assay kit.Measurement of NO production with DAF-FMThe LMMP was incubated in Hanks’ Balanced Salt solution/Hepes containing the fluorescent nitric oxide probe4-amino-5-methylamine-2’,7’-difluorofluorescine (DAF-FM). After loading with DAF the LMMP was placed in DAF-free medium and exposed to either vehicle or OT (10-7M).N-nitro-L-arginine methyl ester (L-NAME) was added before OT to inhibit nitric oxide synthase (NOS) activity. LMMPs were fixed and examined with a confocal lasers canning microscope.ImmunohistochemistryA flattened LMMP preparation was rinsed with Krebs buffer and fixed in4%paraformaldehyde for30min at room temperature, followed by incubation with5%donkey serum for1h. The preparation was incubated with the primary antibody for16-18h in a humid box at4℃.After primary incubation, the tissues were washed with PBS and subsequently exposed to the appropriate secondary antiserum for2h at room temperature. The preparations were observed using confocal microscopy.Data analysis Descriptive statistics were presented as mean±SEM and evaluated using one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s multiple comparison test or Students’ t-test using Sigmaplot statistical software. For all cases, n denotes the number of animals and P<0.05was considered to be statistically significant.ResultsEffect of NOS inhibitors on OT induced down regulation of the response to BKThe specific blocker of nNOS, NPLA (10"6M) increased the response to BK when OT was present by230%.Effect of OT on NO production in jejunumAdding10"7M OT to the serosal superfusate increased NO production within LMMP. Pre-incubating the tissue for10min with either atosiban or NPLA (10-6M) abolished this effect. We also used DAF-FM fluorescence to detect NO content within the LMMP preparation. When the preparation was incubated with OT (10-6M) for10min, the production of NO signicantly increased, whereas the incubation ofL-NAME (10-4M) attenuated the effect of OT.Co-expression of Calbindin28K, nNOS and OTR in LMMPWe counted the number of myenteric neurons expressing Calbindin28K, nNOS or OTR.74.2%Calbindin28K positive intrinsic sensory neurons were immunopositive for both nNOS and OTR.Effect of ω-Agatoxin IVAand ω-conotoxin GVIA on the OT induced inhibition of the response to BKPre-incubating the jejunum with the specificP-type Ca2+channel blocker ω-Agatoxin IVA (3×10-8M) or the specific N-type Ca2+channel blocker ω-conotoxin GVIA (10-7M) partially reversed the inhibitory effect of OT.Effect of glibenclamide on the OT induced inhibition of the response to BKIn the presence of10"6M glibenclamide and OT, the mesenteric nerve peak response to BK was significantly greater than when only OT was present. ConclusionsOT activated nNOS in ENS,then the released NO opened the KATP channels in the spinal afferent fibers, the membrane was hyperpolarized to inhibit the sensitivity of the afferents response to BK. OT may be a new target for the treatment of algesia. Part Ⅲ:Effect of Oxytocin on TNBS-Induced Colitis and Visceral pain in RatsAimsInflammatory bowel disease (IBD) is chronic and relapsing inflammatory conditions in the gut, whose pathogenesis is still undefined. Increasing evidences indicate the immune system has an important effect on the initiation and progression of IBD, furthermore the involvement of mast cells play a role in the pathogenesis of various inflammatory conditions of the gastrointestine, including IBD. Oxytocin (OT) performs series of anti-inflammatory actions and OT receptors (OTRs) have been found to be expressed in some mast cells. Sowe hypothesized that OT alleviates TNBS-induced colitis and the visceral pain reflex by inhibiting the degranulation of mast cells.MethodsInduction of colitis by2,4,6-trinitrobenzene sulfonic acid (TNBS)The rats fasted overnight before the induction of colitis. Under anaesthetized with10%chloral hydrate (0.3g kg-1i.p.),a polyethylene catheter was inserted through rectal into the colon so that the tip was introduced to a point about8cm from the anus.30mg TNBS dissolved in25%ethanol, or saline as a control, was introduced into the lumen of the colon through the catheter.General conditionAfter the treatment of TNBS, the general condition of rats was obvsered every day, including the mental status, activity, diet, hair and stool.The body weight was measured before enema and7days later.Macroscopic evaluation of colonic injuryOn the7th day after the TNBS enema, the animals were decapitated and a midline laparotomy was performed, and the adhesion and macroscopic colonic damage was scored. All measurements were made by at least two observers blinded to the experimental procedures. After scoring, the wet weight of the distal8cm of the colon was also measured.Microscopic evaluation of colonic injuryThe histologic samples were cut into4mm-thick sections and stained with hematoxylin-eosin (HE). A quantitative evaluation of damage was scored by measuring the extent of intact, necrotic and regenerating mucosa in the same slides. All measurements were made by at least two observers blinded to the experimental procedures.Measurement of myeloperoxidase (MPO) activityAfter a piece of tissue was excised from the colon and homogenized. MPO activity in homogenates of colon tissue was measured by using a kit according to the instructions.Measurement of mast cellSections of4μm were obtained from different levels and stained with0.5%toluidine blue. The number of mucosal mast cells in10randomly selected high-power fields (×400) was counted, and the number per10fields was calculated. All measurements were made by at least two observers blinded to the experimental procedures.Immunohistology of OTROTR was stained with Ultra Sensitive TM SP (Goat) IHC Kit. Paraffin sections of4μm were dehydrated and incubated by anti-OTR antibody,4℃overnight. Then the sections were rinsed with PBS.After incubated by agents in the kit, the section was colored by a DAB kit. All sections were dehydrated by graded concentrations of alcohol. The preparations were placed on a glass slide, cover-slipped with neutral resins and then observed under microscopy.Measurement of OTPreparations of0.1g distal colon per rat were collected and rinsed with0.1M PBS, homogenized in1ml of PBS an stored overnight at-20℃. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged at5000rpm for5min, at2-8℃. The supernatant was removed and assayed immediately by an OT kit.Visceral Pain Behavioral AssessmentAsdescribed as Part Ⅰ.Measurement of released histamineColonic tissues were rapidly immersed in hard plastic tubes containing2ml of Hanks’ solution (37℃) and continuously oxygenated (95%O2/5%CO2). After25min incubation,200μl of the bathing solution was removed. All samples were centrifuged and supernatant were aliquoted and stored at-70℃until the assay. At the end of the release experiment, the tissue were blotted and weighed. Histamine was detected from triplicate aliquots of the supernatants using a highly selective enzyme-linked immunoassay according to the manufacturer’s directions. Histamine was measured using a microtiter plate reader and normalized to the weight of the tissues.Data analysisIn each experiment, n indicates the number of experiments. P<0.05was considered to be significant different. Data under the various experimental conditions was presented as means±standard error of the mean (SEM) and evaluated using One-way analysis of variance (ANOVA) followed by post hoc Dunnett’s multiple comparison test or Students’ t-test by the software package of Sigmaplot.ResultsEffects of OT ongeneral status in experiment animalThe degree of inflammation shown as the general status was significantly increased in the TNBS+Saline group rats and the increased body weight of the rats after TNBS enema was less than that of the control group. After given OT (1mg kg-1d-1) by enema for7days, the inflammation was attenuated, and the increased body weight was greater compared with that of the TNBS+Saline group.Effects of OT on TNBS-induced adhesion and macroscopic colonic damageThe adhesion score of the TNBS+Saline group was distinctly higher than that of the control group rats, which was significantly reduced by the treatment of OT. The mucosa of rats in the control group was integrity and smooth; the colonic mucosa in the TNBS+Saline group rats was hyperemia and edema, ulcer or lesion stenosis; the colonic mucosal damage in the TNBS+OT group rat was significantly decreased.The colon weight of the TNBS+Saline group rats was significantly increased than that of the control group rats, which was also reduced by OT treatment.Effects of OT on histologic score of colitis induced by TNBSThe histologic score of colitis was examined7days after administration of TNBS. The colon damage was represented by epithelial exfoliation, hemorrhage, edema, infiltration of polymorphonuclear leukocytes, necrosis and mucosal regeneration. The histologic damage score of the TNBS+Saline group was increased than that of the control group rats. The damage and the score were both significantly attenuated by OT administration.Effects of OT on MPOThe MPO activity in TNBS+Saline group was greater than that of the control group, which was reduced after treatment of OT for7days.Expression of OTR in colon mucosal mast cells and the concentration of OTMast cells and OTR were stained by toluidine blue and immunohistochemical kit respectively. The results showed that OTR was expressed in the mast cells. Interestingly, the concentration of OT in colon was significantly higher in TNBS group than that of the vehicle group.Effect of OT on visceral hyperalgesia in the colitisCompared with the AWR score of the vehicle group, the AWR score of TNBS group was significantly increased. Then we tested the effect of OT on the AWR score in TNBS-induced colitis model rats. The AWR score was distinctly decreased by the treatment of OT, which was partly reversed by treatment of OTR inhibitor atosiban (0.5mg kg-1).Effects of OT on mast cell numberIn the TNBS+Saline group, the number of mast cells was markedly decreased compared with the control group, which was partly reversed by OT treatment.Effects of OTon colonic histamine release The concentration of released histamine was significantly increased in the TNBS+Saline group compared with that of the control group, which was reduced by the treatment of OT.ConclusionOTR was expressed on the colonic mucosa mast cells. In the TNBS-induced colitis, OT alleviated the inflammation and visceral pain relax by inhibiting mast cells degranulation. Therefore, OT signaling system may be a new target for the treatment of visceral hypersensitivity.