| Partâ… :Jejunal afferent nerve sensitivity to chemical and mechanical stimuli in C57BL/6 mice in vitroObjective:Visceral afferent hypersensitivity is now a widely accepted mechanism which could explain many of these clinical symptoms associated with functional bowel disease and inflammatory diseases of the gut.Vagal and spinal afferents represent the information superhighways that convey sensory information from the gut to the central nervous system.These afferents are sensitive to both mechanical and chemical stimuli.We aimed to investigate the mesenteric afferents firing response to 5-HT,Bradykinin(BK) and ramp distension in vitro in mice and produced the dose- response curve of afferent nerve to chemical and mechanical stimuli.Method:C57BL/6 mice were anesthetized with isoflurane and a 2 cm segment of jejunum was excised with the mesentery attached and placed in an organ bath. Multi-unit afferent nerve recordings were measured in a custom-made organ bath consisting of two chambers i.e.a perfusion and a recording chamber.The jejunum was cannulated at both ends,placed in an organ bath and superfused with Krebs solution(32℃,10 ml/min;gassed with O2/CO2 mixture).Under the microscope the mesenteric nerve was separated out of the neurovascular bundle and then connected with bipolar platinum recording electrodes.The electrodes were connected to a CED single channel 1902 preamplifier/filter then the output from 1902 were sent to a power Micro 1401 interface system(CED),saved on the hard drive of a laptop computer to get the original afferent firing signal using Spike 2 software.The afferent nerve response to 5-HT(125μM,250μM,500μM),BK (0.25μM,0.5μM,1μM) and distension was investigated.5-HT and BK was applied in the organ bath acutely for a period of 2 minutes and then washed out.5-HT,distension and BK was performed on one jejunal segment in tum.Only one concentration of 5-Ht and BK was administrated on the jejunal segment, which was chosen randomly.For continuous ramp distension,the outlet cannula in the intestinal lumen was clamped,while perfusion with Kreb's buffer was continued at 10 ml h-1.With this method the gut segment was distended to 60 cmH2O in approximately 90 seconds.After each stimulus an interval of at least 15 minutes was allowed to reach baseline discharge before the next stimulus was administered.In preliminary cross-over experiments,we determined that responses to stimuli are independent of the order in which they are applied.The baseline discharge frequency(imp/second) was determined by averaging the afferent nerve discharge during the 2 minutes recording period prior to administration of test stimuli.The afferent nerve response to chemical stimulation with 5-HT and BK was evaluated as the mean increase in peak impulse frequency per second above baseline discharge frequency during a 3 second period of maximum afferent firing and the mean firing rate 30 seconds,1 minute,2 minutes following drugs administrated in organ bath above mean baseline discharge as well.The response to ramp distension was evaluated by quantifying the peak impulse frequency per second over a 3 sec period and the mean frequency increase at 10 cm H2O increments of intraluminal pressure until 60 cmH2O were reached.Data were presented as mean±SEM and were compared by one-way ANOVA.Results:Continuous afferent firing was present at baseline which consisted of spikes with different amplitudes and waveforms.Baseline discharge was calculated 2 minutes before stimuli,which was not different before the stimuli.1.Afferent nerve discharge increased promptly following 5-HT into the organ bath without obvious latency.The peak afferent firing rate to 5-HT was 42±4.4 imp/second above baseline at 125μM,64±4.0 imp/second above baseline at 250μM,76±2.6 imp/second above baseline at 500μM.At concentration 125μM the mean afferent discharge rate increased 24.6±2.7 imp/second 30s following 5-HT administration,20.4±2.2 imp/second 1 minute following 5-HT administration and 12.5±2.3 imp/second 2 minute following 5-HT administration. At concentration 250μM the mean afferent discharge rate increased 29.1±3.2 imp/second 30s following 5-HT administration,25.9±2.7 imp/second 1 minute following 5-HT administration and 18.3±3.6 imp/second 2 minute following 5-HT administration.At concentration 500μM the mean afferent discharge rate increased 35.3±3.2 imp/second 30s following 5-HT administration,29.5±1.9 imp/second 1 minute following 5-HT administration and 21.2±2.1 imp/second 2 minute following 5-HT administration.2.During mechanical stimulation by ramp distension a pressure dependent increase in afferent nerve discharge was observed.At the maximum pressure of 60 cmH2O,peak afferent firing was 80±3.3 imp/second above baseline,the mean frequency increase was 70.8±5.4 imp/second.3.BK superfusion in the organ bath was followed by an obvious and robust increase in afferent nerve discharge.The increase of peak firing to BK was 65±2.7 imp/second at concentration of 0.25μM,89±7.3 imp/second at concentration of 0.5μM,112±8.9 imp/second at concentration of 1μM.At concentration 0.25μM the mean afferent discharge rate increased 44.8±5.imp/second 30s following BK administration,32.5±3.2 imp/second 1 minute following BK administration and 20.3±3.3 imp/second 2 minute following BK administration.At concentration 0.5μM,the mean afferent discharge rate increased 53.8±6.1 imp/second 30s following BK administration,40.7±3.1 imp/second 1 minute following BK administration and 24.6±2.2 imp/second 2 minute following BK administration. At concentration 1μM,the mean afferent discharge rate increased 62.9±4.9 imp/second 30s following BK administration,45.2±2.2 imp/second 1 minute following BK administration and 28.6±2.4 imp/second 2 minute following BK administration.Conclusions:The afferent firing displayed a pressure dependent increase during ramp distension.5-HT and bradykinin can stimulate serosal afferents by a direct action on the receptor in the gut in vitro with a dose -dependent model.Partâ…¡:Jejunal afferent nerve sensitivity change and mechanism during indomethaicn induced inflammtionObjective:Patients with chronic inflammatory bowel disease often have little symptoms,although extensive morphological alterations of the intestinal mucosa are present.We aimed to characterize visceral sensitivity of the inflammed small intestine with an indomethaicn induced inflammatory bowel disease model in mice and to identify possible regulatory mechanisms.Methods:C57BL/6 mice received 2 injections of 60 mg kg-1 indomethacin or vehicle subcutaneously within 48 h in order to trigger intestinal inflammation Inflammation was quantified by a macroscopic inflammation score which included evaluation of adhesions,intestinal length,hyperemia and ulcers. Microscopic assessment of intestinal inflammation was performed on slides stained with haematoxylin and eosin.One day after the second injection the animal was anesthetized with isoflurane and a 2 cm segment of jejunum was excised with the mesentery attached and placed in an organ bath.Afferent sensitivity was recorded in vitro by extracellular multi-unit afferent nerve recordings from the mesenteric nerve bundle.The recording method and the stimuli were same as the first part.Intestinal motility was assessed by intraluminal pressure recordings. Afferent nerve recordings were obtained on day 3 after the beginning of different forms of pre-treatment leading to 4 subgroups of animals(each n=6).1) Mice injected with indomethacin to induce intestinal inflammation;2) Mice injected with vehicle(100%ethanol) on day 1 and 2 at 8 a.m.as controls for indomethacin induced inflammation;3) Mice injected with indomethacin to induce intestinal inflammation plus chronic pre-treatment with the selective iNOS inhibitor L-N6-(1-iminoethyl)-lysine(L-NIL)(3 mgkg-1,five times i.p.);4) Mice injected with indomethacin to induce intestinal inflammation plus L-NIL (30μM) administered acutely in the organ bath 10 minutes prior to application of the test stimuli.The maximum impulse rate per second following serosal administration of 5-HT(250μM),Bradykinin(0,5μM) or mechanical ramp distension of the intestinal loop was recorded.The method to collect the data is same as the first part.Statistical analysis was performed by One-Way-ANOVA and Two-Way-ANOVA.All data were expressed as mean±SEM.Results:Following indomethacin pretreatment,animals developed acute intestinal inflammation in the entire small intestine characterized by obvious intestinal dilation,marked reduction in small intestinal length and adhesions between small intestinal loops.The macroscopic score was 0.24±0.20 at vehicle group,0.34±0.33 at vehicle plus L-NIL,4.84-0.74 at inflammation group(P＜0.05,versus vehicle group ) and 4.0±0.71 at inflammation plus L-NIL treatment group(P＜0.05, versus vehicle group).Histology score got the similar result,1.64±0.60 at vehicle group,2.0±0.58 at vehicle plus L-NIL,4.6±0.51 at inflammation group(P＜0.05, versus vehicle group ) and 4.0±1.08 at inflammation plus L-NIL treatment group (P＜0.05,versus vehicle group)Intraluminal pressure recordings revealed a decrease of the amplitudes of spontaneous contractions during inflammation(2.514±0.3 versus 0.664±0.1 cmH2O in controls;P＜0.01).Baseline discharge was not different among the various subgroups.In vehicle controls maximum firing response to 5-HT was 65±7.5 imp/second above baseline,which was reduced to 32±3.3 imp/second in animals with indomethacin induced inflammation(p＜0.05).This reduction in the response to 5-HT was preserved when animals were either chronically pretreated with L-NIL(55±7.5 imp/second) or when L-NIL(73±7.2 imp/second) was administered into the organ bath acutely.When compared to controls,mechanical sensitivity was reduced during intestinal inflammation for luminal distension pressures exceeding 20 cmH2O (P＜0.05).At the maximum pressure of 60 cmH2O,afferent firing increase was 38±3.0 imp/second in inflamed intestinal segments and 95±5.9 imp/second in controls(P＜0.05).This hypo-mechanic sensitivity was absent following the iNOS inhibitor L-NIL given either i.p.or in the organ bath.The maximum impulse rate following bradykinin was 55±7.9 imp/second in the inflamed intestine compared to 97±7 imp/second in control animals(P＜0.05). This reduction in the inflamed intestine was not present during chronic treatment with the iNOS inhibitor L-NIL(112±16 imp/second) or acute administration of L-NIL in the organ bath(108±13.7 imp/second).Conclusions:Afferent sensitivity is decreased by an iNOS dependent mechanism during intestinal inflammation which appears to be independent of the inflammatory response.This suggests that iNOS dependent nitrogen monoxidum production alters afferent sensitivity during inflammation by interfering with signal transduction to afferent nerves rather than by attenuating the inflammatory response.Partâ…¢:Colonic afferent nerve sensitivity change and mechanism during DSS induced colitis Objective:Intestinal inflammation sensitizes extrinsic afferents innervating the gut.We aimed to study the underlying mechanisms in a colitis model of inflammatory bowel disease and specifically hypothesized that mast cells and the cyclooxygenase pathway are involved.Methods:C57BL/6 mice received drinking water with 3%dextran-sulfate sodium (DSS) for 7 days to induce colitis.Control animals drank untreated water.On day 8 animals were anesthetized and inflammation was assessed by disease active index(weight loss,colon length,colon weight/length ratio,spleen weight,stool consistency,presence or absence of fecal blood).The colon next to the proximal colon was stained with haematoxylin and eosin to perform microscopic assessment of intestinal inflammation.The proximal colon was harvested with the mesenteric arc attached,while the ileum,coecum and distal colon were removed. A 2 cm segment of the proximal colon was cannulated at both ends,placed in an organ bath and superfused with Krebs solution(containing 1μM nifedipine to eliminate contractions).Extracellular afferent nerve recordings were secured from the mesenteric nerve(same as the first part) and the discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software.Uninflamed segments served for control recordings,while subgroups of colitis segments were treated with either the mast cell stabilizer Doxantrazole (10-4 M) or the cyclooxygenase inhibitor Naproxen(10-5M) in the organ bath (each group n=6).The maximum impulse rate per second following serosal administration of bradykinin(0,5μM) or mechanical ramp distension of the intestinal loop was recorded.The discharge of the mesenteric afferent nerve was evaluated as the total discharge of all the nerve fibres separating by single-unit analysis.Data are given as mean±SEM and were analyzed by one -way ANOVA and two-way ANOVA.Results: Mice exposed to 7 days of DSS developed acute inflammation characterized by decreased body weight,loose feces/diarrhea and visible fecal blood.The length of colon was significantly shortened.The ratio between colon length and colon weight and the spleen weight(%of body weight) increased compared with the control mice.The microscopic score is 6.0±0.3 and 1.0±0.4 at DSS-induced colitis mice and control mice(p＜0.05).The baseline peak frequency of all the single units in colitis segment was significantly higher in colitis segment(11.00±3:2.44 imp/second) than in control tissue(5.00±0.78 imp /second,P＜0.05).Pre-treatment with doxtranzole or naproxen did not change the hyper-spontaneous firing.In colitis segments,single unit afferent nerve discharge to bradykinin(0.5μM) was increased by 47.17±6.71(28 single units) compared to 22.67±5.83 imp/second(26 single units) in recordings from uninflamed control tissue (P＜0.05).Afferent discharge was similar to control levels following administration of either doxantrazole or naproxen(23.17±2.34 in 32 single units and 31.17±4.96 imp/second in 28 single units,P＜0.05 compared to untreated colitis segments).Mechanosensitivity during luminal ramp distension(0 to 80 cmH2O) was enhanced reaching 23.50±3:4.77 imp/second at 80 cmH2O during colitis compared to 13.5±1.59 in uninflamed controls(P＜0.05).Adding doxantrazole or naproxen to the organ bath during recordings from colitis segments,reduced afferent discharge to 13.17±0.87 and 13.83±3.07 imp/second at 80 cmH2O which was reduced compared to untreated colitis segment(P＜0.05).Conclusions:In this colitis model of inflammatory bowel disease,mesenteric afferents were sensitized to bradykinin and mechanical stimuli.The underlying mechanism responsible for this sensitisation seems to involve mast cell mediators and prostanoids. |
PDF Full Text Request