肠炎后肠系膜传入神经敏感性变化及其机制的研究
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摘要
肠炎后肠系膜传入神经敏感性变化及其机制的研究
第一部分:正常小鼠空肠肠系膜传入神经对化学以及机械刺激的敏感性
目的:
内脏传入神经高敏感与功能性肠病和炎性肠病引起的的临床症状有关。迷走神经和脊神经传入纤维末梢对肠道局部的机械和化学刺激敏感,可以把肠道局部的感觉信息传入中枢。在本部分实验中我们建立离体记录小鼠肠系膜传入神经放电的实验装置,观察小鼠肠系膜传入神经对5-羟色胺、缓激肽以及机械刺激的放电反应,制备肠系膜传入神经对5-羟色胺和缓激肽反应的剂量效应曲线。
方法:
C57BL/6小鼠,异氟烷吸入麻醉,腹部正中切口,取一段2厘米长带有完整肠系膜的空肠。用特制的由灌流槽和记录槽两部分组成的器官浴槽记录肠系膜传入神经放电。将空肠段放入灌流槽,以克氏液持续灌流空肠(灌流液温度32摄氏度,10毫升/分钟,95%02/5%CO2混合气体持续通气)。空肠两端分别插管固定,插管的头端通过三通分别与微量灌流泵和压力传感器相连,插管的尾端与大气相通,由空肠头端向尾端以10ml/h的速度以克氏液灌流肠腔。经过灌流槽和记录槽之间的孔隙将肠系膜袢拉入记录槽固定,在显微镜下从肠系膜血管束中仔细分离肠系膜神经,将分离好的神经与双极铂金电极的其中一个电极相连,另一个电极连接直径与神经接近的结缔组织。由电极引导出的神经放电信号和压力传感器引导的压力信号分别输入1902单通道放大器,放大后的电信号以及肠内压信号均被输入1401 A/D转换器,1401 A/D转换器直接与记录电脑相连,将信号输入电脑中的Spike2软件显示电信号和肠蠕动信号,整个实验过程中在电脑中持续记录,存盘后对原始数据进行下线分析(off-line anaylsis)。
得到稳定的基础放电信号后,分别观察器官浴槽中加入5-羟色胺(125μM、250μM,、500μM)、缓激肽(0.25μM、0.5μM、1μM)和机械压力刺激(肠内压升高60cmH_2O)后肠系膜传入神经放电的改变。对于同一个标本,只加入一个浓度剂量的5-羟色胺和缓激肽,每次实验时随机选择二者的浓度直接加入器管浴槽中。,5-羟色胺和缓激肽的在器官浴槽内的作用时间为两分钟。升高肠内压时,将肠腔内灌流管的尾端夹闭,而头端按照原速度持续灌流,经过大约90秒后肠内压升高60cmH_2O。施加刺激的顺序为5-羟色胺、升高肠内压和缓激肽。两次刺激之间至少有15分钟的平衡时间,待神经基础放电稳定后再施行下一个刺激。
根据动作电位的波形和波幅在Spike2软件的帮助下可以将神经放电的信号与干扰信号分开,分别计算每秒钟的最大放电频率(峰放电频率)以及刺激作用持续时间内的平均放电频率。以施加刺激前两分钟的放电频率为基础放电频率,加入5-羟色胺和缓激肽后神经放电的改变分别以最大峰放电频率的增加以及加药后30秒、1分钟以及两分钟内平均放电频率的增加表示。肠内压升高时,放电频率的改变以肠内压每升高10cmH_2O时峰放电频率和平均放电频率的增加表示。所有数据以mean±SEM表示,各组之间行单因素方差分析。
结果:
传入神经表现为自发性的连续放电,电信号包括不同波幅和波形的动作电位,施加刺激前2分钟各组之间基础放电频率无差异。
1.5-羟色胺加入器官浴槽后,传入神经放电频率迅速上升,未看到明显潜伏期。传入神经峰放电频率和正常相比分别增加42±4.4 imp/second(125μM),64±4.0 imp/second(250μM),76±2.6 imp/second(500μM)。器官浴槽内5-羟色胺终浓度为125μM时,平均放电频率在加药30秒后增加24.6±2.7 imp/second,1分钟后增加20.4±2.2 imp/second,2分钟后增加12.5±2.3 imp/second。终浓度为250μM时,平均放电频率加药30秒后增加29.1±3.2 imp/second,1分钟后增加25.9±2.7 imp/second,2分钟后增加18.3±3.6 imp/second。终浓度为500μM时,平均放电频率30秒后增加35.3±3.2 imp/second,1分钟后增加29.5±1.9 imp/second,2分钟后增加21.2±2.1 imp/second。不同浓度的5-HT以及相同浓度不同作用时间5-HT诱导的传入神经放电频率的增加均有显著性差异。
2.肠内压升高时传入神经有压力依赖性的放电频率增加,肠内压升高60cmH_2O时,峰放电频率比基础放电增加80±3.3 imp/second,肠内压升高从50cmH_2O到60cmH_2O的持续时间内平均放电频率增加70.8±5.4 imp/second。
3.器官浴槽中加入缓激肽后,传入神经放电频率显著增加。缓激肽作用后传入神经峰放电频率和正常相比增加65±2.7 imp/second(0.25μM),89±7.3 imp/second(0.5μM),112±8.9 imp/second(1μM)。缓激肽浓度为0.25μM时,平均放电频率在加药30秒后增加44.8±5.imp/second,1分钟后增加32.5±3.2 imp/second,2分钟后增加20.3±3.3 imp/second。终浓度时0.5μM时,平均放电频率30秒后增加53.8±6.1 imp/second,1分钟后增加40.7±3.1 imp/second,2分钟后增加24.6±2.2 imp/second。终浓度是1μM时,平均放电频率30秒后增加62.9±4.9 imp/second,1分钟后增加45.2±2.2 imp/second,2分钟后增加28.6±2.4 imp/second。
结论:
建立了离体记录小鼠肠系膜传入神经放电的模型。传入神经放电表现压力依赖性的增加。5-羟色胺和缓激肽可以直接作用于传入神经末梢的受体刺激浆膜侧传入神经,放电频率的增加呈现剂量依赖性模式。
第二部分:吲哚美辛诱导肠炎后小鼠空肠肠系膜传入神经敏感性的改变及其机制
目的:
有些慢性肠炎病人虽然形态学检查显示肠粘膜有广泛的炎性表现,但却没有明显的腹痛症状。在本部分实验中我们选择吲哚美辛诱导的炎性肠病模型,观察肠炎后空肠肠系膜传入神经敏感性的改变,并进一步探讨其机制。
方法:
C57BL/6小鼠,分别在实验的第一天和第二天早上8点皮下注射吲哚美辛两次(60 mg kg~(-1))诱导肠炎,对照组接受相同容积的酒精注射。实验的第三天早上9点,动物在异氟烷麻醉状态下做腹正中切口,肉眼观察肠系膜粘附、肠道长度、肠充血以及溃疡形成情况,对炎症程度行大体评分。制备空肠石蜡切片,常规HE染色后镜下进行炎症评分。取2厘米长带有完整肠系膜的空肠置于器官浴槽中,分离肠系膜神经记录传入神经放电。引导方法与第一部分相同,在记录肠系膜神经放电的同时观察肠蠕动性的改变。实验标本至少平衡15分钟待信号稳定后才正式开始实验,施加刺激顺序为5-羟色胺(250μM)、升高肠内压(60cmH_2O)和缓激肽(0.5μM)。实验分为四组,分别记录施加化学和机械刺激后肠系膜传入神经放电的改变(n=6)。1)吲哚美辛注射诱导肠炎组;2)注射酒精的对照组;3)皮下注射吲哚美辛诱导肠炎,同时慢性腹腔注射诱导性一氧化氮合酶抑制剂L-N6-(1-iminoethyl)-lysine(L-NIL)(3 mgkg~(-1),1次/12小时,注射5次);4)吲哚美辛诱导肠炎,施加化学和机械刺激前10分钟在浴槽中灌流含有L-NIL(30μM)的克氏液。记录浆膜侧施加5-羟色胺、缓激肽以及肠内压升高时传入神经峰放电频率的增加。施加刺激前两分钟的峰放电频率作为基础放电频率。所有数据以mean±SEM表示,各组数据之间用单因素的方差分析进行统计学分析(肠蠕动和化学刺激诱导神经放电数据),不同实验组肠内压变化引起传入神经放电频率的增加用双因素方差分析,P＜0.05认为有显著性差异。
结果:
1.吲哚美辛注射后,小肠呈急性肠炎改变:肠腔扩张,小肠明显缩短,肠袢粘连明显。各组的大体评分分别为对照组0.2±0.20,对照组加L-NIL腹腔注射组0.3±0.33,单纯炎症组4.8±0.74(P＜0.05,vs vehiclegroup),炎症加腹腔注射L-NIL组4.0±0.71(P＜0.05,versus vehiclegroup)。组织学评分的结果与大体评分类似,对照组1.6±0.60,对照加L-NIL腹腔注射组2.0±0.58,炎症组4.6±0.51(P＜0.05,versus vehiclegroup),炎症加腹腔注射L-NIL组4.0±1.08(P＜0.05,versus vehiclegroup)。
2.肠炎时肠道自发性收缩幅度和对照相比明显下降(炎症组:2.51±0.3cmH_2O,对照组:0.66±0.1 cmH_2O;P＜0.01),炎症后肠道失去正常的位相性收缩模式。
3.各实验组间基础峰放电频率无显著性差异。
5-羟色胺诱导对照组空肠肠系膜传入神经最大放电频率增加65±7.5imp/second,而炎症组传入神经峰放电频率的增加下降至32±3.3imp/second(P＜0.05)。经L-NIL预处理后,吲哚美辛诱导肠炎后导致的传入神经对5-羟色胺反应性的下调被完全扭转,炎症加慢性L-NIL腹腔注射后峰放电频率的增加为55±7.5 imp/second,炎症加急性L-NIL器官浴槽内灌流峰放电频率的增加为73±7.2 imp/second。
与对照组相比,炎症组空肠肠系膜传入神经的机械敏感性在肠内压高于20 cmH_2O时明显下调(P＜0.05)。肠内压增加到最高值(60 cmH_2O)时炎症组传入神经放电频率的增加为38±3.0 imp/ssecond,而对照组为95±5.9imp/second(P＜0.05)。腹腔内慢性注射L-NIL或者器官浴槽内L-NIL灌流均可以扭转炎症后传入神经机械敏感性的下降。
浆膜侧施加缓激肽后,炎症标本的最大峰放电频率增加55±7.9imp/second,和对照组相比明显下降(97±7 imp/second,P＜0.05)。炎症后传入神经对缓激肽反应的低敏感性可以被腹腔注射L-NIL(112±16imp/second)或者器官浴槽内急性应用L-NIL(108±13.7 imp/second)扭转。
结论:
肠炎时肠系膜传入神经表现出诱导性一氧化氮合酶依赖性的敏感性下调,而这种敏感性的下调看起来不依赖于炎症反应本身。提示肠炎时诱导性一氧化氮合酶依赖性的一氧化氮生成可以通过直接影响传入神经的信号转导来调节神经敏感性。
第三部分:DSS诱导结肠炎后结肠肠系膜传入神经敏感性的改变及其机制
目的:
肠炎可以诱导支配肠道的肠外在传入神经的敏感化。本部分的研究中我们利用炎症性肠病中的大肠炎模型研究肠炎时结肠传入神经敏感性的改变及其机制。我们推测结肠炎时结肠传入神经的敏感性增加,肥大细胞和环加氧酶可能参与了这一过程。
方法:
C57BL/6小鼠以3%右旋糖酐硫酸酯钠(dextran-sulfate sodium,DSS)代水饮用,连续饮用7天诱导大肠炎,对照组正常饮水。第八天将动物麻醉,根据疾病活性指数对炎症进行大体评分(体重、结肠长度、结肠重量/长度比值、脾脏重量、粪便连续性、是否有血便)。切除一段带有完整肠系膜的近端结肠用于传入神经放电记录,与其相毗邻的一段结肠制备石蜡切片,常规HE染色,在显微镜下行组织学评分。将行电生理学记录的近端结肠置于器官浴槽中,以克氏液持续灌流(含有1μM尼莫地平,抑制肠收缩)。肠系膜传入神经的引导方法与第一部分类似。待放电信号稳定后,先后施加机械刺激(肠腔扩张)和化学刺激(0.5μM),观察各实验组动物传入神经放电敏感性的改变。对照组肠段仅用克氏液灌流,来自大肠炎组的结肠标本分别用克氏液、含有肥大细胞稳定剂Doxantranzole(10~(-4)M)或者含有环加氧酶抑制剂萘普生(10~(-5)M)的克氏液灌流(每组样本数均为6)。在Spike2软件的帮助下,根据神经放电动作电位的不同波形和波幅,可以进一步对全神经放电信号进行离线分析,分解出单根神经纤维,并记录分离出的所有神经纤维的刺激前后总的放电频率,计算施加机械和化学刺激后各实验组传入神经最大放电频率的改变。所有数据以mean±SEM表示,各组数据之间以单因素方差分析进行统计学处理,P＜0.05为有显著性差异。
结果:
1.饮用7天DSS后,小鼠有明显的急性肠炎改变,体重下降、粪便变松软或者腹泻、有肉眼可见的血便。结肠明显缩短、结肠长度/重量比值以及脾脏重量(占体重的百分比)明显增加。炎症组和对照组的炎症镜下评分分别为6.0±0.3和1.0±0.4(P＜0.05)。
2.结肠炎组的结肠肠系膜传入神经的基础自发性放电频率比对照组明显升高(炎症组:最大放电频率11.00±2.44 imp/second,正常组:最大放电频率5.00±0.78 imp/second,P＜0.05)。用Doxantranzole或者萘普生预处理炎症后标本,对基础放电无影响。
3.结肠炎标本,共分离出28条单根神经纤维,应用缓激肽后28条神经纤维的最大放电频率增加47.17±6.7limp/second,而正常组分离出26条神经纤维,最大峰放电频率增加22.67±5.8 imp/second,明显低于炎症组(P＜0.05)。炎症组标本用Doxantrazole或者萘普生预处理后,最大峰放电频率的增加和正常组相比不再有显著性差异(Doxantrazole预处理后:23.17±2.34imp/second,分离出32条神经纤维;萘普生预处理后,31.17±4.96 imp/secind,分离出28条神经纤维)。
肠炎时肠系膜传入神经的机械敏感性明显升高,肠内压升高80 cmH_2O时,炎症组和正常组最大峰放电频率的增加分别为23.5±4.77 imp/second和13.5±1.59imp/second(P＜0.05)。用Doxantrazole或者萘普生预处理结肠炎标本后,肠内压升高到80 cmH_2O时,最大峰放电频率的增加下降至13.17±0.87imp/second和13.83±3.07imp/second(P＜0.05,versus单纯炎症组标本)。
结论:
炎症性肠病的大肠炎模型中,肠系膜传入神经对缓激肽和机械刺激的敏感性上调,敏感性的增加揭示了炎性肠病和肠易激综合征患者腹痛的外周机制。肥大细胞介质和环加氧酶途径产生的前列腺素类物质具有上调传入神经敏感性的作用。
PartⅠ:Jejunal afferent nerve sensitivity to chemical and mechanical stimuli in C57BL/6 mice in vitro
Objective:
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 O_2/CO_2 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 cmH_2O 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 H_2O increments of intraluminal pressure until 60 cmH_2O 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 cmH_2O,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.
PartⅡ:Jejunal afferent nerve sensitivity change and mechanism during indomethaicn induced inflammtion
Objective:
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 cmH_2O 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 cmH_2O (P＜0.05).At the maximum pressure of 60 cmH_2O,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.
PartⅢ: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~(-5)M) 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 cmH_2O) was enhanced reaching 23.50±3:4.77 imp/second at 80 cmH_2O 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.
第一部分:正常小鼠空肠肠系膜传入神经对化学以及机械刺激的敏感性
目的:
内脏传入神经高敏感与功能性肠病和炎性肠病引起的的临床症状有关。迷走神经和脊神经传入纤维末梢对肠道局部的机械和化学刺激敏感,可以把肠道局部的感觉信息传入中枢。在本部分实验中我们建立离体记录小鼠肠系膜传入神经放电的实验装置,观察小鼠肠系膜传入神经对5-羟色胺、缓激肽以及机械刺激的放电反应,制备肠系膜传入神经对5-羟色胺和缓激肽反应的剂量效应曲线。
方法:
C57BL/6小鼠,异氟烷吸入麻醉,腹部正中切口,取一段2厘米长带有完整肠系膜的空肠。用特制的由灌流槽和记录槽两部分组成的器官浴槽记录肠系膜传入神经放电。将空肠段放入灌流槽,以克氏液持续灌流空肠(灌流液温度32摄氏度,10毫升/分钟,95%02/5%CO2混合气体持续通气)。空肠两端分别插管固定,插管的头端通过三通分别与微量灌流泵和压力传感器相连,插管的尾端与大气相通,由空肠头端向尾端以10ml/h的速度以克氏液灌流肠腔。经过灌流槽和记录槽之间的孔隙将肠系膜袢拉入记录槽固定,在显微镜下从肠系膜血管束中仔细分离肠系膜神经,将分离好的神经与双极铂金电极的其中一个电极相连,另一个电极连接直径与神经接近的结缔组织。由电极引导出的神经放电信号和压力传感器引导的压力信号分别输入1902单通道放大器,放大后的电信号以及肠内压信号均被输入1401 A/D转换器,1401 A/D转换器直接与记录电脑相连,将信号输入电脑中的Spike2软件显示电信号和肠蠕动信号,整个实验过程中在电脑中持续记录,存盘后对原始数据进行下线分析(off-line anaylsis)。
得到稳定的基础放电信号后,分别观察器官浴槽中加入5-羟色胺(125μM、250μM,、500μM)、缓激肽(0.25μM、0.5μM、1μM)和机械压力刺激(肠内压升高60cmH_2O)后肠系膜传入神经放电的改变。对于同一个标本,只加入一个浓度剂量的5-羟色胺和缓激肽,每次实验时随机选择二者的浓度直接加入器管浴槽中。,5-羟色胺和缓激肽的在器官浴槽内的作用时间为两分钟。升高肠内压时,将肠腔内灌流管的尾端夹闭,而头端按照原速度持续灌流,经过大约90秒后肠内压升高60cmH_2O。施加刺激的顺序为5-羟色胺、升高肠内压和缓激肽。两次刺激之间至少有15分钟的平衡时间,待神经基础放电稳定后再施行下一个刺激。
根据动作电位的波形和波幅在Spike2软件的帮助下可以将神经放电的信号与干扰信号分开,分别计算每秒钟的最大放电频率(峰放电频率)以及刺激作用持续时间内的平均放电频率。以施加刺激前两分钟的放电频率为基础放电频率,加入5-羟色胺和缓激肽后神经放电的改变分别以最大峰放电频率的增加以及加药后30秒、1分钟以及两分钟内平均放电频率的增加表示。肠内压升高时,放电频率的改变以肠内压每升高10cmH_2O时峰放电频率和平均放电频率的增加表示。所有数据以mean±SEM表示,各组之间行单因素方差分析。
结果:
传入神经表现为自发性的连续放电,电信号包括不同波幅和波形的动作电位,施加刺激前2分钟各组之间基础放电频率无差异。
1.5-羟色胺加入器官浴槽后,传入神经放电频率迅速上升,未看到明显潜伏期。传入神经峰放电频率和正常相比分别增加42±4.4 imp/second(125μM),64±4.0 imp/second(250μM),76±2.6 imp/second(500μM)。器官浴槽内5-羟色胺终浓度为125μM时,平均放电频率在加药30秒后增加24.6±2.7 imp/second,1分钟后增加20.4±2.2 imp/second,2分钟后增加12.5±2.3 imp/second。终浓度为250μM时,平均放电频率加药30秒后增加29.1±3.2 imp/second,1分钟后增加25.9±2.7 imp/second,2分钟后增加18.3±3.6 imp/second。终浓度为500μM时,平均放电频率30秒后增加35.3±3.2 imp/second,1分钟后增加29.5±1.9 imp/second,2分钟后增加21.2±2.1 imp/second。不同浓度的5-HT以及相同浓度不同作用时间5-HT诱导的传入神经放电频率的增加均有显著性差异。
2.肠内压升高时传入神经有压力依赖性的放电频率增加,肠内压升高60cmH_2O时,峰放电频率比基础放电增加80±3.3 imp/second,肠内压升高从50cmH_2O到60cmH_2O的持续时间内平均放电频率增加70.8±5.4 imp/second。
3.器官浴槽中加入缓激肽后,传入神经放电频率显著增加。缓激肽作用后传入神经峰放电频率和正常相比增加65±2.7 imp/second(0.25μM),89±7.3 imp/second(0.5μM),112±8.9 imp/second(1μM)。缓激肽浓度为0.25μM时,平均放电频率在加药30秒后增加44.8±5.imp/second,1分钟后增加32.5±3.2 imp/second,2分钟后增加20.3±3.3 imp/second。终浓度时0.5μM时,平均放电频率30秒后增加53.8±6.1 imp/second,1分钟后增加40.7±3.1 imp/second,2分钟后增加24.6±2.2 imp/second。终浓度是1μM时,平均放电频率30秒后增加62.9±4.9 imp/second,1分钟后增加45.2±2.2 imp/second,2分钟后增加28.6±2.4 imp/second。
结论:
建立了离体记录小鼠肠系膜传入神经放电的模型。传入神经放电表现压力依赖性的增加。5-羟色胺和缓激肽可以直接作用于传入神经末梢的受体刺激浆膜侧传入神经,放电频率的增加呈现剂量依赖性模式。
第二部分:吲哚美辛诱导肠炎后小鼠空肠肠系膜传入神经敏感性的改变及其机制
目的:
有些慢性肠炎病人虽然形态学检查显示肠粘膜有广泛的炎性表现,但却没有明显的腹痛症状。在本部分实验中我们选择吲哚美辛诱导的炎性肠病模型,观察肠炎后空肠肠系膜传入神经敏感性的改变,并进一步探讨其机制。
方法:
C57BL/6小鼠,分别在实验的第一天和第二天早上8点皮下注射吲哚美辛两次(60 mg kg~(-1))诱导肠炎,对照组接受相同容积的酒精注射。实验的第三天早上9点,动物在异氟烷麻醉状态下做腹正中切口,肉眼观察肠系膜粘附、肠道长度、肠充血以及溃疡形成情况,对炎症程度行大体评分。制备空肠石蜡切片,常规HE染色后镜下进行炎症评分。取2厘米长带有完整肠系膜的空肠置于器官浴槽中,分离肠系膜神经记录传入神经放电。引导方法与第一部分相同,在记录肠系膜神经放电的同时观察肠蠕动性的改变。实验标本至少平衡15分钟待信号稳定后才正式开始实验,施加刺激顺序为5-羟色胺(250μM)、升高肠内压(60cmH_2O)和缓激肽(0.5μM)。实验分为四组,分别记录施加化学和机械刺激后肠系膜传入神经放电的改变(n=6)。1)吲哚美辛注射诱导肠炎组;2)注射酒精的对照组;3)皮下注射吲哚美辛诱导肠炎,同时慢性腹腔注射诱导性一氧化氮合酶抑制剂L-N6-(1-iminoethyl)-lysine(L-NIL)(3 mgkg~(-1),1次/12小时,注射5次);4)吲哚美辛诱导肠炎,施加化学和机械刺激前10分钟在浴槽中灌流含有L-NIL(30μM)的克氏液。记录浆膜侧施加5-羟色胺、缓激肽以及肠内压升高时传入神经峰放电频率的增加。施加刺激前两分钟的峰放电频率作为基础放电频率。所有数据以mean±SEM表示,各组数据之间用单因素的方差分析进行统计学分析(肠蠕动和化学刺激诱导神经放电数据),不同实验组肠内压变化引起传入神经放电频率的增加用双因素方差分析,P＜0.05认为有显著性差异。
结果:
1.吲哚美辛注射后,小肠呈急性肠炎改变:肠腔扩张,小肠明显缩短,肠袢粘连明显。各组的大体评分分别为对照组0.2±0.20,对照组加L-NIL腹腔注射组0.3±0.33,单纯炎症组4.8±0.74(P＜0.05,vs vehiclegroup),炎症加腹腔注射L-NIL组4.0±0.71(P＜0.05,versus vehiclegroup)。组织学评分的结果与大体评分类似,对照组1.6±0.60,对照加L-NIL腹腔注射组2.0±0.58,炎症组4.6±0.51(P＜0.05,versus vehiclegroup),炎症加腹腔注射L-NIL组4.0±1.08(P＜0.05,versus vehiclegroup)。
2.肠炎时肠道自发性收缩幅度和对照相比明显下降(炎症组:2.51±0.3cmH_2O,对照组:0.66±0.1 cmH_2O;P＜0.01),炎症后肠道失去正常的位相性收缩模式。
3.各实验组间基础峰放电频率无显著性差异。
5-羟色胺诱导对照组空肠肠系膜传入神经最大放电频率增加65±7.5imp/second,而炎症组传入神经峰放电频率的增加下降至32±3.3imp/second(P＜0.05)。经L-NIL预处理后,吲哚美辛诱导肠炎后导致的传入神经对5-羟色胺反应性的下调被完全扭转,炎症加慢性L-NIL腹腔注射后峰放电频率的增加为55±7.5 imp/second,炎症加急性L-NIL器官浴槽内灌流峰放电频率的增加为73±7.2 imp/second。
与对照组相比,炎症组空肠肠系膜传入神经的机械敏感性在肠内压高于20 cmH_2O时明显下调(P＜0.05)。肠内压增加到最高值(60 cmH_2O)时炎症组传入神经放电频率的增加为38±3.0 imp/ssecond,而对照组为95±5.9imp/second(P＜0.05)。腹腔内慢性注射L-NIL或者器官浴槽内L-NIL灌流均可以扭转炎症后传入神经机械敏感性的下降。
浆膜侧施加缓激肽后,炎症标本的最大峰放电频率增加55±7.9imp/second,和对照组相比明显下降(97±7 imp/second,P＜0.05)。炎症后传入神经对缓激肽反应的低敏感性可以被腹腔注射L-NIL(112±16imp/second)或者器官浴槽内急性应用L-NIL(108±13.7 imp/second)扭转。
结论:
肠炎时肠系膜传入神经表现出诱导性一氧化氮合酶依赖性的敏感性下调,而这种敏感性的下调看起来不依赖于炎症反应本身。提示肠炎时诱导性一氧化氮合酶依赖性的一氧化氮生成可以通过直接影响传入神经的信号转导来调节神经敏感性。
第三部分:DSS诱导结肠炎后结肠肠系膜传入神经敏感性的改变及其机制
目的:
肠炎可以诱导支配肠道的肠外在传入神经的敏感化。本部分的研究中我们利用炎症性肠病中的大肠炎模型研究肠炎时结肠传入神经敏感性的改变及其机制。我们推测结肠炎时结肠传入神经的敏感性增加,肥大细胞和环加氧酶可能参与了这一过程。
方法:
C57BL/6小鼠以3%右旋糖酐硫酸酯钠(dextran-sulfate sodium,DSS)代水饮用,连续饮用7天诱导大肠炎,对照组正常饮水。第八天将动物麻醉,根据疾病活性指数对炎症进行大体评分(体重、结肠长度、结肠重量/长度比值、脾脏重量、粪便连续性、是否有血便)。切除一段带有完整肠系膜的近端结肠用于传入神经放电记录,与其相毗邻的一段结肠制备石蜡切片,常规HE染色,在显微镜下行组织学评分。将行电生理学记录的近端结肠置于器官浴槽中,以克氏液持续灌流(含有1μM尼莫地平,抑制肠收缩)。肠系膜传入神经的引导方法与第一部分类似。待放电信号稳定后,先后施加机械刺激(肠腔扩张)和化学刺激(0.5μM),观察各实验组动物传入神经放电敏感性的改变。对照组肠段仅用克氏液灌流,来自大肠炎组的结肠标本分别用克氏液、含有肥大细胞稳定剂Doxantranzole(10~(-4)M)或者含有环加氧酶抑制剂萘普生(10~(-5)M)的克氏液灌流(每组样本数均为6)。在Spike2软件的帮助下,根据神经放电动作电位的不同波形和波幅,可以进一步对全神经放电信号进行离线分析,分解出单根神经纤维,并记录分离出的所有神经纤维的刺激前后总的放电频率,计算施加机械和化学刺激后各实验组传入神经最大放电频率的改变。所有数据以mean±SEM表示,各组数据之间以单因素方差分析进行统计学处理,P＜0.05为有显著性差异。
结果:
1.饮用7天DSS后,小鼠有明显的急性肠炎改变,体重下降、粪便变松软或者腹泻、有肉眼可见的血便。结肠明显缩短、结肠长度/重量比值以及脾脏重量(占体重的百分比)明显增加。炎症组和对照组的炎症镜下评分分别为6.0±0.3和1.0±0.4(P＜0.05)。
2.结肠炎组的结肠肠系膜传入神经的基础自发性放电频率比对照组明显升高(炎症组:最大放电频率11.00±2.44 imp/second,正常组:最大放电频率5.00±0.78 imp/second,P＜0.05)。用Doxantranzole或者萘普生预处理炎症后标本,对基础放电无影响。
3.结肠炎标本,共分离出28条单根神经纤维,应用缓激肽后28条神经纤维的最大放电频率增加47.17±6.7limp/second,而正常组分离出26条神经纤维,最大峰放电频率增加22.67±5.8 imp/second,明显低于炎症组(P＜0.05)。炎症组标本用Doxantrazole或者萘普生预处理后,最大峰放电频率的增加和正常组相比不再有显著性差异(Doxantrazole预处理后:23.17±2.34imp/second,分离出32条神经纤维;萘普生预处理后,31.17±4.96 imp/secind,分离出28条神经纤维)。
肠炎时肠系膜传入神经的机械敏感性明显升高,肠内压升高80 cmH_2O时,炎症组和正常组最大峰放电频率的增加分别为23.5±4.77 imp/second和13.5±1.59imp/second(P＜0.05)。用Doxantrazole或者萘普生预处理结肠炎标本后,肠内压升高到80 cmH_2O时,最大峰放电频率的增加下降至13.17±0.87imp/second和13.83±3.07imp/second(P＜0.05,versus单纯炎症组标本)。
结论:
炎症性肠病的大肠炎模型中,肠系膜传入神经对缓激肽和机械刺激的敏感性上调,敏感性的增加揭示了炎性肠病和肠易激综合征患者腹痛的外周机制。肥大细胞介质和环加氧酶途径产生的前列腺素类物质具有上调传入神经敏感性的作用。
PartⅠ:Jejunal afferent nerve sensitivity to chemical and mechanical stimuli in C57BL/6 mice in vitro
Objective:
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 O_2/CO_2 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 cmH_2O 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 H_2O increments of intraluminal pressure until 60 cmH_2O 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 cmH_2O,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.
PartⅡ:Jejunal afferent nerve sensitivity change and mechanism during indomethaicn induced inflammtion
Objective:
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 cmH_2O 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 cmH_2O (P＜0.05).At the maximum pressure of 60 cmH_2O,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.
PartⅢ: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~(-5)M) 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 cmH_2O) was enhanced reaching 23.50±3:4.77 imp/second at 80 cmH_2O 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.
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