【作者】 杨静；

【导师】 李延青；

【作者基本信息】 山东大学 ， 内科学， 2011， 博士

【摘要】 背景炎症性肠病(Inflammatory bowel disease, IBD)和肠易激综合征(Irritable bowel syndrome, IBS)患者常伴有反复发作性腹痛,严重影响患者的生活质量。目前认为内脏高敏感是IBD和IBS患者腹痛发生的主要病理生理机制,其发生机制尚有待明确。内脏感觉通过胞体位于脊髓背根神经节(dorsal root ganglia, DRG)的脊髓内脏传入纤维传递到次级感觉神经元,进而投射到中枢神经系统。内脏敏感性的变化主要可归结为两个因素,外周敏感化和中枢敏感化。各种原因引起的感觉神经末梢和初级传入神经元的致敏可以诱导中枢敏感化。临床上约1/3的IBS患者可追溯到有既往肠道感染史,这类病人称为感染后肠易激综合征(post-infectious IBS, PI-IBS).急性肠道感染后可遗留感觉、运动等肠道功能紊乱。对PI-IBS患者的临床研究发现其结肠标本上皮通透性增加,炎性细胞和炎症介质增多。炎症对感觉神经元具致敏作用。炎症后肠传入神经的致敏可能在IBD和PI-IBS患者内脏高敏感的发生发展中起到重要作用。明确参与这一致敏机制的关键分子,将为临床治疗提供新的靶点,从而有助于腹痛症状的缓解。脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)是神经营养物质家族中的一员,在中枢和外周神经系统均有表达,既往研究认为,BDNF可调控突触可塑性和维持神经内环境的稳定,进而参与学习、记忆等多项功能。近年来研究发现BDNF也可参与调控痛觉的传导。BDNF可在DRG细胞中表达,在多种炎症性躯体痛模型中证实BDNF表达上调,应用BDNF抗体可抑制躯体痛觉过敏。BDNF不仅在神经系统分布,在外周也广泛表达,研究报道BDNF可分布于心脏、肺、膀胱和结肠等多个内脏器官的上皮层,但BDNF的受体TrkB和P75受体不在上皮层表达,而是选择性表达在神经系统内。因此推测内脏上皮细胞合成的内源性BDNF通过作用于支配内脏的神经系统进行调节。本研究中,我们首次结合BDNF基因敲除小鼠探讨BDNF对小鼠内脏敏感性的影响。并应用结肠内灌注三硝基苯磺酸(trinitro benzene sulfonic acid, TNBS),诱导建立内脏高敏感,进一步分析BDNF是否参与炎症后肠传入神经致敏。目的研究脑源性神经营养因子在肠炎后结肠高敏感中的作用。方法1.实验动物分组本研究所用实验动物根据BDNF基因型和是否造成炎症状态共分为四组,BDNF+/+(野生型)/生理盐水对照组,BDNF+/+/TNBS炎症组,BDNF+/-/生理盐水对照组,BDNF+/-/TNBS炎症组。2.三硝基苯磺酸灌肠(trinitro benzene sulfonic acid, TNBS)造结肠炎模型：小鼠灌肠前24小时禁食不禁水,0.1mL 1.75mg经肛门注入降结肠,导管末端距肛门4cm,结束后小鼠保持倒立姿势30s。对照组应用同等体积的生理盐水。3.结肠病理组织学检查：小鼠灌肠7天后,过量麻醉处死,取降结肠远端3cm的一半,甲醛固定,石蜡包埋后行HE染色和组织学评分。4.髓过氧化物酶(myeloperoxidase, MPO)活性检测：使用部分降结肠远端标本,称重、匀浆,按照MPO试剂盒说明操作。5.酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA):取支配结肠的胸腰部(thoracolumbar, TL)DRG (T10-L1)和腰骶部Lumbosacral, LS)DRG(L6-S1)和部分降结肠远端标本,裂解并蛋白定量后,按照ELISA试剂盒的说明操作。BDNF浓度表示为ng/g全蛋白。6.直结肠扩张(Colorectal distension,CRD)法检测结肠敏感性。小鼠乙醚轻度麻醉后,将特制的2cm长度球囊轻插入降结肠,球囊近端距肛门约0.5cm。(1)当小鼠充分苏醒并适应后,分别在0,15,30,45,60,80mmHg压力下进行结肠球囊扩张。每次扩张持续20s,间隔4分钟。在递增的压力下,记录各个压力时的腹壁收缩反射(abdominal withdrawal response, AWR)评分。所有测量均重复3次。AWR评分系统：0：对球囊扩张无反应；1：身体静止不动或头部运动减少；2：腹部肌肉收缩；3：腹部抬高；4：骨盆抬起,身体呈弓形。(2)连接球囊的导管和压力表、注射器扎紧,通过三通管连接。注射器内抽入空气,当小鼠充分苏醒并适应5分钟后,注射空气,逐渐增加压力以测量初始感觉阈值和疼痛阂值,所有测量均重复3次。小鼠接受直肠扩张时,如身体静止不动,头部运动减少时,记录此压力值,为小鼠的初始感觉阈值。如腹部肌肉收缩时,记录此压力值,为小鼠的疼痛阈值。结果1.对于不同基因型的小鼠,TNBS均可诱导明显的结肠炎症,组织学切片表现为节段性溃疡形成,肠黏膜中度充血水肿,大量炎性白细胞浸润和部分上皮坏死脱落。2.组织学评分：BDNF+/+/TNBS炎症组与BDNF+/-/TNBS炎症组的组织学评分无显著性差异。3.髓过氧化物酶(MPO)活性评分,BDNF+/+/TNBS炎症组与BDNF+/-/TNBS炎症组之间无显著性差异。4. ELISA法检测支配结肠的背根DRG内BDNF表达水平(1)非炎症状态下,BDNF+/-小鼠的TL与LS DRG的BDNF表达水平均约为野生型一半。(2) TNBS诱导的炎症导致不同基因型小鼠炎症后TL与LS DRG内BDNF蛋白表达水平均显著性升高。(3) BDNF+/-炎症小鼠上调的BDNF表达水平显著低于野生炎症小鼠上调的BDNF表达水平。(4) BDNF+/-/炎症小鼠BDNF水平仍相对低于野生无炎症小鼠。5. ELISA法检测远端结肠BDNF表达水平(1)非炎症状态下,BDNF+/-小鼠的BDNF表达水平均约为野生型一半。(2) TNBS诱导的炎症导致不同基因型小鼠炎症后结肠组织BDNF蛋白表达水平均显著性升高。(3) BDNF+/-炎症小鼠上调的BDNF表达水平显著低于野生炎症小鼠上调的BDNF表达水平。(4) BDNF+/-/炎症小鼠BDNF水平和野生无炎症小鼠之间无显著性差异。6.CRD检测(1)比较不同扩张压力下小鼠的AWR评分。非炎症状态,在60mmHg以下BDNF+/-小鼠与野生小鼠对球囊扩张的内脏反应性无显著性统计学差异。而在≥60 mmHg以上扩张压力时,BDNF+/-小鼠表现出显著性降低的内脏反应性。在野生小鼠组,TNBS诱导炎症导致内脏反应性明显升高,表现为在≥30mmHg压力时的内脏高反应性,而在<30mmHg压力时炎症组与盐水对照组内脏反应性无显著性差异。在BDNF+/-小鼠组,TNBS诱导炎症也可导致内脏反应性升高,但要在≥45mmHg以上压力时,TNBS炎症方表现出相对于盐水对照组显著性升高的反应性。在<45mmHg压力时BDNF+/-TNBS炎症组与盐水对照组无显著性差异。炎症状态下,BDNF+/-炎症小鼠对球囊扩张后的内脏反应性相对于野生型炎症小鼠显著性降低,但仍高于野生盐水组的内脏反应性。(2)比较各组小鼠的初始感觉阈值和疼痛阈值非炎症状态下,BDNF+/-小鼠和野生小鼠之间无差异。炎症后,BDNF+/-小鼠和野生小鼠相对于其各自的盐水对照组的初始感觉和疼痛阈值均降低。BDNF+/-/TNBS鼠相对于野生/TNBS小鼠的初始感觉阈值和疼痛阈值较高；但仍低于野生盐水对照组小鼠。炎症后BDNF+/-炎症小鼠的内脏敏感性相对于野生型炎症小鼠显著性降低,但仍高于野生无炎症小鼠和BDNF+/-无炎症小鼠的内脏敏感性。提示BDNF的靶向敲除对炎症后内脏高敏感有部分抑制作用。结论1.无炎症状态下,与BDNF+/+小鼠比较,BDNF+/-小鼠结肠和DRG的BDNF表达水平降低,但结肠敏感性无显著性变化,仅在球囊扩张60mmHg以上时BDNF+/-小鼠表现为低反应性。2.应用三硝基苯磺酸建立结肠炎模型,发现炎症后结肠及支配结肠的DRG内BDNF表达增加,同时伴有结肠高敏感。杂合子性BDNF敲除小鼠在肠炎后BDNF表达上调受抑制,同时结肠高敏感受到部分抑制,提示BDNF参与肠炎后结肠高敏感的发生。背景炎症性肠病(Inflammatory bowel disease, IBD和肠易激综合征(Irritable bowel syndrome, IBS)患者常伴有尿频、尿急、排尿痛等膀胱高敏感并发症,这些并发症加重了对患者生活质量的负面影响,但发病机制尚不清楚。此外,临床发现大约三分之一的间质性膀胱炎患者可并发腹痛、腹泻等消化道症状。临床上患者结肠和膀胱功能紊乱的伴发性提示二者可能具有相同或相近的感觉传导通路。目前研究观点认为盆腔脏器功能紊乱的重叠性可能来自于背根神经节(Dorsal root ganglia, DRG)水平的神经元性集合(convergence)和或“对话’’(cross-talk)。支配结肠的DRG位于脊髓T10-L1和L6-S1两侧,支配膀胱的DRG位于脊髓T13-L1和L6-S1两侧,研究发现二者的神经元胞体在T13-L1和L6-S1存在一定比例的重合,重合神经元发出的神经纤维可分叉投射(dichotomizing)到两个不同的脏器,从而提供传入神经纤维的“集合”模式。一脏器因炎症等局部损伤致敏,将信号传入DRG,活化的神经元可将另一支配的脏器信号放大传递至中枢,从而使中枢产生对另一脏器感觉的错误认识。神经性“对话”观点侧重于支配不同脏器的毗邻神经元之间通过神经递质或细胞因子释放导致的交互影响。这一观点已为动物学研究证实,结肠炎可兴奋支配膀胱的DRG神经元,使其钠离子内流增多,表现为膀胱对盐水扩张的耐受性下降。无论神经元“集合”或“对话”观点,相关研究都着重于建立一个脏器的高敏感模型进而探讨参与调节其他脏器牵涉性敏感的关键分子。研究报道结肠炎模型DRG内降钙素基因相关肽(Calcitonin gene-related peptide, CGRP)上调并参与膀胱牵涉性高敏感的调节。脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)可由DRG细胞合成,与CGRP同属神经肽类物质并参与痛觉的调控。我们第一部分的研究证明结肠炎后,结肠和支配结肠的DRG内BDNF的表达上调,与结肠高敏感密切相关。因此我们推测BDNF可能也参与结肠-膀胱牵涉性的高敏感。目的研究脑源性神经营养因子在肠炎后膀胱牵涉性高敏感中的作用。方法1.实验动物分组本研究所用实验动物根据BDNF基因型和是否造成炎症状态共分为四组,BDNF+/+(野生型)/生理盐水对照组,BDNF+/+/TNBS炎症组,BDNF+/-/生理盐水对照组,BDNF+/-/TNBS炎症组。因膀胱敏感性检测需体外暴露性操作,且支配膀胱的DRG节段与结肠不完全相同,本部分研究与第一部分为完全独立的两部分。2.三硝基苯磺酸灌肠(trinitro benzene sulfonic acid, TNBS)造结肠炎模型：小鼠灌肠前24小时禁食不禁水,O.1mL 1.75mg经肛门注入降结肠,导管末端距肛门4cm,结束后小鼠保持倒立姿势30s。对照组应用同等体积的生理盐水。3.膀胱病理组织学检查：小鼠灌肠7天后,过量麻醉处死,取膀胱,一半甲醛固定,石蜡包埋后行HE染色和组织学评分。4.髓过氧化物酶(myeloperoxidase, MPO)活性检测：膀胱组织称重、匀浆,按照MPO试剂盒说明操作。5.酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA):取部分膀胱和支配膀胱的TL DRG(T13-L1)和LS DRG(L6-S1)裂解并蛋白定量后,按照ELISA试剂盒的说明操作。BDNF浓度表示为ng/g全蛋白。6.短时自主性排尿试验灌肠7天后,将小鼠置于底部铺有滤纸的大烧杯中1小时,烧杯口盖以铁丝网,小鼠在滤纸上可自由移动。1小时后,滤纸取出,使用UVP成像系统计数<0.2cm2的小直径排尿点,计算短时自主性排尿的频率。7.膀胱容积性测定小鼠以1.2g/kg的氨基甲酸酯腹腔注射麻醉,PE10导管通过尿道插入小鼠膀胱。预温至37℃的0.9%的生理盐水以每小时1mL的速度将膀胱充盈,直至出现排尿。膀胱容积性被认为是可激发排尿反应的充盈容量。8.膀胱内压测量实验小鼠腹腔注射麻醉,中下腹部逐层剖开,暴露出膀胱,,将PE50导管在膀胱顶部一小切口缓缓插入,以氨基丙烯酸酯粘胶固定。导管另一端通过三通管连接至灌流泵和压力换能器上。2分钟后,预温至37℃的生理盐水以20μl/min的速度持续灌流2小时。(1)排尿间隔时间：记录排尿间的三次间隔时间,以秒为单位。(2)排尿初始压力：通过连接压力换能器,记录开始排尿时的压力。结果1.灌肠结束7天后,TNBS可诱导明显的结肠炎症。但各组小鼠膀胱组织学切片均结构正常完整,无炎症指征,组织学评分均为0。4组小鼠之间膀胱MPO活性无显著性差异。表明TNBS结肠灌注诱导肠炎后,膀胱无炎症表现。2.BDNG在DRG内蛋白表达水平：(1)非炎症状态下,BDNF+/-小鼠支配膀胱的TL与LS DRG的BDNF表达水平均约为野生型一半。(2)TNBS诱导的炎症导致不同基因型小鼠支配膀胱的TL与LS DRG内BDNF蛋白表达水平均显著性升高。(3)炎症后BDNF+/-小鼠上调的BDNF表达水平仍显著低于野生小鼠上调的BDNF表达水平。(4)BDNF+/-/炎症小鼠与野生无炎症小鼠之间BDNF表达水平无显著性差异。3.BDNG在膀胱内蛋白表达水平：(1)非炎症状态下,BDNF+/-小鼠膀胱的BDNF表达水平均约为野生型一半。(2)TNBS诱导的炎症导致不同基因型小鼠膀胱的BDNF蛋白表达水平均显著性升高。(3)炎症后BDNF+/-小鼠上调的BDNF表达水平仍显著低于野生小鼠上调的BDNF表达水平。(4)BDNF+/-/炎症小鼠的BDNF表达水平显著低于野生无炎症小鼠。4.膀胱敏感性测定：(1)非炎症状态下BDNF杂合子小鼠和野生小鼠膀胱敏感性无显著性差异。表现为：短时自主性排尿实验：1小时内滤纸上小直径排尿点计数无显著性差异。膀胱容积性实验：可激发排尿的充盈容量无显著性差异。膀胱内压实验：杂合子小鼠的排尿间隔时间和初始排尿压力在数值上略高于野生鼠,但统计学无显著性差异。结果提示BDNF部分敲除对正常无肠炎状态的膀胱反应性无显著性作用。(2) TNBS诱导炎症导致野生小鼠和杂合子小鼠的膀胱反应性均明显升高,表现为相对于其各自的盐水对照组,肠炎小鼠的短时自主性排尿频率增加,膀胱容积性降低,排尿间隔时间降低和初始排尿压力降低。结果提示TNBS诱导的肠炎可导致明显的膀胱牵涉性高敏感。(3) BDNF部分敲除的肠炎杂合子小鼠膀胱反应性低于肠炎野生小鼠,表现为肠炎杂合子小鼠短时自主性排尿频率较低,膀胱容积性较高,排尿间隔时间较长和初始排尿压力较高。(4)肠炎杂合子小鼠的膀胱反应性仍高于野生盐水对照组小鼠。结果提示BDNF部分敲除对肠炎后的牵涉性膀胱高反应性具部分抑制作用。结论我们应用三硝基苯磺酸建立结肠炎模型,发现肠炎后膀胱无炎症表现,支配膀胱的DRG和膀胱内BDNF表达增加,同时伴有膀胱高敏感。而杂合子性BDNF敲除小鼠在肠炎后BNDF表达上调受到抑制,同时膀胱高敏感受到部分抑制,提示BDNF参与膀胱牵涉性高敏感的发生。背景内脏高敏感被认为是炎症性肠病(Irritable bowel disease, IBD)和感染后肠易激综合征(Post-infectious Irritable bowel syndrome, PI-IBS)患者腹痛发生的重要病理生理机制。研究认为炎症后结肠外周传入神经致敏在IBD和PI-IBS患者内脏高敏感发生中起重要作用。内脏感觉通过胞体位于背根神经节(Dorsal root ganglia, DRG)的脊髓传入神经纤维传导,因此炎症后支配结肠的DRG内分子的变化成为内脏高敏感发生机制研究的重要靶点。TRP (Transient receptor potential)家族因对机械、温度、化学刺激的感知和调节作用近年来备受瞩目。研究表明TRP家族的多个成员如辣椒素受体1(TRP vanilloid-1, TRPV1) and辣椒素受体4 (TRPV4)等广泛参与内脏高敏感性的调控。瞬时感受器电位锚蛋白-1(transient receptor potential ankyrin-1, TRPA1)属于TRP受体超家族一员,选择性表达在感知伤害性刺激的小直径感觉神经元上,并与TRPV1高度共表达。TRPA1被证实参与对伤害性低温和机械性刺激的调控,并可被多种外源性复合物、缓激肽及氧化应激产物等激活。TRPA1的药物性阻断可明显降低组织损伤或炎症带来的躯体高敏感。TRPA1对伤害性刺激的调控及其与TRPV1的共表达提示它在结肠高敏感中或许同样具调控作用。但既往研究都着重于TRPA1在躯体高敏感中的调节作用。本研究首次假设TRPA1受体可能参与结肠炎症后的内脏高敏感。本研究应用三硝基苯磺酸(trinitro benzene sulfonic acid, TNBS)灌肠造结肠炎模型,检测支配肠道的背根神经节内TRPA1蛋白水平及炎症后结肠敏感性的改变。应用TRPA1特异性反义寡核苷酸靶向抑制TRPA1,研究TRPA1表达受抑制后对炎症性内脏高敏感的影响。因TRPA1与TRPV1高度共表达,同时检测靶向抑制TRPA1后TRPV1表达的变化,以明确TRPA1在内脏高敏感中的调节作用。方法1.实验分组：120只成年雄性Wista大鼠(250-300g)被随机分成正常组(n=60)和TNBS诱导结肠炎组(n=60)组。为排除注射核苷酸自身对蛋白表达的可能影响,在反义核苷酸组和盐水溶剂组之外加设错配核苷酸组。因此正常组和TNBS组又进一步各自分成溶剂对照组,反义核苷酸(antisense oligodeoxynucleotides, AS-ODN)干扰组和错配核苷酸(mismatch oligodeoxynucleotides, MM-ODN)组三个亚组,每个亚组n=20,其中8只大鼠用来进行结肠对球囊压力扩张的机械敏感性、背根神经节蛋白水平的测定及病理学检查,6只大鼠用来检测结肠对异硫氰酸丙烯酯(allyl isothiocyanate, AITC)的化学敏感性,6只大鼠用来检测结肠对辣椒素的化学敏感性。2.结肠炎灌肠造模：20mg TNBS溶于50%乙醇中,经肛门插入连接注射器的灌胃针(距肛门7cm)注入降结肠。缓慢拔出灌胃针,用手将大鼠尾巴抬高保持倒立3分钟。对照组给予同等量的生理盐水。3.结肠内脏敏感性的检测：对结肠内刺激的内脏动力性反应(Visceromotor response, VMR)是评价结肠敏感性客观标准。结肠内机械性刺激如球囊扩张或者化学性刺激后,腹外斜肌的肌肉放电记录可将VMR进行量化分析。(1)腹壁肌电埋藏：成年雄性Wistar大鼠(250-300g)以苯巴比妥钠按50mg/kg腹腔注射麻醉,将一特制电极缝合在一侧腹股沟韧带上方、距中线1.5 cm的腹外斜肌上,电极游离端经皮下隧道埋于颈后皮下。(2)机械敏感性测定：氟烷吸入麻醉后,大鼠置于特制塑料筒中,将导管连接的球囊经肛门插入,球囊末端距肛门5cm,用胶带将导管缠在大鼠尾巴根部,固定球囊。取出埋于大鼠颈后皮下的电极,并将电极两端连接BL-420E电生理记录仪。待大鼠适应环境并完全清醒后,分别在0,20,40,60,80mmHg压力下进行结肠扩张。每次扩张持续30s,间隔4分钟。用BL-420E生物信号采集处理和分析腹壁肌电活动。(3)化学敏感性测定：同上对大鼠进行固定及连接电极后,100μl0.25% AITC或者100μl0.05%辣椒素持续10s灌肠,用BL-420E生物信号采集处理和分析刺激结束后45s内腹壁肌电活动。4. TRPA1反义核苷酸干扰：反义核苷酸5’-TCTATGCGGTTATGTTGG-3’,错配核苷酸5’-ACTACTACACTAGACTAC-3’。脊髓L6-S1水平鞘内植入PE-10导管,连接微渗透性压力泵,核苷酸或溶剂以1μ1/小时的速度持续给药3天。5. Western-blotting测定：从各组大鼠提取双侧L6-S2背根神经节,Western-blotting检测TRPA1及TRPV1蛋白含量水平的变化。6.病理组织学测定：取距肛门约3 cm处结肠组织1 cm×0.5 cm，以40 g/L甲醛溶液固定,常规石蜡包埋,连续切片,片厚5μm,做HE染色。结果(1)结肠炎症后支配结肠的L6-S2背根神经节TRPA1与TRPV1蛋白水平明显上调,同时伴有对结肠球囊扩张和化学物质刺激的反应性升高。(2)腰骶部水平鞘内注射TRPA1特异性反义核苷酸能有效抑制炎症状态与非炎症状态下TRPA1的蛋白表达,对炎症状态和非炎症状态的TRPV1蛋白表达均无明显作用。(3)TRPA1反义核苷酸对炎症状态下结肠球囊压力扩张反应性有抑制作用,但在非炎症状态下无明显作用。(4) TRPA1反义核苷酸可显著性抑制炎症状态与非炎症状态下对AITC的化学敏感性,但在炎症状态和非炎症状态下对TRPV1激动剂辣椒素的化学敏感性均无明显抑制作用。结论TRPA1参与结肠炎症后内脏高敏感性的调节。更多还原

【Abstract】 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.更多还原