结肠炎模型小鼠内脏痛及穴位敏化的外周神经机制探讨  

Peripheral neural mechanism of visceral pain and acupoint sensitization in2,4,6-trinitrobenzene sulfonic acid-induced colitis model mice

作　　者：高昕妍[1] 张楠 刘坤[1] 奚晗清 刘允 何勋[1] 韩数 朱兵[1] GAO Xin-yan;ZHANG Nan;LIU Kun;XI Han-qing;LIU Yun;HE Xun;HAN Shu;ZHU Bing(Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Engineering Medicine,Beihang University,Beijing 100191)

机构地区：[1]中国中医科学院针灸研究所,北京100700 [2]北京航空航天大学医学科学与工程学院,北京100191

出　　处：《针刺研究》2023年第12期1183-1192,共10页Acupuncture Research

基　　金：国家自然科学基金面上项目(No.81973963、81973967)。

摘　　要：目的:从背根节(DRG)神经元水平说明内脏病变与相应体表穴位敏化产生的神经生物学机制。方法:皮肤伊文思蓝(EB)外渗实验:BALB/c小鼠随机分为对照组和结肠炎组,每组4只。2,4,6-三硝基苯磺酸直结肠灌注7 d制备结肠炎模型。采用尾静脉注射EB检测体表神经源性炎性反应,观察渗出点的位置及面积。痛行为实验:C57BL/6J小鼠随机分为对照组和结肠炎组,每组8只,造模方法同上,观察下背部和足部Von Frey丝机械刺激诱发的回避或缩足反应次数。小鼠在体DRG钙成像实验:Pirt-GCaMP6s转基因小鼠随机分为对照组和结肠炎组,每组12只,造模方法同上,暴露腰(L)6或L4 DRG,在共聚焦荧光显微镜下观察神经元对直结肠扩张刺激(CRD)、下背部或后爪机械刺激的反应。结果:与对照组比较,结肠炎组小鼠下背部及后爪神经源性炎性EB渗出较多(P<0.05);同时结肠炎组小鼠下背部、后爪对机械刺激的回避或缩足反应次数增加(P<0.01,P<0.05);CRD 60 mm Hg诱发内脏痛引起的L6 DRG神经元激活数量占总数的百分比均较对照组显著增加(P<0.01),其中中型神经元数量增加更为明显(P<0.01)。与对照组相比,结肠炎组小鼠L6 DRG神经元对下背部毛刷刺激反应荧光强度增加(P<0.001),不同直径神经元的荧光强度均增强(P<0.01,P<0.001,P<0.05)。于小鼠后爪施加毛刷、钳夹压力刺激,均引起与结肠不同水平的L4 DRG神经元反应总体数量百分比较对照组显著增加(P<0.01,P<0.05)。结论:结肠炎可以引起同节段和近节段体表穴位敏化,同时DRG神经元激活数量和反应性增加。其中与直结肠同水平的L6 DRG神经元表现为神经元激活数量百分比和钙荧光信号强度增加,而与内脏邻近水平的L4 DRG神经元表现为激活数量百分比增加,提示可能存在不同的外周神经元敏化机制。ObjectiveTo explore the neural m echanism of visceral pain and related som atic(acupoints)sen⁃sitization by using in vivo calcium im aging of dorsal root ganglia(DRG)neurons.MethodsEight BALB/c m ice were random ly divided into control and m odel groups,with 4 m ice in each group.The colitis m odel was induced by colorectal perfusion of 2,4,6-trinitrobenzene sulfonic acid(TNBS)once daily for 7 days.M ice of the control group received colorectal perfusion of norm al saline once daily for 7 days.The location and area of the som atic neurogenic inflam m a⁃tion(cutaneous exudation of Evans blue[EB])of the 2 groups of m ice were observed after intravenous injection of EB.For pain behavioral tests,sixteen C57BL/6J m ice were random ly divided into control and m odel groups,with 8 m ice in each group,and a Von Frey filam ent was used to stim ulate the referred som atic reactive regions in colitis m ice,and the num ber of avoidance and paw withdraw reaction within 10 tests was recorded.For in vivo DRG calcium im aging tests,24Pirt-GCaM P6s transgenic m ice were random ly and equally divided into control group and colitis m odel group.The responses of the neurons in L6 or L4 DRG to colorectal distension(CRD),lower back brushing,or m echanical stim ula⁃tion at the hindpaw were observed using confocal fluorescence m icroscope.ResultsCom pared with the control group,the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis m odel group(P<0.05),and the avoidance or paw withdraw num bers induced by Von Frey stim ulation at the lower back and hindpaw were increased(P<0.01,P<0.05),indicating that colitis induced regional skin(acupoints)sensitization in the lower back and hindpaw regions.Com pared with the control group,the percentage of L6 DRG neurons activated by 60 m m HgCRD in the colitis m odel m ice were apparently increased(P<0.01),the activated neurons m ainly involved the m edium-sized DRG neurons(P<0.01).In Pirt-GCaM P6s transgenic m ice,following brushing the skin of the rec

关 键 词：穴位敏化 结肠炎 在体钙成像 背根节 神经元 

分 类 号：R245-3[医药卫生—针灸推拿学]