Abstract：
OBJECTIVE: To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons.
METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope.
RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation 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. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups.
CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.
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。.
METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope.
RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation 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. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups.
CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.
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。.
摘要：
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