孙海燕 李平 何农 薛富善
Effect of pre-emptive intrathecal administration of L-NAME on CGRP expression in spinal dorsal horn in a rat model of neuropathic pain  
SUN Hai-yan, LI Ping , HE Nong, et al. Department of Anesthesiology, Plastic Surgery Hospital, Peking Union Medical College and Chinese Academy of Medical Science, Beijing 100041, China<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Abstract

Objective  To investigate the effect of pre-emptive intrathecal (IT) administration of L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor on calcitonin gene-related peptide (CGRP) expression in the dorsal horn of spinal cord in a rat model of neuropathic pain. Methods Ninety-six female adult SD rats weighing 220-310 g were randomly divided into four groups with 24 animals in each group: group A received IT 0.9% NaCl 15 min before sham operation; group B received IT 0.9% NaCl 15 min before right sciatic nerve ligation; group C received IT L-NAME 250 mg in 10μl 15 min before sham operation and group D received IT L-NAME 250μg in 10μl 15 min before sciatic nerve ligation. The animals were anesthetized with intraperitoneal pentobarbital 50 mg?kg-1. A PE-10 catheter was placed in subarachnoid space with the tip of the catheter reaching the lumbar enlargement region. The animals were allowed to recover for 3-4 days and only those waking normally were used in the study. Right sciatic nerve was exposed and four loose ligatures were placed on the sciatic nerve according to the method described by Bennet. In sham operation the sciatic nerve was exposed but not ligated. The animals were sacrificed on the 1 st, 4 th, 7 th and 14 th day after operation. The lumbar segment of spinal cord was immediately removed. The CGRP expression in the ligated-side dorsal horn was assessed with immunohistochemistry technique. Results In group B and D CGRP expression in the ligated side dorsal horn was significantly increased on the 4th, 7th and 14th day after operation as compared with that in group A. There was no significant difference in the CGRP expression in the ligated side dorsal horn between group A and C as well as between B and D. Conclusion Pre-emptive ITadministration of L-NAME cannot inhibit the increase in CGRP expression in dorsal horn induced by peripheral nerve injury, suggesting that the neuropathic pain mediated by NO is not related to the release of calcitonin gene-related peptide.
　Key words：Neuralgia; NG-nitroarginine methyl ester; Nitric oxide; Calcitonin gene-related peptide; Injections, spinal

　　神经性疼痛是临床工作中的一种常见难治性疼痛。近年来认为，该类疼痛可能与伤害性感受信息经脊髓神经元多突触传递而诱发的“中枢敏化作用”有关^[1]，而一氧化氮（NO）作为体内一种重要的细胞信使分子，在调节脊髓神经元“可塑性”方面发挥着重要作用^[2]，但是NO在脊髓水平介导神经性疼痛的确切机制尚不清楚。本研究拟观察鞘内预注射非特异性一氧化氮合酶抑制剂NG-硝基-L-精氨酸-甲基酯（L-NAME）后神经性疼痛大鼠脊髓背角隆钙素基因相关肽（CGRP）的表达，探讨NO在脊髓水平调控伤害性感受信息传递的机制。

材料和方法<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

　　动物选择和分组 雌性成年SD大鼠（中国协和医科大学动物实验中心提供）96只，体重220～310g。随机分成4组，每组24只。A组：假手术组，鞘内注射0.9%氯化钠10μl，15min后实施假手术；B组：CCI组，鞘内注射0.9%氯化钠10μl，15min后结扎坐骨神经；C组：预处理后假手术组，鞘内注射L-NAME 10μl（250μg, Sigma公司，美国），15 min后实施假手术；D组：预处理后CCI组，鞘内注射L-NAME 10μl（250μg），15min后结扎坐骨神经。
神经性疼痛模型的建立 腹腔内注射戊巴比妥钠50mg/kg，待麻醉满意后，采用Meller法^[3]切开大鼠的枕寰筋膜，将PE-10导管置入其蛛网膜下隙，插入深度为7.5cm。术后3～4d，选取行走正常、翻正反向良好且能以上肢扶笼站立的大鼠用于实验。选择鞘内置管后无神经损伤体征的大鼠，腹腔注射戊巴比妥钠50mg/kg麻醉。在无菌条件下，按照Bennett等^[4]的方法，于大鼠右下肢股中部分离暴露坐骨神经，用4-0铬制羊肠线结扎4道，制成慢性结扎损伤（CCI）的神经性疼痛模型。实施假手术时，仅暴露坐骨神经，不结扎。
　　脊髓切片的制备 各组动物在鞘内注射氯化钠（或L-NAME）后，再继续注入0.9%氯化钠10μl冲管。每组动物在麻醉苏醒后，置于适宜环境中饲养，于术后第1、4、7、14天分别处死6只。大鼠处死后，立即开胸，经升主动脉插管，灌注0.9%氯化钠100ml，继之以含4%多聚甲醛的0.1mmol/L磷酸缓冲液500ml灌注固定。取脊髓腰膨大（L1-6节段）置于20%蔗糖溶液中浸泡过夜，石蜡切片机连续冠状切片，片厚5μm，置于0.01mmol/L磷酸缓冲液内保存。
　　免疫细胞化学染色 切片放入含0.3%TritonX100的0.01mol/L 磷酸缓冲液中，室温浸泡30min，然后进行免疫组化染色，具体步骤如下：（1）兔抗CGRP血清（1：1000，Peninsula公司，美国）孵育1h；（2）Biotin标记的羊抗兔IgG血清（1：400）孵育30min；（3）Streptavadin-HRP复合物溶液（1：400，DAKO公司，日本）孵育30min；（4）0.33%二氨基联苯胺（Aldrich公司，英国）/H2O2溶液呈色20min。各步骤间均用0.01mol/L磷酸缓冲液彻底清洗切片。最后进行裱片、晾干、脱水、透明和封片处理。
　　结果观察及统计学处理 每只大鼠随机选取5张切片，应用北京航空航天大学图像中心开发研制的彩色病理图像分析系统（版本4.0），对右侧脊髓背角内CGRP免疫阳性反应物质进行分析，以每张切片中CGRP免疫阳性面积百分率（阳性单位）反映CGRP的表达。计量资料以均数±标准差（x±s）表示，应用Excel 5.0统计学软件，组间比较采用单因素方差分析，P＜0.05为差异有显著性。

结果

　　在低倍镜下，脊髓背角内CGRP免疫阳性纤维犹如一顶小帽扣在背角浅层，在高倍镜下可见较密集的末梢膨体，阳性纤维交织成网状。
　　与A组比较，B组、D组右侧脊髓背角的CGRP表达在术后第4、7、14天明显增多（P＜0.05＝，C组术后各时间点差异无显著性（P＞0.05）。与B组比较，D组右侧脊髓背角CGRP表达在术后各时间点差异无显著性（P＞0.05）。与C组比较，A组右侧脊髓背角CGRP表达在术后各时间点差异无显著性（P＞0.05），B组、D组术后第4、7、14天明显升高（P＜0.05＝。各组右侧脊髓背角CGRP表达在术后第1天差异无显著性（P＞0.05）。见表1。

讨论<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

　　在神经性疼痛形成的过程中，脊髓NMDA受体的活化发挥着关键作用^[1]。研究显示，激动NMDA受体可引起Ca²⁺内流，从而激活细胞内钙敏感性一氧化氮合酶（NOS），促进NO生成^[2]。通过增加细胞内cGMP的含量，NO可诱发神经元的“wind-up”效应，显著增强脊髓内的突触传递。
　　CGRP是体内一种与伤害性刺激传入和整合具有密切关系的兴奋性神经肽。在脊髓水平，CGRP的释放及其受体激活是伤害性感受信息传递和调控过程中的一个重要环节。行为学研究显示，鞘内预先注射CGRP可增强炎症或神经性疼痛状态并引起痛觉过敏^[5]，而应用CGRP受体拮抗剂可减轻炎症或神经损伤引起的痛觉过敏^[6]。电生理学研究亦表明，CGRP参与了外周炎症状性疼痛时脊髓背角神经元的致敏^[7]。而且应用离子渗透技术给予CGRP，可明显改变脊髓背角神经元的兴奋性^[8]。
　　研究发现，结扎坐骨神经后3d左右，大鼠就可出现典型的慢性疼痛表现，并能持续到术后28～35d^[4]。免疫组织化学研究证实，周围神经损伤后脊神经节内的CGRP合成明显上调，并且向脊髓背角神经元的兴奋性。本实验亦发现，结扎坐骨神经干可使大鼠脊髓背角内的CGRP表达明显增强，并且在长达14d的观察期内一直保持在较高水平，这进一步提示CGRP可能通过易化脊髓水平的伤害性感受信息传递而促进神经性疼痛的形成。
　　既往采用脊髓切片灌流技术研究发现，NO供体硝普钠可促进脊髓背角释放CGRP^[10]，而且辣椒辣素介导的CGRP释放作用亦需要NO的参与来完成^[11]。还有研究证实，大鼠脊髓胶状质中合成NO的岛细胞可调节CGRP的释放^[12]。而且在脊神经节内，大部分NOS阳性细胞与CGRP、P物质共存^[13]。这些研究均提示，NO在脊髓水平介导神经性疼痛的作用可能与促进CGRP释放有关。但是，本实验结果显示，鞘内预注L-NAME未能有效抑制坐骨神经结扎所诱导的脊髓背角内CGRP释放增加并不是由NO引起的。上述研究与本实验的结果不同，可能与实验动物、实验模型、研究方法和NOS阻滞剂的种类及浓度等诸多因素有关。
　　综上所述，鞘内预注射L-NAME不能抑制周围神经损伤所导致的脊髓背角内CGRP表达，提示NO介导神经性疼痛的作用与促进CGRP释放无关。
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