触液核NGF/ERK信号通路参与CCI大鼠神经病理性疼痛

NGF/ERK pathway of the cerebrospinal fluid-contacting nucleus participates in neuropathic pain of CCI rats

目的:观察神经生长因子(nerve growth factor, NGF)在坐骨神经慢性压迫性损伤(chronic constriction injury, CCI)大鼠触液核的表达以及拮抗触液核NGF对CCI大鼠痛行为学和触液核p-ERK表达的影响。方法:侧脑室注射CB-HRP逆行示踪触液核,结合免疫荧光双标观察正常大鼠是否表达NGF和p-ERK。第一步:SPF级雄性SD大鼠,体重200~300 g,按数字表法随机分为5组(每组6只):sham组,CCI 1、3、7、14天组。术前1天、术后1天、3天、7天、14天测定机械痛阈(mechanical withdrawal threshold, MWT)和热痛阈(thermal withdrawal latency, TWL)后处死并取触液核进行Western Blot观察NGF和p-ERK的表达变化情况。第二步:将SD大鼠随机分为6组(n=6):CCI+NGF抗体组、CCI+PBS组、CCI+对照抗体组、sham+NGF抗体组、sham+PBS组、sham+对照抗体组。CCI大鼠术后第6天在立体定位仪下侧脑室分别注射NGF抗体、PBS及对照抗体,测定MWT和TWL后处死并取触液核进行Western Blot观察p-ERK的表达变化情况。结果:正常大鼠触液核表达NGF和p-ERK,CCI组大鼠的MWT和TWL明显低于sham组,侧脑室注射NGF抗体后CCI+NGF抗体组大鼠的痛阈较CCI+PBS组和CCI+对照抗体组明显提高,并且其p-ERK表达较其他两组明显降低。结论:触液核NGF/ERK信号通路可能参与了大鼠神经病理性疼痛,侧脑室注射NGF抗体可能通过拮抗触液核NGF/ERK信号通路减轻大鼠神经病理性疼痛。

Objective:To investigate the expression of nerve growth factor(NGF)in cerebrospinal fluid-contacting nucleus(CSF-CN)in neuropathic pain rats,the effect of intracerebroventricular injection of NGF antibody on pain behavior and the expression of p-ERK in the CSF-CN of chronic constriction injury(CCI)rats.Methods:We first labeled the distal CSF-CN using CB-HRP,combined with double-labeled immunofluorescence technique to verify whether NGF and p-ERK was expressed in normal rats.Male Sprague-Dawley rats weighing 200-300 g were randomly assigned into five groups(6 in each group),sham group,CCI day 1,day 3,day 7 and day 14 group,through the method of random number table.The expression of NGF and p-ERK were observed by Western blot after the mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were measured on day 1 before and day 1,day 3,day 7,and day 14 after CCI.We next randomly assigned the rats into six groups:CCI+NGF antibody,CCI+PBS,CCI+control IgG,sham+NGF antibody,sham+PBS,sham+control IgG.NGF antibody,PBS or control IgG was injected in lateral ventricle in each rat on day 6 of CCI.After MWT and TWL were tested,the CSF-CN portion of rat brain was isolated to observe the expression of p-ERK.Results:NGF and p-ERK were expressed in CSF-CN of normal rats.The MWT and TWL of CCI rats were significantly lower than those in sham rats.The pain threshold of rats in CCI+NGF antibody group was significantly higher than that in CCI+PBS group and CCI+control antibody group,and the expression of p-ERK was significantly lower than that in the other two groups.Conclusion:The NGF/ERK pathway in the CSF-CN may be involved in the modulation of neuropathic pain.Intracerebroventricular injection of NGF antibody may alleviate neuropathic pain in CCI rats by antagonizing NGF/ERK signaling pathway.