大鼠脊髓背角HCN通道的分布及在部分坐骨神经结扎术后的表达变化

Distribution pattern and expression of HCN channels in spinal dorsal horns of rats with partial sciatic nerve ligation

目的研究超极化激活环核苷酸门控阳离子通道4种亚型(HCN1～4)在大鼠脊髓背角的分布特点,以及其在部分坐骨神经结扎术(PSNL)所致的慢性神经病理性疼痛动物模型中的表达变化。方法首先,选取SD大鼠(150～250 g)制作L_(4-5)段脊髓横切片,用免疫组织化学方法观察HCN1～4在大鼠脊髓背角的分布特点。其次,采用荧光定量PCR技术,检测大鼠L_(4-5)段脊髓背角组织HCN1～4 mRNA的表达特点。另取同龄大鼠,将其随机分为模型组(PSNL组)和假手术组(sham组),每组4只。测定两组大鼠术前,术后1、3、7、10及14 d术侧机械痛阈值(PWT),并于术后14 d取其脊髓背角组织检测HCN1～4 mRNA的表达变化。结果免疫组织化学结果显示,HCN1～4通道蛋白在脊髓背角均有分布,但在Ⅰ～Ⅱ层分布最多,Ⅲ～Ⅵ层分布较少。荧光定量PCR结果显示,生理情况下脊髓背角HCN2 mRNA的表达量最高,其次是HCN1,而HCN3和HCN4表达量较少。与sham组相比,PSNL组PWT显著降低,且术后第14天HCN2 mRNA的表达量显著增加(P<0.05),而HCN1、HCN3及HCN4表达量的变化差异无统计学意义(P>0.05)。结论 HCN1～4在正常大鼠脊髓背角具有特异性表达,但仅HCN2 mRNA在PSNL神经病理性疼痛模型后显著增加。

Objective To investigate the distribution pattern and expression of HCN1-4 subtypes in rat spinal dorsal horn(SDH) in neuropathic pain induced by partial sciatic nerve ligation(PSNL). Methods Transverse slices of L4-L5 segments from Sprague Dawley(SD) rats weighing 150-250 g were prepared to study the distribution of HCN1-4 channels in rat SDH by using the immunohistochemistry technique. The mRNA level of HCN1-4 in SDH at L4-L5 segments was measured through quantitative real-time PCR(qRT-PCR). Eight SD rats of same age were randomly divided into PSNL model and sham group(4 rats in each group). Ipsilateral paw withdrawal thresholds(PWT) were assessed before and on the 1st, 3rd, 7th, 10th and 14th day after the surgery. SDH were collected to study the levels of HCN1-4 mRNA on the 14th day. Results Immunohistochemical results showed that the proteins of HCN1-4 subunits were observed in rat SDH, which were largely expressed in laminae Ⅰ-Ⅱ, with less expression in laminae Ⅲ-Ⅵ. Results of qRT-PCR showed that in normal conditions, the mRNA expression of HCN2 was the highest, followed with HCN1, while the expression of HCN3 and HCN4 was relatively low. Furthermore, the PWT in the PSNL group was significantly lower than that of the sham group after surgery. The level of HCN2 mRNA was significantly increased in the SDH on Day 14th(P<0.05), while no significant change of HCN1, HCN3 or HCN4 expression was revealed(P>0.05). Conclusion All the subtypes of HCN channels are specifically expressed in the SDH, but only the level of HCN2 mRNA is upregulated in the PSNL neuropathic pain model.