外周神经损伤小鼠不同脊髓节段小胶质细胞和星形胶质细胞的激活状态

Activation of microglia and astrocytes in different spinal segments after peripheral nerve injury in mice

脊髓小胶质细胞和星形胶质细胞在外周神经损伤引起的痛行为敏化中发挥重要作用。但是,神经损伤引起的胶质细胞激活在脊髓背角的空间分布尚不清楚。本文采用坐骨神经慢性压迫(chronic constriction injury,CCI)模型,研究了神经病理性疼痛小鼠之颈、胸、腰、骶段脊髓背角胶质细胞的激活状态。采用von Frey方法测定CX3CR1^(YFP)、GFAP^(YFP)转基因小鼠和野生型(wild type,WT)小鼠CCI模型组和假手术组小鼠后爪对机械刺激反应的阈值。免疫荧光标记方法用于检测脊髓背角小胶质细胞和星形胶质细胞的激活状态。结果显示,野生型、CX3CR1^(YFP)和GFAP^(YFP)小鼠在CCI术后3天,术侧后爪均出现明显的触诱发痛,即von Frey刺激引起的缩腿反应阈值(paw withdrawal threshold,PWT)与非手术侧和假手术组动物后爪相比显著下降,并且一直持续到术后第14天;CCI术后14天,CX3CR1^(YFP)小鼠术侧腰段脊髓背角CX3CR1^(YFP)-GFP免疫荧光强度明显增高(P<0.01,n=6),其他各节段无明显改变;GFAP^(YFP)小鼠术侧胸段、腰段和骶段脊髓背角GFAP^(YFP)-GFP免疫荧光强度均显著增高;小胶质细胞和星形胶质细胞的激活情况在WT小鼠Iba-1和GFAP免疫荧光组织化学染色中得到进一步证实;CX-3CR1^(YFP)-GFP和GFAP^(YFP)-GFP免疫荧光信号分别与小胶质细胞标志物Iba-1和星形胶质细胞标志物GFAP共定位;WT小鼠CCI术后3天,术侧胸、腰和骶段脊髓背角Iba-1表达水平均明显上调,但GFAP上调仅发生在脊髓腰段。上述结果表明,较为广泛的脊髓小胶质细胞和星形胶质细胞激活分别发生在痛觉敏化的早期和维持期,提示脊髓小胶质细胞和星形胶质细胞的激活可能分别与继发性痛觉敏化的产生和维持相关。

Spinal microglia and astrocytes play an important role in mediating behavioral hypersensitive state following peripheral nerve injury. However, little is known about the expression patterns of activated microglia and astrocytes in the spinal dorsal horn. The aim of the present study was to investigate the spatial distribution of microglial and astrocytic activation in cervical, thoracic, lumbar and sacral segments of spinal dorsal horn following chronic constriction injury （CCI） of sciatic nerve. The hind paw withdrawal threshold （PWT） of wild type （WT）, CX3CR1vFP and GFAPvFP transgenic mice to mechanical stimulation was determined by von Frey test. Immunofluorescence staining was used to examine the spatial distribution of microglial and astrocytic activation in the spinal dorsal horn. Following CCI, all the WT, CX3CR1vrP and GFAPvFe mice developed robust allodynia in the ipsilateral paw on day 3 after CCI, and the allodynia was observed to last for 14 days. In comparison with sham groups, the PWTs of CCI group animals were significantly decreased （P 〈 0.01, n = 6）. On day 14 after CCI, CX3CR1YFP-GFP immunofluorescence intensity was significantly increased in the ipsilateral lumbar spinal dorsal horn of the CX3CRlVFPmice （P 〈 0.01, n = 6）, but no detectable changes were observed in other spinal segments. Increased GFAPVPP-GFP immunofluorescence intensity was observed in the ipsilateral thoracic, lumbar and sacral spinal segments of the GFAPvFP mice on day 14 after CCI. Iba-1 and GFAP immunofluorescence staining in WT mice showed the same result of microglia and astrocyte activation on day 14 after CCI. CX3CR1YFP-GFP and GFApYFP-GFP immu- nofluorescence signal was colocalized with microglial marker Iba-1 and astrocytic marker GFAP, respectively. Interestingly, on day 3 after CCI, Iba-l-immunoreactivity was significantly increased in the ipsilateral thoracic, lumbar and sacral spinal segments of WT mice, whereas the significant upregulation of GFAP-immunoreactivity restrictedly occurred in the ipsilateral lumbar spinal segment. These results suggest that microglial and astrocytic activation may be involved in the development and maintenance of secondary allodynia in mice with neuropathic pain.