Objectives: The activation of astrocytes is an important process in the formation of chronic pain. This study aims to observe the activation of A1 reactive astrocytes in the medullary dorsal horn in the rat model of trigeminal neuralgia, and to explore the mechanism of central sensitization caused by A1 reactive astrocyte.
Methods: The adult male rats were randomly divided into a sham group and a chronic constriction injury of infraorbital nerve (ION-CCI) group. The facial mechanical pain threshold and thermal withdrawal latency were measured before the operation and on the 1st, 3rd, 7th, 10th, and 14th day after the operation. After pain behavior observation, the expression of glial fibrillary acidic protein (GFAP) in the medullary dorsal horn was observed by immunohistochemistry and immunofluorescence colocalization of GFAP, complement 3 (C3)/S100A10, and 4', 6-diamidino-2-phenylindole (DAPI) was analyzed. Primary astrocytes were cultured and randomly divided into a naive group and a DHK group. The DHK group was treated with 1 mmol/L of astrocyte activation inhibitor dihydrokainic acid (DHK). Fura-2/AM was used to stain the astrocytes and the calcium wave of the 2 groups under the stimulation of high potassium was recorded and compared. The expression of C3 was detected by Western blotting.
Results: The facial mechanical pain threshold and thermal withdrawal latency of the ION-CCI group were significantly lower than those of the sham group (both P<0.05). There were a large number of GFAP positive astrocytes in the medullary dorsal horn of the ION-CCI group. The fluorescence intensity of GFAP in the ION-CCI group was higher than that in the sham group (P<0.05). GFAP and C3/S100A10 were co-expressed in astrocytes. Compared with the sham group, the fluorescence intensity of C3 and the protein expression of C3 in the ION-CCI group were increased (both P<0.05). The expression of C3 in ION-CCI group was significantly increased (P<0.05). Compared with the naive group, the C3 protein expression was significantly decreased in the DHK group (P<0.05). The intensity of calcium fluorescence was increased after high potassium stimulation in both groups. Furthermore, the peak and increase amplitude of calcium fluorescence in the naive group were much higher than those in the DHK group (both P<0.05).
Conclusions: A1 reactive astrocytes in the medullary dorsal horn of trigeminal neuralgia model rats are increased significantly, which may participate in central sensitization of trigeminal neuralgia by impacting astrocyte calcium wave.
目的: 星形胶质细胞激活是慢性疼痛中的重要环节，本研究旨在观察三叉神经痛大鼠模型中延髓背角A1型反应性星形胶质细胞活化情况，并探究其引起中枢敏化的机制。方法: 将雄性SD大鼠随机分为假手术(sham)组和眶下神经慢性缩窄性损伤(chronic constriction injury of infraorbital nerve，ION-CCI)组。分别于造模前1 d及造模后第1、3、7、10、14天进行大鼠面部机械痛阈、热敏潜伏期测定。疼痛行为学检测完成后，采用免疫荧光染色检测大鼠延髓背角胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)的表达，对GFAP、补体3(complement 3，C3)/S100A10、4’, 6-二脒基-2-苯基吲哚(4’, 6-diamidino-2-phenylindole，DAPI)进行免疫荧光共定位分析。取原代星形胶质细胞培养，并将其随机分为空白对照组和二氢红藻氨酸(dihydrokainic acid，DHK)组。DHK组加入1 mmol/L的星形胶质细胞激活抑制剂DHK，使用钙离子荧光探针Fura-2/AM对2组细胞进行钙荧光染色，连续观察高钾刺激下2组细胞的钙波活动差异。采用蛋白质印迹法检测C3的蛋白质表达水平。结果: ION-CCI组大鼠机械痛阈和热敏潜伏期均明显低于sham组(均P<0.05)。ION-CCI组延髓背角存在大量GFAP表达阳性的星形胶质细胞，GFAP荧光强度明显强于sham组(P<0.05)。GFAP和C3/S100A10共定位于延髓背角星形胶质细胞。与sham组比较，ION-CCI组C3的荧光强度和蛋白质表达均明显增加(均P<0.05)。与空白对照组比较，DHK组C3蛋白质表达水平明显下调(P<0.05)。在第60秒给予高钾刺激后，空白对照组和DHK组的钙荧光强度均明显增加，且空白对照组的钙荧光峰值和钙荧光升高幅度均高于DHK组，差异均有统计学意义(均P<0.05)。结论: 三叉神经痛模型大鼠延髓背角A1型反应性星形胶质细胞明显增多，其可能通过影响钙波活动参与三叉神经痛的中枢敏化。.
Keywords: A1 reactive astrocyte; calcium wave; medullary dorsal horn; trigeminal neuralgia.