[Effects of intrathecal injection of IRF8 SiRNA on pain threshold and activation of spinal cord microglia in rats with postoperative persistent pain]

Abstract

Objective: To investigate the effects of intrathecal injection of IRF8 SiRNA on the pain threshold and activation of spinal cord microglia in rats with postoperative persistent pain. Methods: One hundred and twenty male Sprague-Dawley rats were randomly divided into sham group (SH, n=12), SMIR group (SM, n=48), SMIR + DEPC group (SD, n=12) and SMIR + irf8 SiRNA group (SS, n=48). In the SM group, the persistent postsurgical pain(PPsP) model was established according to the skin/muscle incision and retraction (SMIR), and the SH group was only incised without retracted. The SD group and SS group received intrathecal catheterization one week before SMIR, the SS group was injected with 20 μl of IRF8 SiRNA solution (dissolved in DEPC-treated water, 150 pmol) intrathecally on the 5^th and 6^th day after SMIR, and the SD group was injected with the same amount of DEPC-treated water. The paw withdrawal threshold (PWT) of each group was measured and recorded before SMIR and on the 1^st, 3^rd, 7^th, 12^th, 22^nd and 33^rd days after SMIR. Western blot was used to detect the expression of Iba-1 in the dorsal horn of spinal cord on the 12^th days after SMIR, and the saphenous nerves in the SH group and SM group were collected to observe their ultrastructural changes under electron microscope. The flow cytometry was used to detect the activation of microglia in spinal cord dorsal horn before SMIR and on the 1^st, 3^rd, 7^th, 12^th, 22^nd and 33^rd days after SMIR in the SM group and SS group. Results: Compared with D0, the PWT of SM group was decreased on the 1^th to 22^nd day after SMIR (P＜0.05 or P＜0.01), and returned to normal level on the 33^rd day after SMIR (P＞ 0.05). Compared with the SH group, the PWT of the SM group was decreased on the 1^th to 22^nd day after SMIR (P＜0.05 or P＜ 0.01). However, compared with the SD group, the PWT of the SS group was increased on the 7^th to 22^nd day after SMIR (P＜0.05 or P＜0.01). Compared with SH group, the PWT of SS group was decreased on the 7^th to 22^nd day after SMIR (P＜0.05 or P＜0.01). The average thickness of saphenous nerve myelin was (377.0 3±69.60) nm in the SH group and (369.50±73.26) nm in the SM group, and there was no significant difference between the two groups (P＞0.05). Compared with the SH group, the expression level of Iba-1 was increased significantly (P＜0.01) in the SM group. Compared with the SD group, the expression of Iba-1 was inhibited (P＜0.05) in the SS group, and compared with the SH group, the expression of Iba-1 was also statistically different (P＜0.05) in the SS group, while the expression of Iba-1 was not statistically significant between the SM group and the SD group (P＞0.05). Compared with D0, the activation ratio of microglia was increased significantly on the 3^rd to 22^nd day after SMIR (P＜0.01) in the SM group , while the activation of microglia reached a peak on 3rd day after SMIR (P＜0.01) in the SS group. After intrathecal administration, the activation rate of microglia in the spinal dorsal horn of the SS group was decreased significantly, and compared with the SM group, it was decreased significantly on the 7^th to 12^th day after SMIR (P＜0.01). Conclusion: The significant and persistent mechanical hyperalgesia in PPsP induced by SMIR was caused non-obvious peripheral nerve injury, which may be mediated by the activation of microglia in the dorsal horn of the spinal cord. IRF8 SiRNA administrated by intrathecal injection could inhibit the activation of microglia and reverse SMIR-induced hyperalgesia.

Keywords: IRF8; hyperalgesia; microglia; persistent postsurgical pain; rat SiRNA; spinal.