Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats

Abstract

Background: Growing evidence has shown that unilateral nerve injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. This phenomenon is known as mirror image pain (MIP). Glial cells have been indicated in the mechanism of MIP; however, it is not clear how glial cells are involved in MIP.

Methods: To observe phenomenon MIP and the following mechanism, 20 adult male Sprague-Dawley rats (weighing 180-220 g) were separated into two groups: Sham Group (n = 10) and left L5 spinal nerve ligated and sectioned (SNL) group (n = 10). Thermal hyperalgesia and mechanical hypersensitivity were measured for both groups to determine if the SNL model had Mirror image of Pain (MIP). Nav1.7 protein expression in DRG was analyzed using immunohistochemistry and western-blotting. And then to observe the effect of fluorocitrate on MIP, 15 rats were separated into three Groups: Sham Group (n = 5); SNL + FC group: intrathecal injection of Fluorocitric acid(FC) 1 nmol/10 μL (n = 5); SNL + NS group: intrathecal injection of 0.9% Normal Saline (n = 5). Behavior testing, immunocytochemistry, and western-blotting using dorsal root ganglion (DRG) from both sides were then conducted.

Results: The results showed pain hypersensitivity in both hind-paws of the SNL animals, Mechanical tests showed the paw withdrawal threshold dropped from 13.30 ± 1.204 g to 2.57 ± 1.963 g at 14 d as will as the ipsilateral paw thermal withdrawal threshold dropped from 16.5 ± 2.236 s to 4.38 ± 2.544 s at 14 d. Mechanical tests showed the contralateral paw withdrawal threshold dropped from 14.01 ± 1.412 to 4.2 ± 1.789 g at 7d will the thermal withdrawal threshold dropped from 16.8 ± 2.176 s to 7.6 ± 1.517 s at 7d. Nav1.7 expression increased and glial cells actived in bilateral side DRG after SNL compared with sham group. After intrathecal injection of fluorocitrate, the glial cell in bilateral DRG were inhibited and the pain behavior were reversed in both hindpaws too.

Conclusions: Fluorocitrate can inhibit the activation of glial cells in spinal cord and DRG, and reduce MIP.

Keywords: DL-fluorocitric acid; Mirror-image pain; Nav1.7 protein; Satellite glial cells.

Figure 1

Behavioral changes after L5 spinal nerve ligation in rats. A: Mechanical hyperalgesia. after L5 spinal nerve ligation in rats. The threshold of contralateral paw of SNL model were dropped within 14 days after surgery ,the lowest is at 7d. ^# P < 0.05 vs ipsilateral paw of sham group, *P < 0.05 vs contralateral paw of sham group; B: Thermal hyperalgesia after L5 spinal nerve ligation in rats. The threshold of ipsilateral paw of SNL model were dropped within 28 days after surgery ,the lowest is at 14d. The threshold of contralateral paw of SNL model were dropped within 14 days after surgery ,the lowest is at 7d. ^# P < 0.05 vs ipsilateral paw of sham group, *P < 0.05 vs contralateral paw of sham group; n = 30.

Figure 2

Expression of Nav1.7 in bilateral ganglion. The expression of Nav1.7 in bilateral ganglion by Immunohistochemistry A: The positive cell was marked by arrow. compared with the sham group, both side ganglion showed the expression of Nav1.7 was increased significantly, especially at 3d, 7d,14d. B: The expression of Nav1.7 in bilateral ganglion by western staining. B1:Compared to the Sham group, Westernblot showed the expression of Nav1.7/ SCN9A in ipsilateral ganglion increased significantly, especially at 7d,14d. B2: Compared to the Sham group, Westernblot showed the expression of Nav1.7/ SCN9A in contralateral ganglion increased significantly, especially at 7d. C: the graph for statistical *P < 0.05 vs Sham group (n = 3).

Figure 3

The activated of satellite cell of bilateral ganglion. A: GFAP expression by immunohistochemistry staining. GFAP expression was increased in the cell which around the neuron in dorsal ganglion of bilateral sides at 7 d, pointed by arrow. B: the statistical graph of A, *p < 0.05 vs sham group. (n = 3) C: western-blot showed pJNK protein was increased in dorsal ganglion of bilateral sides at 7 d, D: the statistical graph of E,*p < 0.05 vs sham group (n = 3). E: double-labelling immunofluorescence showed GFAP positive cell are around the SCN9A positive cell.

Figure 4

Behavioral changes after separately intrathecal injection FC and 0.9% NS of SNL model, red arrow is sign of intrathecal injection. A: changes of mechanical threshold of bilateral side paws: After injection FC, ipsilateral paw of SNL model showed their mechanical threshold were up, # P < 0.05 vs ipsilateral side paw of SNL + NS group. After injection FC,contralateral side paw of SNL model showed their mechanical threshold were up, *P < 0.05 vs contralateral side paw of SNL + NS group. B: changes of thermal threshold of bilateral side paws, after injection FC, ipsilateral paw of SNL model showed their thermal threshold were up # P < 0.05 vs ipsilateral paw of SNL + NS group. After injection FC,contralateral paw of SNL model showed their thermal threshold were up *P < 0.05 vs contralateral paw of SNL + NS group n = 6.

Figure 5

GFAP expression in bilateral sides DRG after intrathecal injection of SNL rats at 7 d. A: GFAP Immunohistochemistry show positive staining of satellites cell aroud neurons. B: average integral optical density of GFAP immunohistochemical positive area, *P < 0.05 vs SNL + NS group n = 5.

Figure 6

Nava1.7 expression in bilateral sides DRG after intrathecal injection of SNL rats at 7 d. A: Nava1.7 Immunohistochemistry show positive staining of neurons. B: average integral optical density of Nava1.7 immunohistochemical positive area, *P < 0.05 vs SNL + NS group n = 5.