Anti-allodynic effect of intrathecal processed Aconitum jaluense is associated with the inhibition of microglial activation and P2X7 receptor expression in spinal cord

Abstract

Background: For their analgesic and anti-arthritic effects, Aconitum species have been used in folk medicine in some East Asian countries. Although their analgesic effect is attributed to its action on voltage-dependent sodium channels, they also suppress purinergic receptor expression in dorsal root ganglion neurons in rats with neuropathic pain. In vitro study also demonstrated that the Aconitum suppresses ATP-induced P2X7 receptor (P2X7R)-mediated inflammatory responses in microglial cell lines. Herein, we examined the effect of intrathecal administration of thermally processed Aconitum jaluense (PA) on pain behavior, P2X7R expression and microglial activation in a rat spinal nerve ligation (SNL) model.

Methods: Mechanical allodynia induced by L5 SNL in Sprague-Dawley rats was measured using the von Frey test to evaluate the effect of intrathecal injection of PA. Changes in the expression of P2X7R in the spinal cord were examined using RT-PCR and Western blot analysis. In addition, the effect of intrathecal PA on microglial activation was evaluated by immunofluorescence.

Results: Intrathecal PA attenuated mechanical allodynia in a dose-dependent manner showing both acute and chronic effects with 65 % of the maximal possible effect. The expression and production of spinal P2X7R was increased five days after SNL, but daily intrathecal PA injection significantly inhibited the increase to the level of naïve animals. Immunofluorescence of the spinal cord revealed a significant increase in P2X7R expression and activation of microglia in the dorsal horn, which was inhibited by intrathecal PA treatment. P2X7R co-localized with microglia marker, but not neurons.

Conclusions: Intrathecal PA exerts anti-allodynic effects in neuropathic pain, possibly by suppressing P2X7R production and expression as well as reducing microglial activation in the spinal cord.

Keywords: Aconitum jaluense; Allodynia; Microglia; P2X7 receptor.

Fig. 1

Acute effects of intrathecal (i.t.) processed Aconitum jaluense (PA) on the hind paw withdrawal response to von Frey filaments following spinal nerve ligation (SNL). Time course of paw withdrawal threshold (a) shows an increase in PWT in animals treated with i.t. PA, which peaks within 30 min. The anti-allodynic effect lasted 3 h following i.t. PA. The percentage of maximal possible effect (b) of i.t. PA was 65 % at a dose of 300 μg. N = 8 per each treatment. *P < 0.05, **P < 0.01 vs. saline

Fig. 2

Chronic effects of intrathecal (i.t.) PA on allodynic response to von Frey filaments following spinal nerve ligation (SNL). Each group received i.t. PA (N = 8) or saline (N = 8) once daily for 5 days. ‘Pre’ and ‘Post’ on the x-axis represent the PWT measured 10 min before and 30 min following i.t. PA injection, respectively. *P < 0.05 vs. corresponding PWT of the saline group measured prior to injection

Fig. 3

a Changes in P2X7 receptor (P2X7R) expression in the spinal cord ipsilateral to the SNL by Western blot analysis. Bar graphs depicting the relative intensity of P2X7R expression show a gradual increase in P2X7R levels in the ipsilateral dorsal spinal cord. b Effect of intrathecal (i.t.) administration of PA on P2X7R expression in the ipsilateral dorsal spinal cord. Representative blots showing the expression of P2X7R and GAPDH on day 7 following SNL. Intrathecal injection of PA or saline was performed from days 3 to 7. The increase in P2X7R in the ipsilateral dorsal spinal cord was significantly blocked in animals treated with i.t. PA (300 μg). N = 7 per group. *P < 0.05 vs. naive

Fig. 4

a Increased expression of P2X7R mRNA in the ipsilateral dorsal spinal cord following L5 SNL. Gel panels show RT-PCR products from the spinal cord of naïve rats on days 3, 5 and 7 following SNL. P2X7R mRNA is significantly increased after SNL compared to naïve animals. b Intrathecal (i.t.) administration of PA blocked the increase in P2X7R mRNA expression in the ipsilateral dorsal spinal cord on day 7 following SNL. N = 7 per group. *P < 0.05 vs. naive

Fig. 5

Intrathecal (i.t.) PA attenuated the increase in P2X7R and Iba-1 expression in the dorsal horn of the spinal cord seven days following L5 SNL. P2X7R immunoreactivity (green) was increased on the ipsilateral side (b) compared with the contralateral side (a). Expression of Iba1 in microglia (red) within the dorsal horn was significantly increased on the ipsilateral side (f) compared with the contralateral side (e). Both the increase in immunoreactivity to P2X7R and Iba-1 were attenuated by i.t. PA treatment (c, g), which was analyzed and depicted by bar graphs showing mean fluorescence intensity (d, h). N = 4 animals/group for all bars. Scale bar, 25 μm. *P < 0.05 vs. SNL/PA

Fig. 6

Representative sections double labeled for anti-P2X7R (a, e) with anti-Iba1 (b) or anti-Neu-N (f) of the spinal dorsal horn on day 7 following L5 SNL. Right photographs (d, h) show amplified images derived from white open squares in merged images (c, g), indicating that P2X7R is expressed exclusively in microglia. Scale bar, 25 μm