Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

Abstract

Several voltage-gated sodium channels (Navs) from nociceptive nerve fibers have been identified as important effectors in pain signaling. The objective of this study is to investigate the electroacupuncture (EA) analgesia mechanism by changing the expression of Navs in mice dorsal root ganglia (DRG). We injected carrageenan and complete Freund's adjuvant (CFA) into the mice plantar surface of the hind paw to induce inflammation and examined the antinociception effect of EA at the Zusanli (ST36) acupoint at 2 Hz low frequency. Mechanical hyperalgesia was evaluated by using electronic von Frey filaments, and thermal hyperalgesia was assessed using Hargreaves' test. Furthermore, we observed the expression and quality of Navs in DRG neurons. Our results showed that EA reduced mechanical and thermal pain in inflammatory animal model. The expression of Nav1.7 and Nav1.8 was increased after 4 days of carrageenan- and CFA-elicited inflammatory pain and further attenuated by 2 Hz EA stimulation. The attenuation cannot be observed in Nav1.9 sodium channels. We demonstrated that EA at Zusanli (ST36) acupoint at 2 Hz low-frequency stimulation attenuated inflammatory pain accompanied by decreasing the expression of Nav1.7 and 1.8, rather than Nav1.9, sodium channels in peripheral DRG neurons.

Figure 1

Primary inflammation-induced mechanical and thermal hyperalgesia through carrageenan or CFA injection. (a) Electronic von Frey filament test; (b) radial heat assay; (c) and (d) the latency time for forepaw licking and jumping responses after exposure to a hot plate maintained at 50°C. (c) Latency to first forepaw licking, (d) escape times (jumping) within 5 minutes; (e) and (f) the hind paw withdrawal and rearing on exposure to a cold plate kept at 4°C; (e) withdrawal times; (f) rearing times within 5 minutes. Con: saline, Car: carrageenan and CFA: intraplantar injection into hind paw; Car-EA and CFA-EA: after carrageenan or CFA injection, needles inserted at the ST36 acupoint with electrical stimulation at 2 Hz. *P < 0.05, as compared to that of the baseline. **P < 0.01, as compared to that of the baseline. ^# P < 0.05; comparison between inflammation and EA-ST36 groups. ^## P < 0.01; comparison between inflammation and EA-ST36 groups. CFA: complete Freund's adjuvant. Solid lines mean carrageenan-injected group. Dot lines mean CFA-injected group.

Figure 2

Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

Figure 3

Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the CFA-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) CFA-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the CFA-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

Figure 4

Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan- and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan- and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan- and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.

Figure 5

Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3–L5 DRGs in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups. The percentage of Nav protein levels from lumbar DRGs was presented in each group. *P < 0.05, as compared to control group. ^# P < 0.05; comparison between inflammation and EA groups.

Figure 6

Tetrodotoxin-resistant (TTX-R) sodium currents in L3–L5 DRG neurons. (a) Representative TTX-R current traces in Con, CFA, and EA groups. The TTX-R currents were induced by membrane potential depolarized to −40 mV. (b) Mean peak amplitudes of TTX-R currents in each group. **P < 0.01 compared to control group. ^## P < 0.01 compared to CFA group.