The Regulation Of Cannabinoid Receptors Activated By Endocannabinoid 2-AG In The Neuropathic Pain Induced By Spared Nerve Injury

Objective: To investigate whether endocannabinoids 2-AG has the effect of inhibiting neuropathic pain induced by spared nerve injury, and whether AM281 and AM630 can reverse the inhibition effect of 2-AG through selective blocking cannabinoid receptor 1(CB1) and CB2 respectively, which can illuminate that cannabinoid receptor plays a key role on the analgesic effect.Materials and methods: Neuropathic pain animal model was established through spared nerve injury. Behavior experiment part, the change of mechanical pain threshold of rats after spared nerve injury was measured by 50% paw withdrawal mechanical threshold. The inhibition effect of 2-AG on neuropathic pain was observed by intrathecal injecting 2-AG and the reverse effect of cannabinoid receptor antagonists on 2-AG was detected by intrathecal injecting AM281 and AM630, respectively. The co-expression changes of cannabinoid receptor subtypes and astrocytes, the expression changes of glutamine synthetase(GS), which was observed by immunofluorescence morphology experiment and Western blot.Results: The mechanical threshold after nerve injury 4 days was significantly decreased(1.57g) and mechanical hyperalgesia also has happened. After intrathecal administration of endocannabinoids, mechanical pain threshold(about 13.67g) compared with SNI group increased significantly(F(1,71)=318.75,P<0.01), and 2-AG analgesic effect can maintain less than 30 min. After CB1 was blocked by AM281, the analgesic effect of 2-AG was significantly reversed(F(1,71)=346.20,P<0.01)and kept for a long time(60min). Meanwhile, after blocking CB2, the analgesic effect of 2-AG was significantly reversed from 45 min to 60 min, 2-AG group and CB2-blocking group was statistically significant different(F(1,71)=10.81,P=0.022<0.05).Three nerve cells in spinal dorsal horn after nerve injury had different degrees of activation, the activation change of astrocytes was the most obvious. The expression level of CB1 and GFAP was significantly higher than that of Naive group, and the level of GFAP/CB1 co-expression was also significantly increased(P<0.05). The activation degree of astrocytes in 2-AG group was inhibited, and the expression levels of CB1 and GFAP/CB1 were also decreased at the same time. GFAP, CB1 and GFAP/CB1 positive cell number in CB1-blocking group and CB2-blocking group were significantly higher than that of 2-AG group(P<0.05). Obvious positive markers of CB2 receptors in the tissue staining did not appear. Western blot experiment found that GS expression levels after nerve injury significantly improved compared with the Naive group(t=20.22,P<0.01), whereas 2-AG was able to inhibit the high expression of GS(t=13.08,P<0.01). Meanwhile, the expression of GS in CB1-blocking group significantly improved compared with the 2-AG group(t=6.91,P<0.01), but the expression of GS in CB2-blocking group didn’t improved significantly compared with the 2-AG group(t=2.81,P=0.186>0.05).Conclusion: The endocannabinoid 2-AG could inhibit neuropathic pain behavior induced by spared nerve injury after intrathecal injection, the analgesic effect was accomplished by activating CB1 and CB2 in astrocytes, inhibiting the expression of GS, and then reducing the release of glutamate in synaptic terminals; and the analgesic effect after CB1 activating was significantly stronger than CB2 activating.