The Analgesic Effect Of Endocannabinoids In Neuropathic Pain And The Role Of Glutamate-glutamine Shuttle

Objective:To confirm the dose-effect relationship of the analgesic effect of 2-arachidonoylglycerol in spared nerve injury model and explore if 2-arachidonoylglycerol alleviate neuropathic pain by suppressing astroglial-neuron glutamate-glutamine shuttle.Methods:Forty-eight adult male Sprague Dawley rats, the body weight between 180 and 220 g, were randomly divided into 8 groups (n=6). Normal group:rats in this group without any intervention; Sham group:the sciatic nerve and its three terminal branches were only exposed and not be ligated nor cut; SNI group:The common peroneal and the tibial nerves were tightly ligated with 4-0 silk sutures and sectioned distal to the ligation, removing 2 mm to 4 mm of the distal nerve stump. Any stretching or contact with the intact sural nerve was avoided; N.S. group:after the SNI model has been established, the rats received intrathecal injection of 15μl normal saline; 2-AG intervention group 1-4:after the SNI model has been established, the rats received intrathecal injection of 2-AG (50nmol、100nmol、200nmol and 400nmol, respectively). Mechanical withdrawal threshold test were conducted one day before the surgery, the following days after SNI procedure and once every 15 min up to 60 min post intrathecal injection. After the last behaviour test, the rats were immediately sacrificed and the L4-6 spinal cords were rapidly collected. The double sign immunofluorescence measurement were performed to confirm the expression of CB1, CB2 and GS in spinal cord of rats in normal group, model group and treatment group. The Western blot were conducted to semi-quantitative analysis the expression of CB1, CB2 and GS protein.Results:The behavioral experiments showed that there is no statistical significance between Normal group and Sham group in rats mechanical withdrawal threshold; compared with Normal group, the rats mechanical withdrawal threshold decreased significantly in SNI group (F(1,71)= 3844.857, P<0.001); there is no statistical significance between SNI group and N.S. group in rats mechanical withdrawal threshold. Intrathecal injection of endocannabionoid 2-AG (50,100, 200,400nmol; Volume:15μl) inhibited the nociceptive behaviors, and with the increase of 2-AG concentration, the analgesic effect increased, and indicated a dose-dependent manner. The linear regression statistical analysis showed the ED50 value of analgesic effect of 2-AG in SNI neuropathic pain model is 227.25nmol.Immunofluorescence assays showed that compared with Normal group, neurons, astrocytes and microglia in the SNI model group were activated at different degrees. In Normal group, CB1 receptors were expressed in neurons, astrocytes and microglia, In SNI group, CB1 receptor expression were significantly enhanced, and mainly co-expressed with astrocytes. After the intervention of 2-AG, expression of neurons, astrocytes and microglia decreased, while the expression of CB1 receptors also weakened. CB2 receptor expression were not observed in the Normal group and SNI group. In normal group, the expression of GS was observed but the expression was low; in the SNI group, the expression of GS significantly enhanced, mainly expressed in astrocytes. After 2-AG intervention, it significantly inhibited the expression and co-expression of GS and GS in astrocytes.Western-blot indicated that CB1 cannabinoid receptor level was higher in Model group than Normal group (P<0.001), intrathecal injection of endocannabionoid 2-AG did not influcene CB1 expression. CB2 receptor expression was not detected in Normal group, but in the Model group CB2 was significantly increase (P<0.001), and 2-AG intervention inhibited the expression of CB2 receptors (P<0.01). GS in the Model group was higher than Normal group (P<0.001); 2-AG intervention significantly inhibits the GS expression (P<0.001).Conclusion:Endocannabinoid 2-AG inhibits SNI induced neuropathic allodynia. One of the mechanisms of the analgesic effect may be mediated by activating cannabinoid CB 1 receptors on astrocytes, inhibiting glutamate-glutamine cycle between astrocytes and neurons, reducing the synaptic terminals release of excitatory neurotransmitter glutamate to achieve. But we can not exclude the possibility of an analgesic effect by CB2 receptors, while the 2-AG analgesic effect by the action of the CB 1 receptors in neurons and microglial cells also can not be excluded.