Role Of Midbrain Periaqueductal Gray P2X₇ Receptor In Electroacupuncture Tolerance Rats

Adenosine 5’-triphosphate(ATP) is a fast excitatory neurotransmitter, which relate to some physiological and pathological processes after binding to its receptors. Currently, the P2 X receptors have been cloned 7 subtypes(P2X1-7), and the P2X7 subtype has been attracted much attention for its unique molecular structure. P2X7 receptor is widely expressed in various tissues and closely related to the mechanisms of inflammation, cancer and various neurological diseases. As an important non-drug analgesic method, electroacupuncture(EA) is widely and effectively used in clinical analgesic treatments. In recent years, the studies have revealed that P2 X receptor participated in the process of EA analgesia. The midbrain periaqueductal gray(PAG) is an important part of endogenous analgesic system, especially the lateral periaqueductal gray(l PAG) as the key structure in pain modulation and analgesic tolerance. However the role and the cellular signal transduction mechanisms of the l PAG P2X7 receptor in EA tolerance are still unrevealed.Objective: To observe the percent changes of the pain threshold and the expression alteration of the l PAG P2X7 receptor in EA tolerant rats. Additionally, the related intracellular signal transduction mechanisms in EA tolerance were also explored.Methods: The experiment includes two parts: 1) In vivo experiments: The EA tolerant model rats were established in healthy male Sprague Dawley(SD) rats by given multiple EA stimulations in the peripheral acupuncture points, and the thermal withdrawal latency(TWL) and mechanical withdrawal threshold(MWT) were tested by using the radiant heat and the electronic von Frey anesthesiometer, respectively. The immunohistochemical technique and Western blot were used to detect the expression of P2X7 receptors in the l PAG on day 6 post-EA. The effects of intra-l PAG injection of different doses of A-740003, a selective antagonist of P2X7 receptor, on TWL and MWT percent changes were studied on EA tolerant rats. 2) In vitro experiments: The cultured primary SD rat l PAG neurons were used for the observation of the P2X7 receptor activation-induced [Ca2+]i level fluctuation by the laser confocal microscopy.Results: In vivo experiments: Compared with the normal group, the TWL, MWT percent changes and the expression of P2X7 receptors in the l PAG in the sham EA group were not statistically significant(P > 0.05). The TWL and MWT percent changes of the EA group were markedly increased(P < 0.01, versus normal group) and then decreased gradually to the level of the normal group(P > 0.05, versus normal group). These results demonstrated that the EA tolerant model rat was established after 6 times of the EA treatment. The immunohistochemical technique and Western blotting showed that the expression of P2X7 receptors in the l PAG was obviously up-regulated in the EA group(P < 0.001, versus normal group). The intra-l PAG microinjection of A-740003 at doses of 10 and 100 nmol increased the MWT and TWL percent changes in the EA tolerant rats. Furtherly, double immunofluorescence results indicated a co-exprssion of the P2X7 receptor and Neu N in the l PAG neurons. On the contrary, there was no co-expression was existed for the P2X7 receptor with the astrocyte specific marker GFAP and microglia specific marker Iba1. In vitro experiments: The laser confocal microscopy results suggested that the levels of [Ca2+]i of the primary cultured l PAG neurons were significantly increased by the P2X7 selective agonist Bz ATP, but the increased levels of the neuron [Ca2+]i were inhibited by the P2X7 receptor specific antagonist A-740003 or Ca2+ free solution.Conclusion: Multiple EA treatments can decrease the percentage of pain thresholds in rats and induce EA tolerance via the activation of P2X7 receptors in the l PAG. This effect can be reversed by a selective antagonist of P2X7 receptor(A-740003). The [Ca2+]i level of l PAG neurons increased markedly by the P2X7 receptor agonist Bz ATP that may be related to extracellular [Ca2+]i influx and this effect is inhibited by A-740003 or Ca2+ free solution.