Role Of Midbrain Periaqueductal Gray P2X3 Receptors In Electroacupuncture-mediated Endogenous Pain Modulatory Systems

The study on pain mechanisms is always a hot topic in clinic and neuroscience research fields. Neuropathic pain is known as a notorious devastating consequence of central or peripheral nerve injure which is characterized by a combination of spontaneous pain, hyperalgesia and allodynia. Increasing evidence has revealed that the extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain for thousands years in Eastern countries but the mechanisms underlying EA analgesia are still uncertain.Objective to⑴study the function of P2X3 receptors in the lateral midbrain periaqueductal gray (lPAG); the relationship between P2X3 receptor expression and neuropathic pain induced by a chronic constriction injure of the right sciatic nerve on Sprague-Dawley(SD) rats. By immunohistochemistry and Western blotting, we observe the P2X3 receptor protein level and expression location in the lPAG and further explore the effect of pretreatment with A-317491, a novel potent and selective antagonist of P2X3 and P2X2/3 receptors, on the analgesic effect of lPAG microinjection of alpha,beta-methylene ATP(α,β-meATP).⑵investigate whether repetitive administration of electroacupuncture (EA) on'zusanli'and'sanyinjiao'acupoints could reduce thermal and mechanical hyperalgesia in a rat neuropathic pain model and to explore the relationship between the antinociceptive effect of EA treatment and the expression of P2X3 receptor in the lPAG.Methods: The experiment was composed by four series:Series 1. Changes of pain thresholds of SD rats after establishment of rat CCI (chronic constriction injury) model. Study the expression changes of P2X3 receptor in the lPAG by immunohistochemical and immunoblotting analysis. Series 2. Observation of effect of pretreatment with A-317491 on the analgesic effect of lPAG microinjection ofα,β-meATP and the effect of A-317491 onα,β-meATP-induced current of the neonatal rat lPAG neurons. Series 3. Influence of EA treatments on the pain thresholds and investigation of change of P2X3 receptor expression in the lPAG posterior to EA or sham-EA treatment and explore the acupoint specificity of EA treatment on CCI rat either. Series 4. Analgesic effect changes of EA treatments in response to lPAG microinjection of P2X3 antisense ODN.Results:Part one⑴The inclined-plane test was used in all experimental rats before algesimetry test to study whether CCI operation, drugs or ODNs microinjection and EA treatment exerted certain adverse effects on rats motor function and proprioception, there was no significant difference on the average maximum angle among all experimental groups.⑵The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured before CCI operation on the right sciatic nerve and at days 2,4,7,10,12,14,18 and 21 respectively after CCI operation. In CCI group, the hind-paw TWL and MWT were decreased gradually and down to the lowest point on day 14 after CCI operation. In the normal and sham-CCI groups, from day 4 to day 21 after CCI operation, the values of TWL and MWT were quite stable.⑶Immunoblots from lPAG homogenates showed an immunopositive band of P2X3 receptors with molecular weight around 55 kDa both in normal, sham-CCI and CCI groups. There was no significant difference between normal and sham-CCI groups. In CCI group, sciatic nerve ligation caused an increase of P2X3 protein level in the lPAG.⑷In CCI groups, P2X3 receptor-specific immunoreactivity was mildly higher and a little more extensive compared with normal group or sham-CCI group at day 14 after CCI operation.Part two⑴Rats were randomly divided into two groups, the rats received intra-lPAG injection with sterile saline (0.3μl) or A-317491 (1.5nmol/0.3μl) once daily for 5 consecutive days. After pre-treatment, TWL and MWT showed no significant difference between above two groups. Microinjection of A-317491 alone cause no long-term effects on pain thresholds of CCI rats .⑵The saline and A-317491 group were then divided into two subgroups before microinjection with PBS orα,β-meATP. lPAG microinjection with vehicle (0.3μl PBS) induced no change of the thermal or mechanical nociceptive threshold. In saline group, lPAG microinjection ofα,β-meATP (30 nmol/0.3μl) significantly elevated nociceptive thresholds compared with beforeα,β-meATP microinjection. The antinociceptive effect ofα,β-meATP was attenuated significantly by A-317491 pretreatment.⑶Whole cell voltage-clamp recording was performed on neonatal rat brain slices to obtain the currents of the lPAG neurons. Our data show that A-317491 attenuated the frequency of theα,β-meATP-induced current in lPAG neurons.Part three⑴Multiple EA treatments had no significant effect on TWL and MWT values of rats in normal plus EA group. On the contrary, multiple EA treatments significantly increased TWL and MWT values of rats in CCI plus EA group.⑵The P2X3 receptor protein level in lPAG was detected after multiple EA treatments, which reached a statistically significant high level in CCI plus EA group but not in normal plus EA group.⑶The pain thresholds of CCI rats were elevated after multiple sham-EA treatments but still remained lower than rats in CCI plus EA group.⑷NA(non-acupoint)-EA treatments had no significant effect on pain thresholds and the P2X3 receptor protein level in the lPAG of CCI plus NA-EA group rats was not changed after NA-EA treatments.Part four⑴TWL and MWT were both increased after 7 times of EA treatment in the antisense ODN or mismatch ODN group rats compared with CCI rats without EA treatment.⑵The antinociceptive effect of EA treatment was attenuated significantly by P2X3 antisense ODN intra-lPAG injection.Conclusion: The expression change of P2X3 receptor in the lPAG and the elevated pain thresholds after intra-lPAG injection ofα,β-meATP indicate that P2X3 receptors in the lPAG play an inhibitory role in the pain modulatory and thus may be a central target for analgesics. EA produces a long lasting analgesic effect on neuropathic pain and increases expression of P2X3 receptors in the lPAG. The present study for the first time addressed the involvement of purinergic signaling system in EA analgesia on neuropathic pain in rat CCI model, as could deepen our understanding of the mechanism of EA analgesia and provide a rational basis for enhancing EA analgesic effect by potentiating the function of purinergic signaling system at supraspinal level.