Role Of Midbrain Periaqueductal Gray D-serine In Electroacupuncture Tolerance Rats

The NMDA receptor(N-methyl-D-aspartate receptor)is a subtype of ionotropic glutamate receptors,which plays a vital functional role in the central nervous system.Only by binding to the “glycine site” on the NR1 subunit,NMDA receptor is effectivly activated and opens the coupled ion channel.The accumulated research have shown that D-serine(D-Ser)is an endogenous ligand of the “glycine site” of the NR1 subunit,which is involved in many physiological and pathological processes via regulating the activation of NMDA receptors,including the modulation of pain information.In recent years,the study of D-Ser function in the central nervous system has become one of the hot spot in the research field of neuroscience.As an important non-drug analgesic method,electroacupuncture(EA)is widely and effectively used in clinical analgesic treatments.With a dual efficacy of acupuncture and electrical stimulation,EA is a combination of traditional acupuncture and electrical stimulation has a satisfied analgesic effect in clinical trials.But multiple electro-acupuncture stimulation,the analgesic effect of EA will gradually reduced and disappeared,manifested as electroacupuncture tolerance(EA tolerance).The midbrain periaqueductal gray(PAG)is a vital part of the endogenous analgesic system,especially the ventrolateral periaqueductal gray(vl PAG)area,the key structure in pain modulation and analgesic tolerance.The studies showed that there exist a cross tolerance between morphine tolerance and EA tolerance.However,it is not clear whether D-Ser is involved in the mechanisms of EA tolerance by regulating the function of NMDA receptor in the vl PAG.The purpose of this study was to investigate whether the vl PAG D-Ser was involved in the formation and maintenance of the EA tolerant mechanisms on rats.Objective: This study aimed to investigate the role of vl PAG D-Ser in the mechanisms of the EA induced tolerance in rats.Methods: Male Sprague-Dawley(SD)rats were randomly divided into 7 groups,namely,normal group,sham-EA group,EA group,EA plus artificial cerebrospinal fluid(a CSF)group and EA plus Fc group.The EA plus Fc group was divided into low,middle and high dose Fc subgroups,and had 0.3?g,0.6?g and 1.2?g Fc intra-vl PAG(intra-ventrolateral PAG)injection respectively.The EA tolerant model rats were established in healthy male SD rats by given multiple EA stimulations in the peripheral acupuncture points,and the thermal withdrawal latency(TWL)and mechanical withdrawal threshold(MWT)values were tested by using the radiant heat and the electronic von Frey anesthesiometer,respectively.The immunohistochemical technique and Western blot analysis were used to detect the expression of SR protein in the vl PAG on day 6 post-EA treatment.The ELISA technique was used for determination the alterations of D-Ser level in the rats vl PAG.The immunohistochemical technique was used to detect the expression of glia fibrillary acidic protein(GFAP)in the vl PAG on day 6 post-EA.The effects of vl PAG micro-injection of fluorocitric acid(Fc),a glial metabolic inhibitor,on MWT and TWLvalues and the D-Ser concentrations were further studied on EA tolerant rats.Results: At each observation time point,compared with normal group and sham EA group,the MWT and TWL percentage change were no significant difference(P > 0.05).The MWT and TWL percentage change were greatly increased in EA group rats(P < 0.001,versus normal group)and then decreased gradually to the level of the normal group(P > 0.05,versus normal group).These results showed that the EA tolerant model rat was established after 6 times of the EA treatment.At the same time,the concentration of D-Ser,the expression of SR protein levels,the number of SR positive cells and GFAP positive cells in the vl PAG was obviously up-regulated in the EA group(P < 0.001,versus normal group).The intra-vl PAG injection of different doses of Fc(0.3,0.6,1.2),a glial metabolic inhibitor,inhibited the activation of the vl PAG astrocyte followed by the concentrations of D-Ser in vl PAG significant decreased,and the percentage of MWT and TWL increased on the EA tolerant rats.All the difference were statistically significant(P < 0.001,versus normal group).Conclusion: Multiple EA treatments-(> 6 times)can decrease the percent change of the pain thresholds in rats and induce EA tolerance via an activation of the astrocyte accompanied by an increased D-Ser release in the vl PAG on rats.