Analysis On Interrelation Between Electroacupuncture-induced Cumulative Analgesic Effect And Neuronal Plasticity And MAPK/ERK Signal Pathway In The Hippocampus

ObjectiveChronic pain is a long-time stimulation and brings great suffering to the patient. There was lot of attention on the analgesia that to release the pain. Hippocampus which not only has the function of learning and memory, but also play a central role in the regulation of pain is a part of the limbic system. Hippocampus involved in the process of acupuncture analgesia. The acupuncture accumulative analgesia has relationship with Ach, orphanin and the synaptic changes in hippocampus. To further study the effect of the acupuncture analgesia on the hippocampus, we observed the changes in discharge frequency stimulation of hippocampal neurons between the normal rats and the chronic constrictive injury (CCI) rats after trains of the electric impulses applied to the left sciatic nerve and electroacupuncture, and the relationship between the cumulative electroacupuncture and the expression of BDNF, MEK/ERK signal pathway and PKA, CREB in hippocampus.Methods:108Wistar rats were randomly divided into normal control(n=31), CCI (n=29), CCI+EA2D(n=16), CCI+EA1W(n=16), CCI+EA2W(n=16). The neuropathic pain model was established by sciatic nerve ligation(CCI). EA(2/15Hz,1-2m A,30min) was applied to "Zusanli"(ST36)-"Yanglingquan"(GB34) for30min, once daily for2days,1week and2weeks, respectively. The thermal pain threshold(PT) of the bilateral paws was detected the next morning after EA. Train of the electric impulses applied to the left sciatic nerve were used as noxious stimuli, discharges of hippocampal neurons were registered by extracellular recording method with metal electrode. The expression of trk2mRNA, PKAmRNA and CREBmRNA in hippocampus were detected by real-time PCR, and hippocampal Raf, MEK, p-ERK1/2, P38, CREB expression were assayed by western blot.Results:1The influence of cumulative electroacupuncture on changes of behavioral performance in each groups1.1The behavioral performance of CCIThe CCI rats spontaneously lifted the surgical limb (left hind limb), curled and retracted the toes24h after operated. In the4days after operated, we observed some behavior abnormalities of eversion of foot, the limb claudication, avoidance a heavy burden on the limb, but not a drag. The heat radiating pain was caused by a slight thermal radiation. The CCI rats had a long time licks and shakes of the limb, but not self-mutilation.1.2Comparison of the thermal pain threshold(PT) in rats of different groupsIn comparison with the normal control group, the PT in CCI rats was increased considerably in4days after operated (P<0.05).In18days after operated, the PT of CCI group was still higher than the normal control group (P<0.05).In the CCI+EA2D group, the PT was higher than the normal control group (P<0.05) and lower than the CCI group (P<0.05). Compare with the CCI and CCI+EA2W group, the RT of CCI+EA1W was down-regulated considerably (P<0.05). The PT of CCI+EA2W group was significant lower than CCI、 CCI+EA2D group (P<0.05). Comparison between CCI+EA2W and CCI groups showed that there was no difference(P>0.05), suggesting a cumulative analgesic effect of repeated EA2The effect of electroacupuncture analgesia on discharge of hippocampal pain-relation neurons(PEN)2.1The effect of noxious stimuli and electroacupuncture on neuronal discharge in the normal control groupThe discharge frequency of hippocampal PEN (n=17) were increased significantly in the noxious stimuli (134.53%±37) and EA group (126.1%±20.05) after given a noxious stimuli (P<0.05) in normal control rats and there was on difference between the two groups (P>0.05). The discharge frequency of hippocampal PEN in noxious stimuli group were markedly higher than the EA group at2,4,6min after given the noxious stimuli (P<0.05). No significant differences were found between the noxious stimuli group and the EA group at8and10min after given the noxious stimuli(P>0.05).It suggested EA played an inhibited role in regulating the discharge of PEN after given the noxious stimuli.The discharge frequency of hippocampal PIN (n=12) were decreased significantly in the noxious stimuli (67.88%±14.71) and EA group (93.35%±15.83) after given a noxious stimuli in normal control rats.The frequency of the noxious stimuli group was evidently lower than EA group (P<0.05). In comparison with the noxious stimuli group, the frequency of EA group were increased considerably (P<0.05) at2,4,6,8,10min after given the noxious stimuli.It suggested EA played an excited role in regulating the discharge of PIN after given the noxious stimuli.2.2The effect of noxious stimuli and electroacupuncture on neuronal discharge in CCI control groupThe discharge frequency of hippocampal PEN (n=14) were increased significantly in the noxious stimuli (204.24%±95.07) and EA group (203.34%±127.56) after given a noxious stimuli in CCI rats and there was on difference between the two groups(P>0.05). The frequency of hippocampal PEN was reached193.54%±80.45in noxious stimuli group and markedly higher than EA group (133.41%±43.19, P<0.05).The frequency of hippocampal PEN in EA group (113.6%+64.66) recovered to the normal levels, and observably lower than noxious stimuli group (188.82%±60.45, P<0.05) at6min after given the noxious stimuli.The discharge frequency of hippocampal PIN (n=4) were decreased significantly in the noxious stimuli66.44%±17.35) and EA group (83.21%±36.84) after given a noxious stimuli (P<0.05) in CCI rats and there was on difference between the two groups(P>0.05). There was no difference between the two groups at2,4min after the noxious stimuli(P>0.05). At6min after the noxious stimuli, the frequency of hippocampal PIN in the noxious stimuli group (59.82%±19.14) was still inhibited and the frequency of EA group (94.22%±41.45) recovered to the normal levels. At8and10min after the noxious stimuli, the frequency of hippocampal PIN reached to70.65%±20.62�'�90.75%±18.50in noxious stimuli group. The discharge frequency of noxious stimuli group and EA group had no significant changes(P>0.05).2.3Comparison of the discharges of the normal control rats and CCI rats after noxious stimuli and electroacupunctureAt2min after the noxious stimuli, there was no difference between the normal control group and the CCI group (P>0.05);4min,6min,8min after noxious stimuli, the discharges of PEN in CCI group markedly higher than the normal control group<P0.05), and the discharge of normal control group and the CCI group had no significant changes (P>0.05) at10min after noxious stimuli. Except for the moment of given a noxious stimuli,2,4,6,8,10min after noxious stimuli, there were remarkable difference between the two group (P<0.05)The discharge frequency of normal control group and the CCI group had no had no significant changes (P)0.05), except for the moment of given a noxious stimuli.2.3The effect of inhibitor of ERK U0126on the changes of discharge induced by electroacupuncture in ratsThe discharge frequency of hippocampal PEN of U0126(n=9) group and EA group had no significant changes (P>0.05) after given a noxious stimuli.The frequency of hippocampal PEN in the U0126group (190.52%±49.01) was higher than the EA group (121.76%±11.41, P<0.05) at2min after the noxious stimuli(after injection the U0126).At4,6,8min after the noxious stimuli, the discharge of the U0126group were still higher than the EA group(P<0.05). There were no difference between the U0126group and the EA group at10min after noxious stimuli (P>0.05).The discharge frequency of hippocampal PIN of U0126(n=6) group and EA group had no significant changes (P>0.05) after given a noxious stimuli At the moment of injection of U0126(2min after noxious stimuli), the U0126group (55.6%±29.67) was lower than the EA group (74.36%±16.62, P>0.05). At4,6,8,10min after noxious stimuli, the U0126group were significantly lower than the EA group(P<0.05)3The effect of electroacupuncture analgesia on hippocampal MEK/ERK signal pathway in different groups3.1The effect of EA on hippocampal BDNF and trk2expression Compare with the normal control group, the expression level of hippocampal BDNF in CCI group was decreased considerably (P<0.05).In comparison with the CCI group, the hippocampal BDNF expression in the CCI+EA2D, CCI+EA1W and CCI+EA2W group was upregulated considerably (P<0.05). The expression of hippocampal BDNF in CCI+EA2W was higher than the CCI+EA2D and CCI+EA1W group(P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal BDNF expression. NO significant difference were found between CCI+EA2D and CCI+EA1W groups in hippocampal BDNF(P>0.05). Compared with normal control group, the expression level of hippocampal trkmRNA in CCI group was decreased significantly (P<0.05). Compare with the CCI group, hippocampal trkmRNA expression in CCI+EA2D, CCI+EA1W and CCI+EA2W group had no significant changes(P>0.05).3.2The effect of EA on hippocampal MER/ERK signal pathway3.2.1The effect of EA on hippocampal Ras-. C-Raf and MEK expression Compared with the normal control group, the expression of hippocampal Ras in CCI group was decreased considerably(P<0.05). Compared with the CCI group, hippocampal Ras expression was up-regulated slightly in CCI+EA2D and CCI+EA1W(P>0.05), significantly in CCI+EA2W (P<0.05). In comparison with the CCI+EA2D and CCI+EA1W group, hippocampal Ras expression in CCI+EA2W group was up-regulated considerably(P<0.05).Compared with the normal control group, the expression level of hippocampal C-Raf was down-regulated markedly (P<0.05). In comparison with the CCI group, the hippocampal C-Raf expression in CCI+EA2D and CCI+EA1W group was increased slightly(P>0.05), and in CCI+EA2W was increased considerably(P<0.05).Compared with the normal group, the expression level of hippocampal MEK in CCI group had no significant changes(P>0.05). No significant differences were found between CCI, CCI+EA2D, CCI+EA1W and CCI+EA2W group(P>0.05).3.2.2The effect of EA on hippocampal ERK1/2expressionCompared with the normal control group, the expression level of hippocampal ERK1/2mRNA and ERK1/2in CCI group had no significant changes(P>0.05). No significant differences were found between those groups in hippocampal ERK1/2mRNA and ERK1/2expression (P>0.05) expect the ERK1expression in CCI+EA2W group was higher than in CCI group (P<0.05). In comparison with the normal control group, the hippocampal p-ERK1/2expression in CCI group were down-regulated considerably (P<0.05). Compared with the CCI group, hippocampal p-ERK1/2expression in CCI+EA2D and CCI+EA1W group were increased significantly (P<0.05). The expression level of hippocampal p-ERK1/2in CCI+EA2W group were higher than in CCI group and in CCI+EA2D group(P<0.05). There was no significant difference between CCI+EA1W and CCI+EA2W groups in hippocampal p-ERK1/2expression(P>0.05). It suggested a cumulative effect of repeated EA in up-regulating hippocampal p-ERK1/2expression.3.3Effect of EA on p38and p-p38expression in the hippocampusCompared with the normal control group, the p38MAPKmRNA expression of hippocampus in CCI group, CCI+EA2D group and CCI+EA1W group were decreased considerably (P<0.05). The expression level of hippocampal p38MAPKmRNA was markedly higher than those in CCI group and CCI+EA2D group (P<0.05), and markedly lower than that in the normal control group(P<0.05). The expression level of hippocampal p38MAPK had no significant changes between the different groups (P>0.05).In comparison with the normal control group, the expression of hippocampal p-p38MAPK in CCI group was decreased slightly (P<0.05). Comparison between CCI group and CCI+EA2W group showed that the expression level of hippocampal p-p38MAPK was significantly lower in the former group than in the latter group (P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal p-p38MAPK expression.3.4Effect of EA on hippocampal PKA mRNA and CREB mRNA, CREB expression Compared with the normal control group, the expression of hippocampal PKA mRNA in CCI group was increased considerably (P<0.05). Compared with the CCI group, the hippocampal PKA mRNA expression in CCI+EA2D and CCI+EA1W group were significantly up-regulated (P<0.05). The expression of hippocampal PKA mRNA in CCI+EA2W was lower markedly than that of in CCI+EA2D and CCI+EA1W group(P<0.05).Compared with the normal control group, the expression of hippocampal CREB mRNA in CCI and CCI+EA2D group was increased considerably (P<0.05).In comparison with the CCI group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased markedly (P<0.05), while comapaed with the CCI+EA2D group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased lightly (P>0.05).It suggested a cumulative effect of repeated EA for1weeks and2weeks in down-regulating the hippocampal CREB mRNA increase expression caused by CCI. However, there was no significant changes between those different groups in the expression level of hippocampal CREB and p-CREB.ConclusionThe present study is representing behavioral, electrophysiological and molecular evidence to observe the relationship between electroacupuncture analgesia and hippocampal neuron plasticity changes and hippocampal ERK signaling pathway. The electrical activity of hippocampal pain-relation neurons had abnormal changes in the CCI model. There was a cumulative effect of repeated EA in relieving the state of the thermal radiation hyperalgesia. The effect of EA analgesic had close relationship with the hippocampal ERK signaling pathway.