Objective:To observe the effects of repeated electroacupuncture(EA) of Zusanli(ST36)- Yanglingquan(GB34) on hypothalamic acetylcholinesterase(AchE) and vesicular acetylcholine(ACh) transporter(VAChT) activities and choline acetyltransferase(ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury(CCI) and/or ovariectomy(OVX) rats so as to reveal its underlying mechanism in cumulative analgesia.Methods:A total of 103 female Wistar rats were randomly divided into normal control(n=15),CCI(n=15),CCI+EA2d(n=15),CCI+EA2W(n=15),OVX+CCI (n=13),OVX+CCI+EA2d(n=15),and OVX+CCI+EA2W groups(n=15).CCI model was established by ligature of the unilateral sciatic nerve with surgical suture.Memory impairment model was established by removal of the bilateral ovaries.Morris water test was conducted to evaluate the OVX rats’ memory- learning ability,and the thermal pain threshold(PT) of the bilateral paws was detected the next morning after EA.EA(2/15 Hz,1 mA) was applied to bilateral ST36-GB34 for 30 min,once daily for 2 days or 2 weeks,respectively.Hypothalamic AChE activity was detected by histochemistry,VAChT immunoactivity was determined by immunohistochemistry,and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction(RT-PCR).Results:In comparison with the normal control group,the AChE activity in hypothalamic arcuate nucleus(ARC) and supraoptic nucleus(SON) regions of CCI group,AChE activity in paraventricular nucleus(PVN),ARC,and SON regions of OVX+CCI group,and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly(P<0.05).Compared with the CCI group,the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably(P<0.05).In comparison with the OVX+CCI group,the AChE activities in PVN,ARC,and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably(P<0.05).The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN,ARC,and SON regions, VAChT immunoactivity in ARC region,and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P<0.05).Similar situations were found in OVX+CCI rats after EA2W.It suggested a cumulative effect after repeated EA of ST36-GB34.Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P<0.05),suggesting a reduction of EA2W effects after OVX.Conclusion:Repeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats,which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals’ neuromemory ability. Objective: To observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36) - Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (M1R) mRNA expression in chronic constrictive injury (CCI) and / or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia. Methods: A total of 103 female Wistar rats were randomly divided into normal control (n = 15 CCI + EA2d (n = 15), CCI + EA2W (n = 15), OVX + CCI (n = 13), OVX + CCI + EA2d (n = 15), and OVX + CCI + EA2W groups (n = 15) .CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats’ memory- learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain the AChE activity in parahaemic nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and The ATP regions of OVX + CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX + CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI + EA2d and CCI + EA2W groups and PVN region of CCI + EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI + EA2W group were up-regulated considerably (P <0.05) .In comparison with the OVX + CCI group, the AChE activities inPVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX + CCI + EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI + EA2W group were evidently superior to those of OVX + CCI + EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05) OVX + CCI rats after EA2W.It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI + EA2W and OVX + CCI + EA2W groups showed that the effects in rats of the former group were evidently better than those of latter suggesting that a reduction of EA2W effects after OVX.Conclusion: Repeated EA can significantly up-regulate AChE and VAChT activities (P <0.05) and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX + CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals’ neuromemory ability.