| Objective: To investigate the mechanism of electroacupuncture regulating circadian rhythm of spontaneous activity in mice and expression of P2X7 receptor in astrocytes in SCN.Methods: Experiment 1: Wild-type C57BL/6J mice were selected and randomly grouped according to the transfer value of active phase by SPSS19.0statistical software.Due to the significant difference in phase transfer between CT8 and CT16 in our previous study,wild-type mice were only randomly divided into CT8 and CT16.Each time point was divided into 3 groups: control group,electroacupuncture group,non-acupoint group,6 animals in each group.After 8-10 days of free running,the mice were guided by the corresponding phase point.(1)Electroacupuncture group: after fixing the animal with a self-made fixer,choose two acupoints.In order to limit the current between the two acupoints,a needle was pricked at each shallow point near the two acupoints,and the needle was pricked subcutaneously.The two needle handles are connected to the positive and negative poles of HANS acupoint nerve stimulator.Non-acupoint group: The non-acupoint point was selected,that is,the point at the intersection of the mouse midaxillary line and the iliac crest at 1cm,and the point was directly pricking about 0.3 inches.Then a needle was taken near the non-acupoint and the needle was pricking subcutaneously.Connect the two needle handles to the positive and negative poles of the electroacupuncture apparatus respectively.The operating parameters of the electroacupuncture apparatus are the same as the electroacupuncture group.(3)Control group: do not do acupuncture and binding,continue to be placed under continuous dark conditions.After grouping at each time point,the mice continued their spontaneous activity for 8-10 days,and behavioral data were recorded and collected.To observe and compare the difference between the two time points of CT8 and CT16 in wild-type mice.Experiment 2: The experimental group was the same as Experiment 1,and each mouse was intraperitoneally anesthetized with 10% chloral hydrate under the condition that the red light was less than 10 Lux after 2h treatment in the corresponding group.After anesthesia,the mice were beheaded and brain tissues containing SCN were taken out.Then,they were quickly placed in a rubber tube containing paraformaldehyde solution for fixation and preservation.After brain tissues were collected from all animals,the expression of P2X7 in astrocytes in each SCN group was detected by immunofluorescence double-standard.Experiment 3:P2X7 knockout genotype mice were selected,and SPSS19.0 statistical software was used to randomly group the mice according to the transfer value of active phase.The genotype mice were divided into 6 time points,namely CT0,CT4,CT8,CT12,CT16 and CT20.Each time point was divided into 3 groups: control group,electroacupuncture group,non-acupoint group,6 animals in each group.The domestication and selection of experimental animals,guided stimulation experiment,grouping processing method and experimental data collection and analysis method are all the same with experiment 1.To observe and compare the differences of the initial activity time phase between different groups and different time points of genotype mice.Results :(1)When at CT8,the phase change of the spontaneous activity rhythm of wild-type mice of the electroacupuncture group was compared with that of the non-acupoint group and the control group,P<0.05,that is,the difference was very significant.At CT16,the rhythm phase change of spontaneous activity of wild-type mice of the electroacupuncture group was compared with those of the non-acupuncture point group and the control group(P<0.05),that is,the difference between the electroacupuncture group and the non-acupuncture point group and the control group was particularly significant.At CT8,wild-type C57 mice could transfer their spontaneous rhythm phase forward for about 0.60 hours on average.At CT16,wild-type C57 mice would drift their spontaneous circadian rhythm backward for about 2.15 hours on average.(2)At CT8,most of the astrocytes in the SCN of wild-type C57 mice in the electroacupuncture group showed activation state,while most of the astrocytes in the SCN of mice in the non-acupoint group and the control group showed resting state.Compared with the non-acupoint group,the average optical density of P2X7 receptor in brain SCN of mice in the electroacupuncture group was significantly increased(P<0.01),and the average optical density of P2X7 receptor in brain SCN of mice in the electroacupuncture group was also significantly increased compared with the control group(P<0.05).Compared with the control group,the average optical density of P2X7 receptor in astrocytes in brain SCN of mice in the electroacupuncture group was significantly increased(P<0.01),and compared with the non-acupoint group,the average optical density of P2X7 receptor in astrocytes in brain SCN of mice in the electroacupuncture group was also higher(P<0.01).(3)At CT16,most astrocytes in SCN of wild-type C57 mice in electroacupuncture group,non-acupoint group and control group showed resting state,and correspondingly,fewer activated astrocytes were found.Compared with the control group and the non-acupoint group,the average optical density of astrocytes in the brain tissue SCN of the electroacupuncture group showed no significant difference(P>0.05).Compared with control group and non-acupoint group,the average optical density of P2X7 receptor in astrocytes in brain SCN of electroacupuncture group showed no significant difference(P>0.05).(4)Under constant dark conditions,the starting time of spontaneous activity of P2X7-/-genotype mice moved forward successively.At CT8,both the electroacupuncture group and the non-acupoint group presented the phenomenon of forward metastasis at the time of spontaneous activity of P2X7-/-mice,but the change trend of the electroacupuncture group was more significant(P<0.05),while the control group showed no obvious change trend.Moreover,the forward drift value of the phase of the spontaneous activity of mice in the electroacupuncture group at CT8 was about 0.04 hours.When at CT16,the circadian phase change tendency of the spontaneous activity of P2X7-/-mice is significantly(P < 0.05),and it showed backward drift of circadian rhythm.which will make the circadian phase of the spontaneous activity of mice transfer backward about 2.73 hours when for electroacupuncture at CT16.And the spontaneous activity phase of P2X7-/-mice of the non-acupoint group and the control group is no significant change(P >0.05).However,after guided stimulation at other time points,pair comparison results of the electroacupuncture group,the control group and the non-acupoint group showed that there was no significant difference in the trend changes of the initial activity time of P2X7-/-mice among each group(P>0.05).(5)At CT8,there was a significant difference between wild-type C57 mice and P2X7-/-genotype mice in the electroacupuncture group in the phase transfer value of spontaneous circadian rhythm of mice(P<0.05),and wild-type C57 mice showed more phase transfer and larger transfer amplitude at the initial activity time.At CT16,wild-type C57 mice and P2X7-/-genotype mice showed no significant difference in the change trend of initial activity time and phase between the electroacupuncture group,the non-acupoint group and the control group.Conclusion:(1)Time-dependent circadian rhythm regulation by electroacupuncture is closely related to the difference in P2X7 receptor expression level in astrocytes in the suprachiasmatic nucleus of the hypothalamus.(2)The P2X7 receptor of astrocytes in SCN may be related to the phase forward transfer by electroacupuncture to regulate spontaneous activity of mice during subjectve daytime,but may not be significantly related to the late phase transfer. |
PDF Full Text Request