| BACKGROUNDThe autonomic nervous svstem(ANS)maintains important life activities of the human body.Its main function is to regulate the activities of the body’s internal organs,blood vessels,smooth muscles and glands,and to participate in the body’s response to internal and external environmental stimuli.The occurrence and development of many diseases are related to autonomic nervous dysfunction.As a simple and effective method,acupuncture is often used in the treatment of diseases related to ANS disorders.The research results related to the mechanism of acupuncture and moxibustion suggested that acupuncture can regulate the function of internal organs and ANS.In recent years,domestic and foreign scholars have done a lot of work in regulation of acupuncture on ANS,but the conclusions obtained from animal experiments are not consistent.Considering that the anesthetics must be applied in the experiment,the effects and duration of different anesthetics on autonomic nerves are different.Then,in the study of the mechanism of acupuncture effect,whether the application of anesthetic will affect the research results?Is the uses of different anesthetics in animal one of the reasons for the different experimental results of the relevant experiments?So far,there are no clear research reports on the scientific problems mentioned above at home and abroad.OBJECTIVEThe aim of the present study was to observe the changes of heart rate variability(HRV)and autonomic nerve discharge in rats before and after twisting acupuncture at Neiguan(PC 6)acupoint under anesthesia of three different anesthetics and to explore the influence of different kinds of anesthetics on the regulation of autonomic nerve effect by acupuncture at Neiguan(PC 6)acupoint.The completion of the study will not only provide a scientific explanation for minimizing the influence of anesthetics in the study of acupuncture regulation of autonomic nerve,but also provides a basis for preliminary research on how to use anesthetics reasonably in different acupuncture mechanisms..METHODSIn this experiment,clean Sprague-Dawley(SD)rats were used as experimental animals.Thirty-six healthy male SD rats were recorded after ECG was recorded in awake state.And then,they were randomly divided into six groups:urethane control group(n=6),urethane acupuncture group(n=6),pentobarbital sodium control group(n=6).=6),pentobarbital sodium acupuncture group(n=6),chloral hydrate control group(n=6)and chloral hydrate acupuncture group(n=6).Each control group was given the above anesthetics respectively,and only recorded after anesthesia stabilization without acupuncture.Each acupuncture group was treated with twisting acupuncture at Neiguan(PC 6)acpoint.The twisting needling manipulation was performed one minute at a time.The method was taken three times and each interval was one minute.After the last twisting needling manipulation,the needle was taken out.The record was continued about 10 minutes.The experiment was carried out at a fixed time(9:00-11:00)every morning in a constant shielding environment at room temperature in order to reduce the influence of environmental factors.Cardiac sympathetic and vagus activity were recorded in vivo by neuroelectrophysiological techniques,and respiratory rate,standard II lead electrocardiogram,heart rate and autonomic nerve discharge were measured by Powerlab physiological recorder.In the later stage.the HRV data were analyzed by short-term frequency domain analysis.The values of heart rate,respiratory rate,HRV and frequency of nerve discharge were recorded into SPSS 19.0 statistical software at three fixed time points,i.e.before acupuncture,during acupuncture and after acupuncture.One-way analysis of variance(ANOVA)analysis was used to analyze the difference.A value of P<0.05 was considered statistically significant.RESULTS1 Comparison of heart rate in different time periods of the rats in each groupThere was no significant difference in heart rate of the rats in each groups when they were awake(all P>0.05),which indicated that the initial state of the rats in each group was basically the same.There was no obvious fluctuation in the control group at each time point after anesthesia.It is suggested that the heart rate can remain stable after anesthesia stability induced by the three anesthetics.The heart rates after anesthesia of the rats in urethane and chloral hydrate control groups were significantly lower than that of the two control groups when they were awake,and the decrease of the heart rate in chloral hydrate control group was significantly lower than that in urethane control group(P<0.01),while The heart rates after anesthesia of the rats in pentobarbital sodium control group was higher than that when awake,but there was no significant difference(P>0.05).It was suggested that both urethane and chloral hydrate anesthesia can slow down the heart rate,while pentobarbital sodium anesthesia may cause a slight increase in heart rate;on the other hand,the heart rate of the rats in the three acupuncture groups increased after acupuncture compared with that after anesthesia,but there was no statistical difference.(all P>0.05).2 Comparison of respiratory frequency of the rats in each groupThere was no significant fluctuation of respiratory frequency of the rats in each control group at each time point after anesthesia,suggesting that the respiratory rate of the three anesthetics can remain stable after anesthesia.After anesthesia,the respiratory frequency of the pentobarbital sodium control group decreased to a lower level(P<0.05),suggesting that pentobarbital sodium anesthesia may cause respiratory inhibition.However,during acupuncture,the respiratory rate of the rats in each acupuncture group showed an increasing trend,but there was no statistical difference(P>0.05).3 Analysis results of HRV indicators3.1 Comparison of VLF values in different time periods of the rats in each group There was no significant difference in VLF values of the rats in each groups when they were awake(all P>0.05).which indicated that the initial VLF values of the rats in each group were basically the same.After anesthesia,the VLF values of each control group decreased to a lower level(P<0.01 or P<0.001).and the VLF values ot each control group showed no significant fluctuations at all time point after anesthesia,suggesting that all three anesthetics can cause the down-regulation of VLF values,and the VLF value can remain stable after the anesthesia stability.Among them,the VLF value of the rats in pentobarbital sodium control group and the chloral hydrate control group decreased more significantly(both P<0.001).During acupuncture,the VLF value were increased significantly both in pentobarbital sodium and urethane acupuncture group,especially in the urethane acupuncture group(P<0.001),while the VLF value in the chloral hydrate acupuncture group showed a downward trend,but there was no statistical difference(P>0.05).It is suggested that twisting acupuncture used in this study can up-regulate the VLF value of the rats after anesthetized with sodium pentobarbital and urethane,while may down-regulation the VLF value of the rats after anesthetized with chloral hydrate.3.2 Comparison of LF values in different time periods of the rats in each groupThere was no significant difference in LF values between each groups when awake(all P>0.05),suggesting that the LF values of the initial state of the rats in each group were basically the same.There was no significant fluctuation in LF values in each control group at each time point after anesthesia,suggesting that the LF values can remain stable after anesthesia stability induced by the three anesthetics.Among them,the LF value after the anesthesia in pentobarbital sodium control group was significantly lower than that of the awake time(P<0.001).Compared with that of the awake time,the LF value after the anesthesia in hydrated chlorine control group showed only a downward trend,but there was no statistical difference(P>0.05),while there was no significant fluctuation in LF value at each time point after anesthesia in urethane control group compared with when it was awake(all P>0.05).It is suggesting that only pentobarbital sodium anesthesia can cause the down-regulation of LF value in HRV.During acupuncture,the LF value in hydrated chlorine acupuncture group showed a decrease trend,but there was no statistical difference(P>0.05).while there was no significant fluctuation in the LF value between the other two acupuncture groups after acupuncture(all P>0.05).3.3 Comparison of HF values in different time periods of the rats in each groupThere was no significant difference in HF values between each groups when awake(P>0.05),indicating that the initial HF values of the rats in each group were basically the same.After anesthesia,the HF values of the rats in each group increased significantly(P<0.05 or P<0.001),and there was no significant fluctuation in HF value in each control group at each time point after anesthesia.It is suggested that the HF value can be up-regulated after anesthesia with the three anesthetics,and can remain a stable state after anesthesia.During acupuncture,the HF values in the urethane acupuncture group and the pentobarbital sodium acupuncture group were significantly lower than those after anesthesia(both P<0.01),especially in the urethane acupuncture group(P<0.001),while the HF value in the chloral hydrate acupuncture group was significantly increased compared with that after anesthesia(P<0.05).It is suggested that the twisting needling manipulation used in this study can down-regulate the HF value of threats in urethane acupuncture group and pentobarbital sodium anesthetized rats,but up-regulated the HF value of the rats anesthetized with chloral hydrate.3.4 Comparison of the ratio of VLF/HF in different time periods of the rats in each groupThere was no significant difference in the value of VLF/HF between the three groups when they were awake(all P>0.05),which indicated that the values of VLF/HF in the initial state of the rats in each group were basically the same.After anesthesia,the value of VLF/HF in each control groups was significantly decreased.(P<0.01 or P<0.001).and there was no significant fluctuation of VLF/HF value in each control group at each time point after anesthesia,suggesting that all three anesthetics can down-regulated the VLF/HF value after anesthesia,and can maintain a stable state after anesthesia.During acupuncture,the ratio of VLF/HF in the urethane acupuncture group was significantly higher than that after anesthesia and that in the control group(both P<0.05).while the ratio of VLF/HF in sodium pentobarbital acupuncture group only showed an upward trend and a downward trend in hydrated chlorine acupuncture group,but there was no statistical difference(both P>0.05).It is suggested that the twisting needling manipulation used in this study can up-regulate the VLF/HF ratio of the rats anesthetized with urethane and pentobarbital sodium,but may down-regulate the VLF/HF ratio of the rats anesthetized with chloral hydrate.3.5 Comparison of the ratio of LF/HF in different time periods of rats in different time periodsThere was no significant difference in the ratio of LF/HF in each group when they were awake(all P>0.05),which indicated that the initial LF/HF of the rats in each group was basically the same.After anesthesia,the ratio of LF/HF in each group showed a downward trend but only the ratio of LF/HF in sodium pentobarbital control group was significantly higher than that when awake(P<0.01).During acupuncture,there was no significant difference in the ratio of LF/HF between that during acupuncture and that after anesthesia(all P>0.05).4 Results of autonomic nerve discharge frequency analysis4.1 Comparison of sympathetic nerve discharge frequency in the rats of each groupThere was no significant fluctuation in the frequency of sympathetic nerve discharge in each control group at each time point after anesthesia(all P>0.05),suggesting that the sympathetic nerve discharges of the three anesthetics remained stable after anesthesia.During acupuncture,the frequency of sympathetic nerve discharge increased slightly in pentobarbital sodium acupuncture group and decreased slightly in the urethane acupuncture group,but therewas no significant difference(both P>0.05)..4.2 Comparison of vagus nerv e discharge frequency in the rats of each groupThere was no significant fluctuation in the frequency of vagus nerve discharge in each control group at each time point after anesthesia(P>0.05),suggesting that the vagus nerve discharges of the three anesthetics can remain stable after anesthesia.During acupuncture,the discharge frequency of vagus nerve in the three acupuncture groups showed an increasing trend,and the discharge frequency of vagus nerve in the chloral hydrate acupuncture group was the most significantly higher than that after anesthesia(P<0.05).It is indicated that the twisting needling manipulation used in this study can increase the discharge frequency of vagus nerve.4.3 Comparison of sympathetic/vagal discharge frequency ratios in the rats of each groupThere was no significant difference in the ratio of sympathetic/vagal discharge frequency between the groups mentioned above(all P>0.05).CONCLUSION1.According to heart rate and HRV changes,both urethane and chloral hydrate anesthesia could slow down the heart rate,decrease the VLF.the ratio of VLF/HF in HRV and up-regulate the HF value.Although pentobarbital sodium anesthesia might cause a small increase in heart rate,it will also cause the down-regulation of VLF and the ratio of VLF/HF and the up-regulation of HF values in HRV.It is suggested that the above three anesthetics may lead to the decrease of sympathetic nerve excitability and the increase of vagus nerve excitability after anesthesia.2.Pentobarbital sodium anesthesia can inhibit respiratory rate to a certain extent and is not suitable for use as an anesthetic in respiratory system-related studies.3.The twisting needling manipulation used in this study can cause the up-regulation of VLF and the down-regulation of HF in HRV of rats anesthetized with urethane and pentobarbital sodium.However,the vagal discharge frequency increased,and the results of HRV index analysis were not consistent with the changes of autonomic nerve discharge.Considering that the VLF,which represented sympathetic excitation,and the HF,which represented parasympathetic excitability,may change too much after anesthesia with the two anesthetics mentioned above,the changes of the above indexes after acupuncture can no longer truly reflect the corresponding activities of autonomic nerve,so urethane and pentobarbital sodium may not be suitable for the analysis of the regulatory effect of acupuncture on autonomic nerve by HRV analysis alone.4.When the rats were anesthetized with chloral hydrate,the twisting needling manipulation used in this study can cause the down-regulation of VLF and LF and the significant up-regulation of the HF in the HRV,meanwhile,the vagus nerve discharge frequency was significantly increased The results of HRV index analysis were consistent with the changes of autonomic nerve discharge.It is suggested that chloral hydrate anesthesia is suitable for the analysis of the regulatory effect of acupuncture on autonomic nerve more.5.When the rats were anesthetized with chloral hydrate,the twisting needling manipulation used in this study can increase vagus nerve excitation. |
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