Mechanism Of Glutaminergic Pathway From Lateral Hypothalamic Area To Fastigial Nucleus In The Alleviation Of Myocardial Ischemia-Reperfusion Injury By Electroacupuncture Pretreatment

Objective:On the basis of clarifying that electroacupuncture（EA）pretreatment of acupoints of heart meridian can effectively alleviate myocardial ischemia-reperfusion injury,to explore whether the glutaminergic neurons in the lateral hypothalamus area（LHA）participate in the protective effect of EA pretreatment.Further clarify the neural projection relationship between the LHA and fastigial cerebellar nucleus（FN）.It is preliminarily proved that the glutaminergic pathway from the LHA to the FN is involved in the mechanism of acupuncture preconditioning to alleviate myocardial ischemia-reperfusion injury,laying a foundation for further clarifying the central nervous mechanism of acupuncture heart meridian regulating cardiac function.Methods:This experiment was divided into three parts.In the first part,we mainly determined that EA pretreatment of heart meridian acupoints can effectively alleviate MIRI.C57BL/6 was randomly divided into sham operation group,model group,EA shenmen acupoint（HT7）group and EA fuliu acupoint（KI7）group（n=15 in each group）.The MIRI model was reproduced by ligation of left anterior descending branch of the coronary artery.In Sham group,only one empty puncture was performed at the corresponding position,without EA;Model group only ligated without EA;EA HT7 group and EA KI7 group were continuously electroacupuncture for 7 days before modeling,which started in 0.5 h after the end of EA treatment.Powerlab multi-channel physiological recorder was used to record the Ⅱ lead electrocardiogram（ECG）of mice in each group and observe the changes of ST segment.The contents of c Tn I,CK-MB and NE in serum were detected according to ELISA instruction,which were used to explain the degree of myocardial injury and the changes of peripheral sympathetic nerve activity.The myocardial infarction area was observed by TTC-Evans blue syaining,and the myocardial histomorphology was observed by HE staining.Three mice in each group were selected to record the discharge of LHA neurons and local field potential（LFP）using Plexon multi-channel acquisition system.Immunofluorescence staining was used to observe the c-fos positive neurons,glutamatergic neurons and GABAergic neurons.The second part aimed to discuss the protective effect of EA preconditioning on the glutaminergic neurons in the LHA against MIRI.C57BL/6 mice were bilaterally injected with virus named r AAV-Ca MKⅡ a-m Cherry-WPRE-h GHp A or r AAV-Ca MKⅡ a-h M3Dq（Gq）-m Cherry-WPRE-h GHp A or r AAV-Ca MK Ⅱ a-h M4Di（Gi）-m Cherry-WPRE-h GHp A in the LHA（200n L/side）.After 21 days,these mice were divided into sham operation group（Sham+m Cherry）,model group（Model+m Cherry）,EA group（Model+m Cherry+EA）,inhibition group（Model+h M4Di）and activation with EA group（Model+h M3Dq+EA）,12 in each group.The EA group and activation with EA group were injected clozapine N-oxide（CNO）intraperitoneally 30 minutes before EA treatment for 7 days,and the other groups were injected CNO intraperitoneally 30 mins before modeling.The methods of model preparation and EA intervention were the same as the first part.Similarly,ECG was used to record the ST segment displacement value and low frequency and high frequency power ratio（LF/HF）of mice in each group.The content of serum c Tn I,CK-MB and NE was detected by ELISA.The myocardial infarction area and myocardial histomorphology were observed by TTC-Evans blue staining and HE staining.In the third part,the LHA-FN^Glu neural pathway was regulated by virus tracing combined with chemical genetic methods to preliminarily explore the mechanism of the neural pathway in EA preconditioning to alleviate MIRI.C57BL/6 mice were bilaterally injected with r AAV-Ca MKⅡa-Cre-WPRE-h GH p A（200n L/side）into the LHA,and r AAV-h Syn-DIO-m Cherry-WPRE-p A/r AAV-h Syn-DIO-h M3Dq-m Cherry-WPRE-p A/r AAV-h Syn-DIO-h M4D-m Cherry-WPRE-p A（100n L/side）into the FN.After21 days,mice were divided into sham operation group（Sham+m Cherry）,model group（Model+m Cherry）,EA group（Model+m Cherry+EA）,inhibition group（Model+h M4Di）and activation with EA group（Model+h M3Dq+EA）,with 12 mice in each group.The EA group and activation with EA group were given CNO intraperitoneally 30 mins before EA treatment for 7 days,and the model was performed within half an hour after the end of the last EA treatment.The methods of model preparation and EA intervention were the same as the first part.3 mice in each group were randomly selected to observe the fiber projection from LHA to Med.ECG was used to record the ST segment displacement value.The content of serum c Tn I,CK-MB and NE was detected by ELISA.The myocardial infarction area was detected by TTC-Evans blue staining.All these indicators tried to clarify the possible mechanism of LHA-FN^Glu neural pathway participating in the alleviation of MIRI by EA pretreatment.Results:1.Protective effect of EA preconditioning of heart meridian on MIRI mice.（1）Changes of ST segment displacement during ischemia/reperfusion in mice in each group: Compared with the sham group,the ST segment displacement of the model group increased significantly（P<0.01,P<0.05）;Compared with the model group,the ST segment displacement value of the EA HT7 group was significantly lower（P<0.01,P<0.01）;Compared with EA HT7 group,the ST segment displacement of EA KI7 group was higher（P<0.01,P<0.01）.（2）Low frequency and high frequency（LF/HF）ratio of mice in each group: during the whole ischemia/reperfusion period,the LF/HF ratio of the model group was significantly higher than that of the sham group（P<0.001）;The ratio of LF/HF in the EA HT7 group was lower than that in the model group（P<0.001,P<0.01）;The ratio of the EA KI7 group was significantly higher than that of the EA HT7 group（P<0.01）,but only after 120 mins of reperfusion was lower than that of the model group（P<0.05）.（3）The content of serum c Tn I,CK-MB and NE in mice in each group: Compared with the sham group,the content of c Tn I,CK-MB and NE in the model group was significantly higher（P<0.01）;Compared with the model group,the expression of c Tn I,CK-MB and NE in EA HT7 group decreased significantly（P<0.01）,while only the expression of NE in EA KI7 group decreased compared with the model group（P<0.01）;Compared with EA HT7 group,the content of the three indicators significantly increased（P<0.01）.（4）TTC-Evans blue staining showed that the myocardial infarction area in the model group was significantly larger than that in the sham group（P<0.01）;Compared with the model group,the infarct area of the EA HT7 group decreased（P<0.01）,and there was no statistical difference in EA KI7 group;Compared with EA HT7 group,the myocardial infarction area increased in EA KI7 group（P<0.01）.（5）HE staining showed that the myocardial cells in the sham group were arranged orderly with few myocardial fractures;In the model group,the myocardial fibers were broken and disordered,the intercellular space was enlarged and edema was obvious,and there was inflammatory cell infiltration;In the EA HT7 group,the myocardial fibers were less broken and the myocardial celle were slightly enlarged;in the EA KI7 group,myocardial fibers were broken to some extent,and interstitial edema was obvious.（6）The total discharge frequency of LHA neurons in each group: Compared with the sham group,the discharge frequency of LHA neurons in the model group increased significantly（P<0.001）;Compared with the model group,the discharge frequency of LHA neurons in EA HT7 group and the EA KI7 group decreased significantly（P<0.001,P<0.05）;Compared with the EA HT7 group,the firing frequency of LHA neurons in EA KI7 group increased significantly（P<0.01）.The energy spectrum showed that the energy of model group was significantly higher than that of sham group;EA KI7 group and EA HT7 group were lower than that of the model group;The energy of the EA HT7 group was lower than that of KI7 group.（7）Immunofluorescence staining showed that the number of c-fos positive neurons in the model group was significantly higher than that in the sham group（P<0.01）;Compared with the model group,the expression of c-fos in the EA HT7 group decreased significantly（P<0.01）;Compared with the EA HT7 group,the expression of c-fos in KI7 group increased significantly（P<0.01）.In the model group,the proportion of glutaminergic neurons in positive c-fos neurons was significantly greater than GABAergic neurons（P<0.001）.2.Protective effect of electroacupuncture preconditioning on myocardial ischemia reperfusion injury mediated by glutaminergic neurons in the lateral hypothalamus area.（1）Changes of ST segment displacement during ischemia/reperfusion in mice in each group: Compared with sham group,ST segment displacement in model group was significantly higher（P<0.001,P<0.05）;Compared with the model group,the ST segment displacement value in the EA group and inhibition group decreased significantly（P<0.01,P<0.01）;Compared with the EA group,the ST segment displacement value in the activation with EA group increased significantly（P<0.01,P<0.01）.（2）Low frequency and high frequency power ratio（LF/HF）of mice in each group:before ischemia,there was no significant difference in LF/HF ratio between groups（P >0.05）.During ischemia/reperfusion,the ratio of LF/HF in model group was higher than that in sham group（P <0.05,P <0.01）;The ratio of LF/HF in inhibition group and EA group was lower than that in model group（P <0.05,P <0.01）;The ratio of LF/HF in activationwith EA group was higher than that in EA group（P<0.01,P <0.05）.（3）The content of serum c Tn I,CK-MB and NE in mice in each group: Compared with sham group,the content of serum c Tn I,CK-MB and NE in model group increased significantly（P <0.01）;Compared with the model group,the contents of serum c Tn I,CK-MB and NE in the EA group and inhibition group decreased significantly（P<0.001）;Compared with the EA group,the content of serum c Tn I,CK-MB and NE in the activation with EA group increased significantly（P <0.01,P <0.01,P<0.001）.（4）Myocardial infarction area of mice in each group: Compared with sham group,the infarction area of model group was significantly increased（P <0.001）;Compared with the model group,the infarction area of the EA group and the inhibition group decreased significantly（P <0.001）;Compared with the EA group,the myocardial infarction area in the activation with EA group increased significantly（P <0.001）.（5）The HE staining results of the myocardium of mice in each group showed that the muscle fibers in the sham group were complete,arranged tightly and orderly,and the myocardial cells were normal;In the model group,most of the myocardial fibers were broken and the myocardial cell interstitium was enlarged;In the inhibition group,the myocardial fibers were arranged orderly and the myocardial cells were slightly enlarged;In the EA group,the myocardial fibers were rarely broken and arranged orderly,and the myocardial cells were slightly edema;in the activation with EA group,there were more myocardial fiber breaks,obvious interstitial edema,and inflammatory cell infiltration.3.The mechanism of the LHA-FN^Gluneural pathway in the alleviation of MIRI by EA preconditioning in mice.（1）Changes of ST segment displacement during ischemia/reperfusion in mice in each group: Compared with sham group,ST segment displacement in model group was significantly higher（P <0.001,P <0.01）;Compared with the model group,the ST segment displacement value in the EA group and inhibition group decreased significantly（P <0.001,P <0.001）;Compared with the EA group,the ST segment displacement value in the activation with EA group increased significantly（P <0.01,P <0.001）.（2）The content of serum c Tn I,CK-MB and NE in mice in each group: Compared with the sham group,the content of serum c Tn I,CK-MB and NE in the model group increased significantly（P <0.001）;Compared with the model group,the contents of serum c Tn I,CK-MB and NE in the EA group and inhibition group decreased significantly（P <0.001）;Compared with the EA group,the content of serum c Tn I,CK-MB and NE in the activation with EA group increased significantly（P <0.001）.（3）TTC-Evans blue staining of myocardium of mice in each group: compared with sham group,the infarct area of model group was significantly increased（P <0.001）;Compared with the model group,the infarction area of the EA group and the inhibition group decreased significantly（P <0.001）;Compared with the EA group,the area of myocardial infarction in the activation with EA group increased significantly（P <0.001）.Conclusions:1.EA preconditioning at heart meridian acupoints can effectively alleviate MIRI and reduce myocardial infarction area.2.Protective effect of electroacupuncture preconditioning on myocardial ischemia reperfusion injury mediated by glutaminergic neurons in the lateral hypothalamus area.3.There are nerve fiber projections between the LHA and FN,and the LHA-FN^Glu neural pathway was involved in EA preconditioning to alleviate MIRI.