| ObjectiveTo determine the location of the cortical zone of swallowing that promotes swallowing activity and the specific neuronal type.On this basis,a new mouse model of dysphagia after cortical stroke was established to evaluate the effect of electroacupuncture at Lianquan on the swallowing function of the mouse model.On the basis of above,the mechanism of the effect of the electroacupuncture at Lianquan point was discussed by using the pyramidal neurons in the contralateral cortical swallowing area as the entry point.MethodsExperiment 1:Location,type and function of cortical neurons associated with swallowing activity in miceThe experiments in this section are carried out in three phases.Six-week-old male C57BL/6 mice were first selected.After routine anesthesia,neck surgery was performed to expose the anterior cervical muscles of the mice.The second abdominal muscles and the mandibular ligament muscles were bluntly separated and connected by a micro-injection pump.The virus injection needle aspirates the PRV-CMV-EGFP virus,and the virus is injected into the mandibular lingual muscle of the mouse,and the injection volume per mouse is 3 ul.Mice were perfused at different time points.Immunofluorescence techniques were used to detect areas of positive virus expression and neuronal types.Then,the second phase of the experiment was started.First,C57BL/6 mice were performed craniotomy and the skull plane was adjusted.The skull above the target brain area was marked and the marked area was drilled by a driller.AAV-CaMKⅡα-hChR2-mCherry virus was injected into the target brain area through a microinjection pump.After the injection,the ceramic ferrule was embeded.After 2 weeks of routine recovery in mice,the efficiency of the tool virus was detected using optogenetic combined with patch clamp technique.Finally,the third stage experiment was carried out.The optical genetic stimulation system and the ceramic ferrule on the mouse head were connected by a fiber jumper,and the recording electrode was inserted into the swallowing muscle group of the mouse,and the reference electrode was inserted into the masseter muscle.Set different light stimulation frequencies and intensities,and use the CED series MICR01401Ⅱ signal acquisition device and Spike2 computer signal analysis system to collect the EMG signals induced by light stimulation in real time.Experiment 2:Establishment of a mouse model of dysphagia after stroke and evaluation of the efficacy of acupuncture at LianquanThis part of the experiment is carried out in two stages.First,20-week-old male C57BL/6 mice were randomly divided into Con group,M1-Isc group,Laser group,RB group and PFC-Isc group.The Con group of mice did not receive any treatment.M1-Isc mice were injected with Rose Bengal B solution to irradiate M1 region to produce ischemic lesions.Laser group mice were not injected with Rose Bengal B solution,only laser irradiation was used to irradiate M1 region.RB group mice were only injected with Rose Bengal B solution and not irradiated with laser.The mice of PFC-Isc group were injected with Rose Bengal B solution to irradiate the PFC region to produce an ischemic lesion.The cerebral blood flow perfusion was first detected by a laser speckle imager.The amount of water consumed by the mice for 4 minutes was then measured.The mice were anesthetized and fixed on the adapter with ear clips.The mice were turned over and placed in a supine position,and a plastic hose was inserted from the side of the mouse to the posterior pharynx with a stereotactic device.The other end of the plastic hose is connected to a micro-syringe and a micro-injection pump.The myoelectric recording line and the ground wire are connected to the collecting instrument,and the distilled water is injected into the posterior pharynx of the mouse through a micro syringe to collect the electromyographic signal induced by the water in real time.Then proceeded to the second phase of the experiment.Male C57BL/6 mice of 20 weeks old were randomly divided into Normal,Isc,Isc+EA and Isc+sham EA groups.The mice in the Normal group were blank control.The mice in Isc group were injected with the Rose Bengal B solution to irradiate the M1 region to produce the ischemic lesion.The Isc+EA group was injected with the Rose Bengal B solution,and the laser was used to irradiate the M1.The mice in Isc+EA group were treated with electroacupuncture at Lianquan point 1 hour after the experiment.The electroacupuncture parameters are 1 mA,2 Hz,15 min.After the Isc+sham EA group was injected with Rose Bengal B solution,the M1 area was irradiated with a laser to create an ischemic lesion,and then subjected to a sham electroacupuncture treatment.The sham electroacupuncture only replaces the acupuncture needle with a toothpick,and the rest procedure was the same as electroacupuncture.All mice were banned for 12 hours before the modeling operation and 12 hours after the modeling operation.Then the drinking water experiment and the water-induced EMG signal were detected.Experiment 3:Activation of pyramidal neurons in the contralateral cortex swallowing area of mice with dysphagia after unilateral cortical dysphagia after electroacupuncture at Lianquan pointThis part of the experiment is carried out in three stages.Male C57BL/6 mice,20 weeks old,were first selected and randomly divided into Isc,Isc+EA and Isc+sham EA.The Isc group mice were sacrificed 2 hours after model establishment,and the Isc+EA and Isc+sham EA mice were treated with electroacupuncture or sham electroacupuncture 1 hour after modeling,and 50 minutes after the end of the intervention.Immunofluorescence technique was used to detect the expression of c-Fos protein in neurons in the contralateral cortex.Then proceed to the second phase of the experiment.Male C57BL/6 mice and GAD67 mice at 20 weeks of age were used.AAV-CaMKIIα-mCherry was injected into the left target brain region of C57BL/6 mice by brain stereotactic injection technique,and the mice were routinely recovered for two weeks after virus injection.Then,C57BL/6 mice were randomly divided into Isc group and Isc+EA group.Similarly,GAD67 mice were randomly divided into Isc group and Isc+EA group.Immunofluorescence technique was used to detect the double-labeling of specific neurons and c-Fos positive neurons in the contralateral cortex.Finally,the third stage experiment was carried out.Male C57BL/6 mice of 20 weeks old were randomly divided into EA(CV23)-M1 group,EA(CV23)-PFC group and sham EA(CV23)-M1 group.The EA(CV23)-M1 group of mice and the sham EA(CV23)-M1 group of virus injection and left ceramic ferrule embedding sites were in the M1 region.Both the virus injection of the EA(CV23)-PFC group and the left ceramic ferrule embedding site were in the PFC region.The ceramic ferrules on the right side of the three groups of mice were embedded in the M1 region.The injected virus was AAV-CaMKII α-Gcamp6s.The mice were routinely recovered for two weeks after surgery.Then,photochemical method was used to damage the cortical swallowing area on the right side of the three groups of mice.After 1 hour,we used a fiber jumper to connect the fiber recording system to the ceramic ferrule on the mouse head.EA(CV23)-M1 group and EA(CV23)-PFC group mice were treated with acupuncture needles and Lianquan points,while sham EA(CV23)-M1 mice were subjected to false needles using a toothpick and recorded in real time by fiber optic recording system.Calcium signal in the target brain area was recorded at real time for 5 minutes before acupuncture,15 minutes after acupuncture and 30 minutes after acupuncture.Experiment 4:Inhibition of acupuncture on activation of vertebral cells in the pyramidal neuron and swallowing area,and observation of the therapeutic effect of acupunctureThis experiment was carried out in two stages.First,20-week-old male C57BL/6 mice were randomly divided into two groups,which were injected with AAV-CaMKIIα-hM4Di-mCherry or AAV-CaMKII α-mCherry virus,and the virus injection area was the left M1 region.The mice were routinely recovered for two weeks after surgery.Two weeks later,the right cortical swallowing area of the two groups of mice was damaged by photochemical methods.Mice injected with AAV-CaMKIIα-hM4Di-mCherry or AAV-CaMKIIα-mCherry virus were randomly divided into Isc group and Isc+EA group,and each group of mice was intraperitoneally injected with CNO at 35 minutes before electroacupuncture.The expression of c-Fos protein in these groups of mice were detected by immunofluorescence technique.Then,proceed to the next stage of the experiment.Male C57BL/6 mice of 20 weeks old were used.They were randomly divided into Normal,hM4Di+CNO,hM4Di+Isc+EA+CNO,hM4Di+Isc+EA,mCherry+Isc+EA+CNO groups.Normal group mice were fed normally without any intervention.The mice in hM4Di+CNO group received only AAV-CaMKII α-hM4Di-mCherry and CNO injection.The mice in hM4Di+Isc+EA+CNO group were injected with AAV-CaMKII α-hM4Di-mCherry firstly,and the virus was fully expressed,and then routine photochemical method was used to perform in the mice.CNO was given at 35 minutes before electroacupuncture treatment.Mice in hM4Di+Isc+EA group were not given CNO before acupuncture,and the rest were treated with hM4Di+Isc+EA+CNO group.The mice of mCherry+Isc+EA+CNO group were injected with AAV-CaMKIIα-mCherry,and the rest was treated with the hM4Di+Isc+EA+CNO group.Finally,water-induced EMG signals and water consumption test were detected.Results1.The cortical coordinates associated with swallowing activity in mice are AP:-0.16,ML:1.01,DV:-1,and the neuron type can be judged as pyramidal neurons.The results of immunofluorescence showed that the expression of neuronal virus in the target brain region was high,and the results of optogenetic combined with patch clamp showed that the virus-infected neurons responded rapidly and stably to blue light stimulation.The results of optogenetic and electromyographic recordings show that light stimulation can induce a significant response of the swallowing muscles.The best stimulation parameter is 8mW,50Hz.2.The results of laser speckle imaging showed that the blood perfusion of the injured side of the Isc group was significantly lower than that of the healthy side(P<0.01).The water consumption test showed that the water consumption of M1-Isc mice was significantly lower than that of Con group(P<0.01),Laser group(P<0.05),RB group(P<0.05)and PFC-Isc group(P<0.05).The results of electromyography showed that the area under the EMG curve of M1-Isc group was significantly smaller than that of Con group(P<0.01),Laser group(P<0.05),RB group(f<0.05)and PFC-Isc group(P<0.05).For the second phase of the experiment,the statistical results of the water consumption test showed that the water consumption of the Isc+EA group was significantly higher than that of the Isc group,and the difference was statistically significant(P<0.05).The results of electromyography showed that the area under the EMG curve of the Isc+EA group was significantly larger than that of the Isc group(P<0.01)and the Isc+sham EA group(P<0.05).3.The immunofluorescence statistics showed that the number of positive neurons for c-Fos protein expression in the contralateral cortex of swallowing area was significantly higher in the Isc+EA group than in the Isc group and the Isc+sham EA group(P<0.05).In the Isc-EA group,the ratio of c-Fos protein positive pyramidal neurons positive to total pyramidal neurons in the contralateral cortical swallowing area was greater than that in the Isc group,and the difference was statistically significant(P<0.05).There was no statistically significant ratio between c-Fos protein-positive interneurons and total interneurons in the contralateral cortical swallowing area of Isc-EA mice.The results of fiber-recorded calcium imaging showed that the calcium signal of the pyramidal cells in the contralateral cortex and prefrontal cortex was significantly increased during the acupuncture process.However,the sham electroacupuncture stimulation had no significant effect on the calcium signal of the pyramidal neurons in the contralateral cortical swallowing area.4.Immunofluorescence results showed that there was no significant difference in the expression rate of c-Fos in hM4Di-positive neurons between Isc group and Isc+EA group in mice receiving AAV-CaMKII α-hM4Di-mCherry virus injection(P>0.05).For the mice receiving AAV-CaMKII α-mCherry virus injection,the expression rate of c-Fos in mCherry-positive cells of Isc+EA group was significantly higher than that of Isc group,and the difference between the two groups was statistically significant(P>0.05).For the second phase of the experiment,the statistical results of the water consumption test showed that the water consumption of the hM4Di+Isc+EA+CNO group was significantly less than that of the hM4Di+Isc+EA group(P.01)and the mCherry+Isc+EA+CNO group(P<0.01).The results of electromyography showed that the area under the EMG signal curve of hM4Di+Isc+EA+CNO group was significantly smaller than that of hM4Di+Isc+EA group(P<0.01)and mCherry+Isc+EA+CNO group(P<0.01).Conclusion1.Pyramidal neurons in the cortical swallowing area are key neurons involved in promoting swallowing activity.2.Using a photochemically located lesion of the cortical swallowing area to establish a reliable mouse model of post-cortical stroke dysphagia,and electroacupuncture at Lianquan can effectively improve the swallowing function of model mice.3.Electroacupuncture at Lianquan can effectively activate pyramidal neurons in the cortex of swallowing.4Electroacupuncture at Lianquan activates the pyramidal neurons in the contralateral cortical swallowing area to compensate for the function of the damaged brain area.This is the key central mechanism for acupuncture treatment of dysphagia after cortical stroke. |
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