Study On Acupuncture At Hand Twelve Jing-Well Points Promotes Awakening Via Ventral Periaqueductal Gray-Hypothalamus Circuit

Objective:Timely awakening of traumatic brain injury（TBI）coma is a key point in neuroscience.It has been proved that acupuncture at hand twelve Jing-well points（HTJW）can safely and effectively improve the acute state of consciousness in sufferers with manifold central nervous injuries,containing TBI,but the arousal mechanism n has not been clarified,which limits its application and extension of it.Our group found that P2RX7 expressing dopamine（DA）in ventral periaqueductal gray（vPAG）region mediates arousal in TBI comatose rats based on acupuncture at HTJW,which can activate the lateral hypothalamus（LH）dopamine receptor D2（DRD2）and orexin（ORX）;activates DRD2,and downregulates histamine（HA）expression in the tuberomamillary nucleus（TMN）of the hypothalamic.The LH and TMN regions are essential components of the ascending reticular activation system（ARAS）,which receives projections from the vPAG DAergic system.Therefore,this study further elucidate the biological mechanism of TBI induced by this method centering on the P2RX7 pathway in vPAG region-hypothalamus projection system.Methods:1.Based on EEG collecting to estimate the acupuncture effect at HTJW on enhancing consciousness level of severe TBI ratsAdult male Sprague Dawley（SD）healthy rats were divided into three groups（n=6）:sham-operated group（Sham）,model group（TBI）,and model+acupuncture at HTJW（Hand Acu）.Sham group only had a cranial window;In the TBI group,a cranial window was opened and an experimental severe TBI model was prepared;The Hand Acu group was immediately administered acupuncture at HTJW on the basis of successful modeling.All rats were monitored for brain electroencephalogram（EEG）.Effect indicators:coma duration,I-VI level consciousness scale score after 3 h,4 h,5 h,and 6 h of TBI,modified Neurological severity scores（mNSS）after 6 h of TBI,and calculate the energy proportion of different EEG frequency bands.2.P2RX7 pathway in the vPAG mediates arousal promoting effects of acupuncture at HTJWSD rats were divided into four groups（n=8）:Sham+DMSO solvent（Sham）,model+DMSO solvent（TBI）,acupuncture at HTJW+DMSO solvent（Hand Acu）,acupuncture at HTJW+P2RX7 antagonist（Hand Acu+anti-P2RX7）.All rats were subjected to TBI modeling at 30 min of injection in the vPAG region,and the Hand Acu and Hand Acu+anti-P2RX7groups were given the acupuncture at HTJW.Effect indicators:coma duration,I-VI level consciousness scale score after 3 h,4 h,5 h,and 6 h of TBI,modified Neurological severity scores（mNSS）after 6 h of TBI.3.The P2RX7 pathway is involved in the regulation of dopamine receptor D2 and orexin neurons in the lateral hypothalamus by acupuncture at HTJWImmunofluorescencestaining（DRD2+c-fos+DAPI,ORX+c-fos+DAPI,DRD2+ORX+DAPI）was used to detect the expression of LH region in hypothalamus of Sham,TBI,Hand Acu and Hand Acu+anti-P2RX7 groups,and to explore the upstream regulation of P2RX7 pathway in vPAG region on TBI-awaking.4.The P2RX7 pathway is involved in the regulation of dopamine receptor D2 and histaminergic neurons in tuberopapillary nuclei of hypothalamus by acupuncture at HTJWImmunofluorescence staining（DRD2+c-fos+DAPI）and immunohistochemical staining（HA）was used to detect the expression of TMN region in hypothalamus of Sham,TBI,Hand Acu and Hand Acu+anti-P2RX7 groups,and to explore the upstream regulation of P2RX7pathway in vPAG region on TBI-awaking.Results:1.Acupuncture at HTJW can cut down the coma duration and promote the consciousness level of TBI coma rats（1）Acupuncture at HTJW can cut down the coma duration and promote the consciousness level of TBI comatose rats.①Coma duration:By comparison with Sham group,TBI group was longer than Sham group(^**P<0.01);Hand Acu group was lower than TBI group(^▲▲P<0.01).②I-VI level consciousness scale score:the consciousness state of TBI group was lower than that of Sham group(^**P<0.01,after 3 h,4 h,5 h and 6 h of TBI);The consciousness state of Hand Acu group was better than that of TBI group(^▲P<0.05,4 hours after TBI;^▲▲P<0.01,5 and 6hours after TBI).③mNSS score:TBI group was higher than Sham group(^**P<0.01);Hand Acu group was lower than TBI group(^▲▲P<0.01).It is suggested that acupuncture at HTJW can promote the awakening of TBI rats and improve their consciousness level.（2）EEG signals reflect that acupuncture at HTJW can improve the coma degree of TBI comatose rats.①According to the EEG color spectrum,the coma duration of the TBI group was increased compared with that of the sham group(^**P<0.01);The coma duration was shorter in the Hand Acu group than in the TBI group(^▲▲P<0.01),indicating that acupuncture at HTJW based on EEG color spectrum can shorten the duration of coma in TBI rats.②（δ+θ）/（α+β）Values:TBI group were higher than Sham group(30-50,100-110 min after TBI:^**P<0.01;50-70,90-100 min after TBI:^*P<0.05).The Hand Acu group were lower than the TBI group(30-40 min after TBI:^##P<0.01;40-50,100-110 min after TBI:^##P<0.05),suggesting that acupuncture at HTJW could improve the brain functional status after TBI.③（δ+θ）/（δ+θ+α+β）Values:TBI group were higher than Sham group(30-60,70-80,90-110 min after TBI:^**P<0.01;60-70,80-90 min after TBI:^*P<0.05).The Hand Acu group were lower than the TBI group（30-50,100-110 min after TBI:^#P<0.05）,suggesting that acupuncture at HTJW can improve coma state after TBI.④（α+β）/（δ+θ+α+β）Values:TBI group were lower than Sham group(30-60,70-80,90-110 min after TBI:^**P<0.01;60-70,80-90 min after TBI:^*P<0.05).The Hand Acu group were higher than the TBI group（30-50,100-110 min after TBI:^#P<0.05）,suggesting that acupuncture at HTJW improves wakefulness after TBI.⑤TBI groupγwave band energies were lower than Sham group(30-110 min after TBI:^**P<0.01).The Hand Acu group was higher than the TBI group(100-120 min after modeling:^##P<0.01).It is suggested that acupuncture at HTJW increases cortical awake related EEG in TBI rats and is one of the characterizations of rat awake.2.P2RX7 pathway of vPAG region mediates acupuncture at HTJW to realize arousal promoting effects①Coma duration:By comparison with the Sham group,TBI group was longer than Sham group(^**P<0.01);The Hand Acu group was shorter than the TBI group(^▲▲P<0.01);It was increased in Hand Acu+anti-P2RX7 group than that in Hand Acu group(^##P<0.01).②I-VI level consciousness scale score:the consciousness state of TBI group was lower than that of Sham group(^**P<0.01,after 3 h,4 h,5 h and 6 h of TBI);The consciousness state of Hand Acu+DMSO group was better than that of TBI group(^▲P<0.05,4 hours after TBI;^▲▲P<0.01,5 and 6 hours after TBI).The consciousness state of Hand Acu+anti-P2RX7 group was decreased compared with that in the Hand Acu+DMSO group(^#P<0.05,4 hours after TBI;^##P<0.01,5 and 6 hours after TBI).③mNSS score:TBI group increased compared with Sham group(^**P<0.01);The Hand Acu group was shorter than the TBI group(^▲▲P<0.01).It was increased in Hand Acu+anti-P2RX7 group than that in Hand Acu group(^##P<0.01).It is suggested that acupuncture at HTJW may promote awakening of TBI rats and improve their level of consciousness,and antagonizing P2RX7 may antagonize this effect.3.Acupuncture at HTJW can activate DRD2 and orexin in the lateral hypothalamus regions,enhance DRD2 expression in orexin neurons,and the injection of P2RX7 antagonists into the vPAG can antagonize the regulatory effect of HTJW.（1）Acupuncture at HTJW can activate dopamine receptor D2 in the lateral hypothalamus,and the regulation of this method can be antagonized by injecting P2RX7 antagonists into the ventral periaqueductal gray.The expressions of DRD2,c-fos,and the co-expression of DRD2 and c-fos in the LH region of the hypothalamus were decreased in the TBI group compared with the Sham group(DRD2:^**P<0.01;c-fos:^*P<0.05;DRD2+c-fos:^**P<0.01);However,it was increased in the Hand Acu group than in the TBI group(^▲▲P<0.01).The decrease in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group(^##P<0.01)indicates that acupuncture at HTJW activates DRD2 in the LH region of the hypothalamus of TBI comatose rats,whereas antagonizing P2RX7 in the vPAG region antagonizes this effect.Counting the DRD2 to c-fos co-expressing cell number in the DRD2 ratio revealed that the Hand Acu group upregulated the DRD2 ratio with neural excitability compared with the TBI group（^▲P=0.017）.DRD2 with neural excitability was downregulated by 18.4%in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group.（2）Acupuncture at HTJW can activate orexin in the lateral hypothalamus,and the regulation of this method can be antagonized by injecting P2RX7 antagonists into the ventral periaqueductal gray.The expressions of ORX⁺,c-fos⁺,and the co-expression of ORX⁺and c-fos⁺in the LH region of the hypothalamus were decreased in the TBI group compared with the Sham group(ORX⁺:^**P<0.01;c-fos⁺:^*P<0.05;ORX⁺c-fos⁺:^*P=0.012);However,it was increased in the Hand Acu group than in the TBI group(^▲▲P<0.01).The decrease in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group(^##P<0.01)indicates that acupuncture at HTJW activates ORX⁺in the LH region of the hypothalamus of TBI comatose rats,whereas antagonizing P2RX7 in the vPAG region antagonizes this effect.Counting the ORX⁺to c-fos co-expressing cell number in the ORX⁺ratio revealed that the Hand Acu group upregulated the ORX⁺ratio with neural excitability compared with the TBI group(^▲▲P<0.01).ORX⁺with neural excitability was downregulated in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group(^##P<0.01).（3）The expression of orexin by dopamine receptor D2 in the lateral hypothalamus region can be upregulated by acupuncture at HTJW,and the regulation of this method can be antagonized by P2RX7 antagonist injection in the ventral periaqueductal gray.The expressions of DRD2⁺,ORX⁺,and the co-expression of DRD2⁺and ORX⁺in the LH region of the hypothalamus were declined in the TBI group by comparison with the Sham group(DRD2⁺:^*P=0.014;ORX⁺:^**P<0.01;DRD2⁺ORX⁺:^*P=0.019);And increased in the Hand Acu group than in the TBI group(^▲▲P<0.01).The decrease in the Hand Acu+anti-P2RX7group compared with the Hand Acu group(^##P<0.01),indicating that acupuncture at HTJW upregulates the expression of DRD2⁺and ORX⁺and the co-expression of DRD2⁺and ORX⁺,whereas antagonizing P2RX7 in the vPAG region antagonizes the effect.The percentage of DRD2⁺positive cells with ORX⁺expression in the LH region of the hypothalamus was calculated as follows:in the Hand Acu group,compared with the TBI group,ORX⁺expression was up-regulated by 29.4%in the number of DRD2⁺positive cells;However,compared with the Hand Acu+anti-P2RX7 group,ORX⁺expression was down regulated by 24.4%in the number of DRD2⁺positive cells.4.Acupuncture at HTJW can activate DRD2 in the hypothalamic tuberomamillary nucleus regions,reduce expression of histamine,and the injection of P2RX7 antagonists into the vPAG can antagonize the regulatory effect of HTJW.（1）Acupuncture at HTJW can activate dopamine receptor D2 in the hypothalamic tuberomamillary nucleus regions,and the regulation of this method can be antagonized by injecting P2RX7 antagonists into the ventral periaqueductal gray.The expressions of DRD2⁺,c-fos⁺,and the co-expression of DRD2⁺and c-fos⁺in the TMN region of the hypothalamus were decreased in the TBI group compared with the Sham group(^**P<0.01);However,it was increased in the Hand Acu group than in the TBI group(^▲▲P<0.01).The decrease in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group(^##P<0.01)indicates that acupuncture at HTJW activates DRD2 in the TMN region of the hypothalamus of TBI comatose rats,whereas antagonizing P2RX7 in the vPAG region antagonizes this effect.The proportion of co-expressing cells of DRD2⁺and c-fos in DRD2⁺was calculated:in Hand Acu group,compared with TBI group,DRD2 with neural excitability in DRD2⁺of hypothalamic TMN area was up-regulated by 14.2%;Compared with the Hand Acu group,the DRD2⁺with neuroexcitability in the TMN area of hypothalamus in the Hand Acu+anti-P2RX7 group was down regulated by 18%.（2）The expression of histamine in the tuberomammillary nucleus of the hypothalamus can be inhibited by acupuncture at HTJW,and the regulation of this method can be antagonized by injecting P2RX7 antagonist into the ventral periaqueductal gray.HA⁺in the TMN region of the hypothalamus:TBI group increased compared with Sham group(^**P<0.01);Hand Acu group downregulated its expression compared with the TBI group(^▲▲P<0.01).The decrease in the Hand Acu+anti-P2RX7 group compared with the Hand Acu group(^##P<0.01),suggesting that acupuncture at HTJW downregulates HA⁺expression in the TMN region of the hypothalamus and antagonizes the effect of P2RX7 pathway in the vPAG region.Conclusion:1.Based on the platform of EEG monitoring,it was confirmed that acupuncture at HTJW can realize arousal promoting effects of TBI coma rats.2.Acupuncture at HTJW can activate dopamine receptor D2 and orexin in the lateral hypothalamus,enhance dopamine receptor D2 expression in orexin neurons,and reduce expression of histamine.3.P2RX7 pathway in ventral periaqueductal gray mediates the arousal promoting effects of acupuncture at HTJW and its regulation on dopamine receptor D2,orexin and histamine neurons in hypothalamus.