Acupuncture At Jmg-Well Points Alleviates Severe Traumatic Brain Injury-induced Coma Via ATP Receptor Expressed Neuron In Ventral Periaqueductal Gray

Objective:Traumatic brain injury?TBI?is a common and serious classification among traumatic diseases in neurosurgery with extremely high mortality and disability.According to the relevant researches,afterwards consciousness and coma duration are clinical prognostic factors for coma patients.Therefore,it is extremely important to find a safe and effective awakening therapy to reduce mortality and disability and return patients to family and society.Acupuncture therapy at Jing-Well points is an emergency medical technique with unique characteristics of traditional Chinese medicine as well as a handy and inexpensive method.And acupuncture at Jing-Well points has been proved effective in improving awakening consciousness induced by central nervous system injuries?CNSIs?in many studies.However,the potential mechanisms of its wake-promoting effect have not been fully elucidated.Therefore,by further elucidating the wake-promoting molecular mechanism,it would be helpful to expand its clinical application and international promotion in TBI-induced coma or other CNSIs-induced disturbance of consciousness.In our previous basic studies,gene chip screening analysis has been used to identify that acupuncture at Jing-Well points for 1 h mainly activated adenosine triphosphate?ATP?signaling pathway,neural projection and protein secretion signal pathway in the brainstem.Among them,the results confirmed that acupuncture at Jing-Well points up-regulated the neural projection related factors such as P2RX7,P2RX3 and TRPV1,and up-regulated the expression of dopamine receptor D3?DRD3?in the brainstem of TBI rats.This study explored its wake-promoting molecular mechanisms by targeting the key neural projection receptors?P2RX7,P2RX3,TRPV1?.Method:1.Study on the effect platform of replication acupuncture at Jing-Well points on improving the conscious state of TBI-induced coma ratsTwenty-four healthy Sprague-Dawley?SD?male rats?8 weeks?were divided into three groups at random,Sham group,model group?TBI group?and TBI+Acupuncture at Hand twelve Jing-Well points group?Hand Acu group?.To preliminarily explore acupoint specificity in the wake-promoting effect of Hand twelve Jing-Well points,this study added the acupuncture at Foot twelve Jing-Well points group?Foot Acu group?,6 rats in each group.Rats in Sham group received a similarly located craniotomy but no percussion injury.TBI models were subjected to TBI elicited by electricity controlled cortical impact?e CCI?.According to the I-VI consciousness scale,grade V and VI are defined as coma in this experiment.In the Sham group and TBI group,the rats received no treatment.Based on TBI,rats in Hand Acu group and Foot Acu group were treated with Hand twelve Jing-Well points and Foot twelve Jing-Well points,twice daily for 48 h.Coma duration and modified neurological severity score?m NSS?are the main index of curative effect to be assessed,and m NSS were observed at 6 h,24 h and 48 h after TBI modeling.2.Study on P2RX3,P2RX7 and TRPV1 mediates acupuncture at Jing-Well points therapy to promote awakening actionThe previous results of this research showed that the acupuncture at Jing-Well points up-regulated the expression of neural projection receptors such as P2RX7,P2RX3,TRPV1 in the brainstem at 1 h.At the same time,it was found that acupuncture up-regulated the content of DRD3 in the brainstem of TBI rats.Next,intracerebral ventricle injection?i.c.v?injection of P2RX7,P2RX3 and TRPV1 antagonist were carried out to observe whether the wake-promoting effect of acupuncture was blocked,and we further explored whether neural projection receptors P2RX7,P2RX3 and TRPV1 mediates acupuncture effect.According to the different solvents of the three antagonists,the rats were randomly assigned into the following7 groups?n=6 rats per group?:TBI+i.c.v saline,TBI+i.c.v saline+Acu,P2RX3 antagonist+Acu;TBI+i.c.v DMSO,TBI+i.c.v DMSO+Acu,P2RX7 antagonist+Acu and TRPV1antagonist+Acu.That is,the three antagonist groups were treated with acupuncture intervention based on ICV injection.3.Study on the exploration of acupuncture at Jing-Well points acting on P2RX7,P2RX3,TRPV1 and dopamine of ventral periaqueductal gray?v PAG?matterWe continued to study which nuclei of brainstem expresses these neural projection receptors and mediates acupuncture at Hand Jing-Well points to promote awakening.Previous reports showed that v PAG is one of the key projection sites of the brainstem reticular ascending reticular activating system?ARAS?.The dopamine neurons of v PAG act as a relay station in arousal conduction,and v PAG expresses neural projection receptors such as P2RX7,P2RX3and TRPV1.Therefore,we further focused to the v PAG nuclei in the brainstem to clarify the regulatory effect of Hand Jing-Well points on P2RX7,P2RX3 and TRPV1 signal pathways.Multiple immunofluorescence staining combined with the Histo FAXS Tissue Analysis System?Tissue Gnostics?to were applied to detect the expression and co-localization of P2RX7,P2RX3,TRPV1 and dopamine in brainstem v PAG nuclei.P2RX7,P2RX3 and TRPV1 in Hand Acu group were co-stained with dopamine to observe whether dopaminergic neurons express neural projection receptors,such as P2RX7,P2RX3,TRPV1.At the same time,P2RX7,P2RX3,TRPV1,dopamine co-staining with c-fos in v PAG nuclei to detect the excitability of the above neurons.Result:1.Acupuncture at Jing-Well points improved the consciousness level and neurologic defects in TBI ratsThe effects of acupuncture in coma duration and neurological deficits in different groups were investigated.The results of this experiment are as follows:?1?After TBI,it was found that the coma duration of TBI group was significantly longer than Sham group(^**P<0.01 v.s.Sham group),and it was suggested that the TBI rats model were replicated successfully.The coma duration of Hand Acu group and Foot Acu group were significantly shorter than TBI group after acupuncture intervention,which indicated that acupuncture at Hand/Foot twelve Jing-Well points shortened the coma duration of TBI rats.And the wake-promoting effect of Hand Acu group better than Foot Acu group.?2?After the rats wake up,the m NSS scoring at 6h,24 h and 48 h following TBI surgery were evaluated.TBI rats displayed obvious neurological deficits at 6 h,24 h and 48 h after surgery(^**P<0.01 v.s.Sham group)compared with those in Sham groups.The neurological deficits were improved by acupuncture at HTPWs at both 24 h?^?P<0.05 v.s.TBI group?and 48 h(^??P<0.01 v.s.TBI group).At 48 h,there was significant improvement by Hand Acu group compared to Foot Acu group in Fig.11?^#P<0.05 v.s.Hand Acu group?.The results above indicated that the therapeutic actions of acupuncture at HTPWs were better than those of FTPWs,thus the subsequent investigations were treated with acupuncture at HTPWs.To sum up,the results of the two effect indexes showed that the effect of the acupuncture at Hand twelve Jing-Well points therapy is more significant,so the later experiments uses it as an intervention measure to explore its wake-promoting mechanism.2.P2RX3,P2RX7 and TRPV1 mediated acupuncture at Jing-Well points to promote awakeningAcupuncture at Hand twelve Jing-Well points showed wake-promoting effect,respectively?^*P<0.05 v.s.TBI group?.The results of intracerebroventricular injection of P2RX7,P2RX3and TRPV1 antagonists showed that P2RX7 antagonist effectively blocked the awakening effect of acupuncture at HTWPs?^?P<0.05 v.s.Hand Acu group?,and the injection of P2RX3or TRPV1 antagonist into the lateral intraventricular area did antagonized the acupuncture effect as well(^**P<0.05 v.s.TBI group,^??P<0.01 v.s.Hand Acu group).This study showed that intracerebroventricular injection of P2RX7,P2RX3 and TRPV1 antagonists effectively blocked the awakening effect of acupuncture at HTPWs,indicating that P2RX7,P2RX3 and TRPV1 signaling may mediate the awakening effect of acupuncture at HTWPs.3.Acupuncture at Jing-Well points activated the expression and the excitability of dopamine and P2RX7 neurons in ventral periaqueductal grayThe results of multiple immunofluorescence staining in this experiment are as follow:?1?Multiple immunofluorescence staining confirmed that P2RX3,P2RX7,TRPV1 and dopamine were mainly expressed in the cell cytoplasm and membrane of neurons in v PAG.And then,most DA neurons located in v PAG were found expressing P2RX3,P2RX7 and TRPV1.?2?Multiple immunofluorescence staining combined with the Histo FAXS Tissue Analysis System?Tissue Gnostics?quantified analysis showed that acupuncture for 1 h increased the expression and excitability P2RX7 neurons,with an increased number of P2RX7?^?P<0.05 v.s.TBI group?and c-fos double positive cells?^?P<0.05 v.s.TBI group?,while the excitability of P2RX3/TRPV1 neurons?co-expressed c-fos?showed an upward trend.Further,our results also have linked the increased excitability of v PAG DA neurons?^*P<0.05 v.s.Sham group,^?P<0.05 v.s.TBI group?by acupuncture.Therefore,these data suggested that acupuncture can increase the expression and excitability of DA and P2RX7 neurons,which may be involved in the awakening effect of acupuncture.Conclusion:1.Acupuncture at twelve Jing-Well points promotes the awakening and improves the neurological deficit symptoms of traumatic brain injury rats.2.P2RX7,P2RX3 and TRPV1 mediates awakening action of acupuncture at Hand twelve Jing-Well points.3.Acupuncture at Hand twelve Jing-Well points activates the dopamine neurons expressing P2RX7 in ventral periaqueductal gray,which may participate in the awakening action of this therapy.