Acupuncture At Hand Twelve Jing-well Points Alleviates Severe Traumatic Brain Injury-induced Coma Via Dopamine Receptor In Hypothalamus

Objective:Traumatic brain injury(TBI)coma is a common acute and critical disease in neurosurgery,which has the characteristics of high morbidity and mortality.Whether the patients with TBI coma can wake up as soon as possible is an important factor affecting the clinical prognosis,and how to promote awakening is a hot and difficult point in the field of neuroscience at home and abroad.Acupuncture at hand twelve Jing-well points(HTJW acupuncture)is a traditional first aid awakening technique in traditional Chinese medicine.Our team used this therapy to treat central nervous system injury and conducted several clinical trials to confirm that this therapy can safely and effectively improve the consciousness level of patients with TBI,stroke and carbon monoxide poisoning in acute stage,but its awakening mechanism has not been elucidated,which limits the clinical application and popularization of this therapy.In the previous study,based on the effective awakening effect of HTJW acupuncture on TBI-induced coma rats,it was found that this therapy could up-regulate the expression of P2RX7 neurons and the excitability of P2RX7 and dopamine neurons in ventral midbrain periaqueductal gray of rats with TBI-induced coma.Through intracerebroventricular injection of antagonist,it was found that the brain nerve projection receptor P2RX7/P2RX3/TRPV1 mediates the HTJW acupuncture to promote awakening.This study focuses on the P2RX7 neurons of v PAG and the dopamine receptor neurons projecting from v PAG dopamine neurons to the hypothalamus to further explain the biological mechanism of the HTJW acupuncture in promoting the recovery of TBI-induced coma rats.Method:1.Duplicating the effect platform of the acupuncture at hand twelve Jing-well points to improve the level of consciousness in rats with TBI coma(1)To replicate the platform of rats with severe TDI coma: Thirty-two healthy adult male rats(8 weeks)were randomly divided into sham operation group(Sham),model group(TBI),TBI+ HTJW acupuncture group(TBI+Hand Acu),sham group + HTJW acupuncture group(Sham+Hand Acu),with 8 rats in each group.In Sham group,only the cranial window was opened without craniocerebral impact.In the TBI group,the severe TBI rat model was made by using the electricity Controlled cortical impact(e CCI)after opening the cranial window.According to the I-VI state of consciousness rating scale,V and VI TBI rats were defined as coma in the experiment.Neither Sham group nor TBI group was treated with Jingwell acupoint bloodletting.Hand Acu group and Sham+Hand Acu group were immediately treated with HTJW acupuncture intervention treatment on the basis of the success of the corresponding model.The main index of curative effect evaluation is to observe the coma duration of experimental rats in real time.(2)To explore the effect of using EEG monitoring to evaluate the effect of HTJW acupuncture on improving the consciousness level of rats with TBI coma based on EEG.Six healthy adult(8 weeks)male SD rats were randomly divided into TBI group(n = 3)and Hand Acu group(n = 3).After successful modeling,EEG monitoring electrodes are installed and connected to the Power Lab biological signal acquisition system for real-time EEG monitoring.Using Mathlab Software to extract the relativepower of Alpha,Beta,Delta and Theta waves.The ratio of Alpha/Theta wave relativepower was calculated to quantify the effect of HTJW acupuncture to promote awakening.2.Study on ventral midbrain periaqueductal gray P2RX7 receptor mediated acupuncture at hand twelve Jing-well points to promote awakeningThe previous results showed that HTJW acupuncture could increase the expression and excitability of P2RX7 in v PAG after modeling 1h.Therefore,this experiment further explored whether the awakening effect of HTJW acupuncture disappeared after injection of P2RX7 antagonist into v PAG nucleus,in order to determine whether the P2RX7 receptor of v PAG nucleus mediated HTJW acupuncture to promote awakening.The experimental animals were divided into 4 groups(n = 6/group): TBI+DMSO group,Hand Acu+DMSO group,TBI+P2RX7antagonist group and Hand Acu+P2RX7 antagonist group.All groups were modeled after injection of 30 min into v PAG nuclei.Among them,Hand Acu+DMSO group and Hand Acu+P2RX7 antagonist group were treated with HTJW acupuncture intervention on the basis of successful injection of v PAG nucleus and modeling.The evaluation index of curative effect was to observe the coma duration and modified neurological deficit score(m NSS)of experimental rats in real time,and to evaluate the neurological deficit of rats 6 hours after modeling.3.Effect of acupuncture at hand twelve Jing-well points on the expression of dopamine receptor in hypothalamusCombined with the previous study on the expression of P2RX7 receptor in DA neurons in v PAG region,and HTJW acupuncture can improve the excitability of DA neurons and P2RX7 neurons in v PAG region.Experiment 2 further confirmed that P2RX7 neurons in v PAG region mediated HTJW acupuncture to promote awakening.It has been reported that the DA neurons of v PAG can be projected directly to the orexin neurons in the lateral hypothalamic(LH)region to maintain arousal.The dopamine regulation of histamine neurons in the hypothalamic tuberomammillary nucleus(TMN)plays an important role in the regulation of sleep arousal.Therefore,the purpose of this study is to further explore the effect of HTJW acupuncture on the expression of dopamine receptor in lateral hypothalamus and tuberomammillary nucleus.RTq PCR was used to detect the gene content of dopamine receptor DRD1/DRD2/DRD3 in hypothalamus 1 hour after modeling and HTJW acupuncture intervention.Immunofluorescence staining and DRD1/DRD2/DRD3+DAPI co-staining were used to detect the expression of dopamine receptor in hypothalamus LH and TMN nucleus.4.Regulation of acupuncture at hand twelve Jing-well points on the excitability of dopamine receptor D2 neurons in lateral hypothalamus and papillary tubercle nucleusThe purpose of this study was to explore the regulatory effect of HTJW acupuncture on DRD2 neurons in hypothalamic LH and TMN nucleus.The rats were divided into three groups:Sham group,TBI group and Hand Acu group,with 4 rats in each group.Multiple immunofluorescence staining was used to detect the co-staining of DRD2+c-Fos+DAPI.To observe the expression of DRD2 in LH and TMN nucleus of hypothalamus after HTJW acupuncture intervention.At the same time,the excitability of DRD2 neurons in LH and TMN was detected by Image J software.In addition,Hand Acu group samples were selected for Orexin+DRD2+DAPI and Orexin+c-Fos+DAPI co-staining to determine whether ORX neurons in hypothalamic LH region expressed DRD2 receptors,and whether HTJW acupuncture could activate ORX neurons to express c-Fos protein.In order to further determine whether HTJW acupuncture can activate ORX neurons,samples from Sham group,TBI group and Hand Acu group(n= 4)were selected for Orexin+c-Fos+DAPI co-staining.In addition,the samples of Sham group,TBI group and Hand Acu group were selected for immunohistochemical staining of histamine neurons in order to observe the effect of HTJW acupuncture therapy on histamine neurons in TMN region.Results:1.HTJW acupuncture therapy can shorten the coma duration of rats with severe TBI comaThe purpose of this study was to observe the effect of HTJW acupuncture therapy on the coma duration of TBI rats.The results are as follows:(1)there was no significant change in the coma duration between the Sham + HTJW acupuncture group and the Sham group,suggesting that HTJW acupuncture had no awakening effect on the coma caused by chloral hydrate.The coma duration of the TBI group was significantly higher than that of the Sham group in the TBI model of rats,suggesting that the TBI rat model was successfully replicated.After the intervention of HTJW acupuncture,the duration of coma was shortened.The above results suggest that HTJW acupuncture can promote the recovery of TBI-induced coma in rats.(2)EEG showed that the EEG of rats in coma was characterized by slow wave and high amplitude,while the frequency of EEG in awake period was faster than that in coma period,and the amplitude was lower than that in coma period.The analysis of EEG data showed that there was no significant change in the ratio of relativepower of Alpha/Theta wave between TBI group and HTJW acupuncture group 30-40 minutes after the intervention of TBI modeling and HTJW acupuncture In TBI group,the ratio of alpha / theta wave relativepower remained low from 40 min to 100 min after modeling.Compared with TBI group,the relativepower ratio of alpha / theta wave was increased by 8.73-19.47% in 40-100 min after HTJW acupuncture intervention.The above results suggest that HTJW acupuncture can improve the consciousness of rats with TBI coma to some extent.2.Ventral midbrain periaqueductal gray P2RX7 receptor mediate HTJW acupuncture to promote awakeningThere was no significant change in coma duration between TBI group and TBI+P2RX7antagonist group,suggesting that P2RX7 antagonist had no significant effect on coma duration in TBI rats.Compared with TBI group,the coma time of TBI rats could be shortened by HTJW acupuncture intervention,and it could also shorten the coma time of TBI rats compared with TBI+P2RX7 antagonist group.Compared with the HTJW acupuncture group,the coma duration of rats in the HTJW acupuncture + P2RX7 antagonist group was higher than that in the HTJW acupuncture group.There was no significant difference in coma time between HTJW acupuncture + P2RX7 antagonist group,TBI group and TBI+P2RX7 antagonist group.In terms of m NSS score: Compared with TBI group and TBI+P2RX7 antagonist group,the 6h m NSS score of HTJW acupuncture group was improved.There was no significant difference in m NSS score between HTJW acupuncture + P2RX7 antagonist group and HTJW acupuncture group.The m NSS score of HTJW acupuncture + P2RX7 antagonist group was lower than that of TBI group and TBI+P2RX7 antagonist group.The results suggest that the P2RX7 receptor signal pathway of v PAG may mediate the awakening effect of HTJW acupuncture.3.HTJW acupuncture can increase the expression of DRD2 in hypothalamic LH and TMN regionsThe results of hypothalamic dopamine receptor gene content detection showed that the contents of DRD1,DRD2 and DRD3 genes in hypothalamus increased significantly 1 hour after HTJW acupuncture intervention,and the difference was statistically significant.The results of immunofluorescence staining showed that there was a large amount of DRD2 expression in LH and TMN region of hypothalamus 1 hour after HTJW acupuncture intervention,but no expression of DRD1 and DRD3.The immunofluorescence quantitative results showed that in the hypothalamic LH region,the DRD2 in the TBI group decreased significantly compared with the sham operation group.Compared with the TBI group,the DRD2 of the HTJW acupuncture group increased significantly after the intervention of HTJW acupuncture.Compared with the Sham group,the DRD2 in the HTJW acupuncture group was significantly higher than that in the Sham group.In the TMN region,compared with the Sham group,the decreasing trend of DRD2 in the TBI group was not obvious.After the intervention of HTJW acupuncture,compared with TBI group,the level of DRD2 in HTJW acupuncture group increased significantly.Compared with the Sham group,the DRD2 of the HTJW acupuncture group showed an increasing trend.The results showed that HTJW acupuncture could up-regulate the expression of DRD2 in hypothalamic LH and TMN regions.4.HTJW acupuncture can improve the excitability of DRD2 neurons in hypothalamic LH and TMN regionsThe results of multiple immunofluorescence staining in LH and TMN regions of hypothalamus showed that after the intervention of HTJW acupuncture,the excitability of DRD2 neurons in LH and TMN regions of hypothalamus increased in rats with TBI coma and there was statistical significance.The Co-staining results showed that ORX neurons in LH region expressed DRD2 receptors,and HTJW acupuncture could activate 81.6%(0.816 ±0.020)of ORX neurons in LH region to express c-Fos protein;and HTJW acupuncture could upregulate the expression of ORX neurons in hypothalamic LH region.The histamine immunohistochemical results of hypothalamic TMN region showed that compared with Sham group,the expression of histamine neurons in hypothalamic TMN region increased significantly in TBI group at 1 hour after modeling.Compared with TBI group,the expression of histamine neurons in hypothalamic TMN region was down-regulated for 1 hour after HTJW acupuncture intervention.The above experimental results suggest that HTJW acupuncture can increase the excitability of DRD2 neurons in hypothalamic LH and TMN regions.Orexin neurons in LH region express DRD2 and HTJW acupuncture can excite orexin neurons.HTJW acupuncture could down-regulate the expression of histamine neurons in hypothalamic TMN region 1 hour after HTJW acupuncture intervention.Conclusion:1.Ventral midbrain periaqueductal gray P2RX7 receptor mediates the effect of acupuncture at hand twelve Jing-well points therapy to promote awakening.2.Acupuncture at hand twelve Jing-well points can increase the expression and excitability of dopamine receptor D2 neurons in hypothalamic LH and TMN nuclei,which may participate in the awakening action of this therapy.3.Acupuncture at hand twelve Jing-well points can up-regulate the number of orexin neurons in hypothalamic LH nucleus and inhibit the expression of histamine neurons inhypothalamic TMN nucleus.But its mechanism of promoting awakening by acupuncture at hand twelve Jing-well points needs to be further verified.