| PurposeWith the increase of traffic,work-related injuries and sports accidents in China,the incidence of craniocerebral trauma has shown a trend of increasing year by year,and severe patients often suffer from coma symptoms.Such patients often need long-term monitoring and treatment to maintain survival,which is the difficulty and hotspot of clinical treatment in the rehabilitation department.Since there is no effective treatment at present,the prognosis is poor and the cost is high,which brings heavy mental shock and huge financial pressure to the patient’s family and also leads to a series of ethical and legal problems.Therefore,the awakening of comatose patients is of great importance.The awakening acupuncture method is a group of acupuncture points that I and my team members have summarized based on long-term clinical practice,and it has shown good efficacy in clinical awakening treatment and preliminary randomized controlled trials.However,its effectiveness has not been fully verified by imaging and other laboratory data,and its mechanism of action has not been discussed.In the present study,the clinical effects were evaluated by analyzing the changes of Glasgow Coma Scale(GCS)before and after the treatment of comatose patients with wakefulness stimulating acupuncture.The animals’consciousness scores and the results of Orexin-A and OX1R expression were used to explore the mechanism of the awakening acupuncture method.Study I(clinical component).Methods(1)Patients in coma after craniocerebral trauma with a Glasgow ComaScale score of 3-8 were selected and divided into a control group of 30 patients and an observation group of 30 patients according to randomization method.(2)The control group was treated with conventional Western medical treatment and conventional acupuncture,and the observation group was treated with conventional Western medical treatment and awakening acupuncture for 4 weeks.Before the treatment and on the 2nd and 4th week after the treatment,the points of eye opening response,speech response and motor response were recorded,the recovery of consciousness,speech and limb function was assessed,and the GCS score was completed.(3)Statistical Analysis MethodsData were statistically analyzed using SPSS23.O,all measures were expressed as mean plus or minus standard deviation((?)±s),comparison of means between the two groups was performed using the independent samples t/t’ test,all count data were expressed as frequency(f),and unordered classification data were tested using theχ~2 test.P<0.05 is considered a significant difference and P<0.01 is considered a highly significant difference.Results(1)Finally 56 patients completed the trial,including 28 in the control group and 28 in the observation group.There was no significant difference between the two groups in terms of GCS score,age,gender and whether they had undergone surgery before treatment(P>0.05),so the two groups were comparable.(2)After 2 weeks of treatment,the GCS score was 9±2.944 in the observation group and 7.04±2.687 in the control group.the observation group was higher than the control group,and the difference was statistically significant(P<0.05).After 4 weeks of treatment,the GCS scores of the observation group and the control group were 11.07±3.242 and 8.21±3.27,respectively.the observation group was higher than the control group,with a highly significant difference(P<0.01).(3)In the observation group,the GCS score was 6 ± 1.633 beforetreatment,9±2.944 after 2 weeks of treatment and 11.07±3.242 after 4 weeks of treatment,the difference was statistically significant(P<0.05)compared with that before treatment.In the control group,the GCS score was 6.25±1.506 before treatment,7.04±2.687 after 2 weeks of treatment and 8.21±3.27 after 4 weeks of treatment,which was statistically significant compared with that before treatment(P<0.05).In both the observation and control groups,the change in GCS score changes significantly with the change in treatment time point,and the time point*group interaction effect was significant(P<0.001),and the difference was statistically significant.(4)In terms of efficacy evaluation,after 2 weeks of treatment,the observation group was awake in 2 cases,markedly effective in 19 cases,effective in 6 cases,invalid in 2 cases,the markedly effective rate was 75%,the total effective rate was 92.9%;in the control group,1 case was awake,4 cases were markedly effective,12 cases were effective,11 cases were ineffective,the significant efficiency was only 17.9%,the total effective rate was 60.7%.At the end of the course of treatment,in the observation group,5 cases were awake,20 cases were markedly effective,2 cases were effective,and 1 case was invalid,with the marked effective rate of 89.3%,and the total effective rate was 96.4%;in the control group,2 cases were awake,12 cases were markedly effective,9 cases were effective,and 5 cases were ineffective,with the marked effective rate of 50%,and the total effective rate was 82.1%.The GCS scores of the observation group and the control group after 2 and 4 weeks of treatment were improved compared with those before treatment,and the above differences were statistically significant(P<0.05).Study Ⅱ(animal experimental component)Methods(1)Clean-grade healthy adult male SD(Sprague-Dawley)rats were selected and randomly divided into a normal group(n=8),a model group(n=8),and an awakening acupuncture group(n=8)after 7 days of acclimatization.The model group and the awakening acupuncture group used a rat brain stereotactic precision percussion instrument(68099II Reward RWD)to produce a coma model of craniocerebral trauma.The awakening acupuncture group was treated with awakening acupuncture immediately after the model was successfully established and 3 hours after the completion of the first acupuncture.All groupsof rats were simultaneously sampled brain tissue 1 hour after the last treatment.and the material was taken 1 hours after the last treatment.(2)Behavioral observation of the level of arousal in each group ofrats.(3)HE staining was performed under light microscope to observe the pathological changes of brain tissue at the site of injury.Western-blot and RT-PCR were used to detect the expression of Orexin-A and OX1R proteinsand genes in the hypothalamus and frontal lobe of the brain in each group of rats.(4)Statistical analysisThe data were analyzed by SPSS23.0 package,and the measures were expressed as mean±standard deviation((?)±s),the differences between groups obeying normal distribution were analyzed by One-Way ANOVA,theLSD-t test was used for two-by-two comparisons when the variance was uneven,the Welch test was used when the variance was uneven,and the non-parametric test was used for data not obeying normal distribution.Counting data were expressed as frequencies,and comparisons between groups were made using the χ~2 test.P<0.05 was taken as a significant difference and P<0.01 as a highly significant difference.Results(1)Behavioral changes in rats.Rats in the sham-operated group,model group,and awakening acupuncture group were evaluated for behavior after the first treatment after successful modeling and before sampling.A total of 12 rats died during the whole experiment and were excluded from the data.The activities of 8 rats in the sham operation group were more or less normal after awakening from anesthesia,and the consciousness status of 4 rats were grade Ⅰ and 4 rats were grade Ⅱ.Some rats in the model group and awakening acupuncture group had irregular breathing,cardiac arrest,twitching of limbs,urinary incontinence,horn retrusion and other symptoms after modeling,after manual CPR and resting,the surviving rats could enter coma.At the point of animal behavior assessment(6 hours),among the 8 rats in the model group,3 rats were grade Ⅵ,3 rats were grade Ⅴ,and 2 rats were grade Ⅳ.In the awakening acupuncture group,there were 3 rats of grade Ⅲ,4 rats of grade Ⅱ,and one subject of grade Ⅰ.The state of consciousness of the three groups was sham operation group>awakening acupuncture group>model group.(2)OX1R and Orexin-A expression in each brain lobe of rats.QPCR and WB results showed that the expression of Orexin-A in frontal,parietal and hypothalamic Orexin-A were significantly decreased in the model group and the awakening acupuncture group compared with the sham operation group,and the difference wasstatistically significant(P<0.05).The expression of Orexin-A decreased in the model group compared with the sham operation group,and the difference was statistically significant(P<0.05).The expression of Orexin-A showed a decreasing trend of sham operation group>awakening acupuncture group>model group.WB results showed that OX1R expression was consistent with Orexin-A expression,and the difference wasstatistically significant(P<0.05).The expression of OX1R showed a decreasing trend in the sham operation group,the awakening acupuncture group and the model group.But inQPCR,the results of OX1R in frontal lobe,parietal lobe and hypothalamus were opposite,showing an increasing trend of sham operation group<awakening acupuncture group<model group.(3)In the sham operation group,the morphology and intercellular space of parietal lobe were normal,the arrangement of nerve cells was orderly,and the shape of nucleus was uniform and regular;in the model group,the intercellular space was widened,the arrangement of nerve cells was disordered,the shape of nucleus was irregular,and part of nucleus was pyknosis;the morphology and intercellular space of parietal lobe in awakening acupuncture group were basically normal,and the arrangement of nerve cells was more disordered than that in sham operation group Most of the nuclei were uniform and regular in shape,and a small number of nuclei were pyknosis.Compared with the sham operation group,the number of neurons in the model group was significantly reduced(P<0.05),and there were more deeply stained neurons(damaged neurons),indicating that the neurons were seriously damaged.Compared with the model group,the number of neurons in the awakening acupuncture group was significantly increased(P<0.05).Conclusion(1)Awakening acupuncture has some awakening effect in patients who are unconscious after craniocerebral trauma.(2)Awakening acupuncture can change the HE staining of brain tissue at the site of injury in comatose rats,and has a protective effect on neuronal damage caused by TBI.Elevated levels of Orexin-A and OX1R in hypothalamus and cerebral parietal and frontal lobes.(3)Awakening acupuncture had an awakening effect on comatose rats after craniocerebral trauma,not only has the effect of promoting awakening,but also can increase the level of Orexin-A in the brain tissue.It is speculated that the improvement of the state of consciousness by awakening acupuncture may be related to the increase of the level of Orexin-A in the brain tissue. |
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