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[1]杜偉斌,陳榮良,俆亦生,等.電針夾脊穴和督脈穴對(duì)急性脊髓損傷家兔后肢神經(jīng)功能的影響及其作用機(jī)制[J].中醫(yī)正骨,2016,28(12):13-20.
　DU Weibin,CHEN Rongliang,XU Yisheng,et al.Effects of electroacupuncture at Point JIAJI(EX-B2)and Point governor vessel on neural function of hindlimbs in rabbits with acute spinal cord injury and the mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(12):13-20.
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電針夾脊穴和督脈穴對(duì)急性脊髓損傷家兔后肢神經(jīng)功能的影響及其作用機(jī)制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期數(shù):
2016年12期
頁(yè)碼:
13-20
欄目:
基礎(chǔ)研究
出版日期:
2016-12-30

文章信息/Info

Title:
Effects of electroacupuncture at Point JIAJI(EX-B2)and Point governor vessel on neural function of hindlimbs in rabbits with acute spinal cord injury and the mechanism of action
作者:
杜偉斌1; 陳榮良1; 俆亦生1; 全仁夫1; 胡華輝2; 鮑關(guān)愛(ài)3
1.浙江省杭州市蕭山區(qū)中醫(yī)院,浙江 杭州 311201;
2.浙江中醫(yī)藥大學(xué),浙江 杭州 310053;
3.浙江省腫瘤醫(yī)院,浙江 杭州 310022
Author(s):
DU Weibin1; CHEN Rongliang1; XU Yisheng1; QUAN Renfu1; HU Huahui2; BAO Guanai3
1.Xiaoshan Hospital of Traditional Chinese Medicine,Hangzhou 311201,Zhejiang,China 2.Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 3.Zhejiang Cancer Hospital,Hangzhou 310022,Zhejiang,China
關(guān)鍵詞:
脊髓損傷;  電針;  穴; 夾脊;  穴位; 督脈;  神經(jīng)干細(xì)胞;  神經(jīng)傳導(dǎo);  兔;  動(dòng)物實(shí)驗(yàn)
Keywords:
spinal cord injuries;  electroacupuncture;  point EX-B2(JIAJI);  points; governor vessel;  neural stem cells;  neural conduction;  rabbits;  animal experimentation
摘要:
目的:觀察電針夾脊穴和督脈穴對(duì)急性脊髓損傷家兔后肢神經(jīng)功能的影響,并探討其作用機(jī)制。方法:采用隨機(jī)數(shù)字表將60只新西蘭家兔隨機(jī)分為假手術(shù)組、模型組、電針組,每組20只。假手術(shù)組咬除T13～L1棘突及全部椎板,暴露0.5 cm寬硬膜,不損傷脊髓; 模型組和電針組采用改良Allens法建立急性脊髓損傷家兔模型,并行脊髓MRI掃描鑒定模型。模型組和假手術(shù)組不給予治療,電針組給予電針刺激受損脊髓節(jié)段上下兩對(duì)夾脊穴及上下督脈穴(筋縮、命門)治療,每次30 min,每日1次,共7次。電針干預(yù)開(kāi)始后1 d、3 d、7 d采用改良Tarlov評(píng)分法評(píng)價(jià)家兔后肢神經(jīng)功能。干預(yù)結(jié)束后每組取10只家兔,以4%多聚甲醛行心臟灌注后剖取損傷脊髓約1 cm,制成切片后經(jīng)HE染色于顯微鏡下觀察脊髓組織形態(tài),并采用免疫組織化學(xué)法觀察脊髓細(xì)胞增殖情況和巢蛋白(Nestin)表達(dá)水平。將每組剩余的10只家兔麻醉,分離坐骨神經(jīng)并注入5 μL的30%辣根過(guò)氧化物酶(horseradish peroxidase,HRP),24 h后取脊髓橫斷面背側(cè)靠近損傷處脊髓,采用HRP逆行示蹤法標(biāo)記紅核神經(jīng)元細(xì)胞數(shù)。結(jié)果:①模型鑒定結(jié)果。MRI片上假手術(shù)組脊髓橫斷面信號(hào)正常,可見(jiàn)清晰的腦脊液通過(guò)。模型組和電針組脊髓橫斷面信號(hào)增強(qiáng),腦脊液受壓; 受傷節(jié)段的脊髓在矢狀位MRI片T2WI上呈明顯的高信號(hào),T1WI上呈低信號(hào); 提示脊髓斷端周圍充血、水腫,證明造模成功。②后肢神經(jīng)功能評(píng)分。電針干預(yù)后不同時(shí)間點(diǎn)Tarlov評(píng)分比較,差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=6.920,P=0.001); 除假手術(shù)組外,干預(yù)后模型組和電針組Tarlov評(píng)分均逐漸升高。3組Tarlov評(píng)分總體上比較,組間差異有統(tǒng)計(jì)學(xué)意義,即存在分組效應(yīng)(F=426.78,P=0.000)。干預(yù)開(kāi)始后1 d,電針組Tarlov評(píng)分高于模型組[(0.30±0.47)分,(0.20±0.41)分,q=7.010,P=0.486],低于假手術(shù)組[(0.30±0.47)分,(4.70±0.47)分,q=30.843,P=0.000)]; 模型組低于假手術(shù)組(q=31.544,P=0.000)。干預(yù)開(kāi)始后3 d,電針組Tarlov評(píng)分高于模型組[(1.55±0.60)分,(1.05±0.53)分,q=2.877,P=0.006],低于假手術(shù)組[(1.55±0.60)分,(4.80±0.41)分,q=20.775,P=0.000]; 模型組低于假手術(shù)組(q=23.651,P=0.000)。干預(yù)開(kāi)始后7 d,電針組Tarlov評(píng)分高于模型組[(2.65±0.59)分,(1.40±0.50)分,q=8.004,P=0.000],低于假手術(shù)組[(2.65±0.59)分,(4.85±0.41)分,q=14.081,P=0.000]; 模型組低于假手術(shù)組(q=22.093,P=0.000)。時(shí)間因素和分組因素之間存在交互效應(yīng)(F=70.786,P=0.000)。③脊髓組織形態(tài)。假手術(shù)組脊髓形態(tài)結(jié)構(gòu)完整,神經(jīng)元結(jié)構(gòu)正常、分布均勻,細(xì)胞膜、細(xì)胞核及組織間隙均正常,尼氏體顯示清晰; 模型組脊髓組織正常結(jié)構(gòu)喪失,可見(jiàn)組織充血、水腫,神經(jīng)元細(xì)胞數(shù)量減少,部分尼氏體消失,灰質(zhì)中部分區(qū)域甚至有較大的空腔; 電針組與模型組相比,脊髓形態(tài)結(jié)構(gòu)基本完整,神經(jīng)元細(xì)胞結(jié)構(gòu)基本正常,空泡變性減少,組織充血、水腫消失。④脊髓細(xì)胞增殖情況。電針干預(yù)開(kāi)始后7 d,200倍顯微鏡下觀察,每個(gè)視野下3組家兔脊髓增殖細(xì)胞數(shù)比較,組間差異有統(tǒng)計(jì)學(xué)意義[(43.67±8.19)個(gè),(18.77±3.55)個(gè),(6.30±2.32)個(gè),F=382.413,P=0.000]; 電針組脊髓增殖細(xì)胞多于模型組(q=20.889,P=0.000)和假手術(shù)組(q=31.348,P=0.000),模型組多于假手術(shù)組(q=10.459,P=0.000)。⑤脊髓細(xì)胞Nestin表達(dá)水平。干預(yù)開(kāi)始后7 d,200倍顯微鏡下觀察,每個(gè)視野下3組家兔脊髓細(xì)胞Nestin陽(yáng)性表達(dá)細(xì)胞數(shù)比較,組間差異有統(tǒng)計(jì)學(xué)意義[(23.37±5.10)個(gè),(10.77±3.26)個(gè),(4.43±1.33)個(gè),F=218.084,P=0.000]; 電針組Nestin陽(yáng)性表達(dá)細(xì)胞數(shù)多于模型組(q=15.770,P=0.000)和假手術(shù)組(q=23.696,P=0.000),模型組多于假手術(shù)組(q=7.926,P=0.000)。⑥紅核神經(jīng)元細(xì)胞數(shù)。干預(yù)開(kāi)始后7 d,200倍顯微鏡下觀察,每個(gè)視野下3組紅核神經(jīng)元細(xì)胞數(shù)比較,組間差異有統(tǒng)計(jì)學(xué)意義(513.40±46.79,417.90±43.03,578.40±43.91,F=32.760,P=0.000); 電針組紅核神經(jīng)元細(xì)胞數(shù)多于模型組(q=6.771,P=0.000)、少于假手術(shù)組(q=4.608,P=0.000),模型組少于假手術(shù)組(q=11.379,P=0.000)。結(jié)論:電針刺激夾脊穴和督脈穴能明顯改善急性脊髓損傷家兔的后肢神經(jīng)功能,其作用機(jī)制可能是通過(guò)促進(jìn)神經(jīng)干細(xì)胞增殖分化和抑制紅核神經(jīng)元細(xì)胞凋亡,從而使損傷節(jié)段神經(jīng)組織修復(fù)及神經(jīng)通路再通。
Abstract:
Objective:To observe the effects of electroacupuncture at Point JIAJI(EX-B2)and Point governor vessel on neural function of hindlimbs in rabbits with acute spinal cord injury and to explore its mechanism of action.Methods:Sixty New Zealand rabbits were randomly divided into sham-operated group,model group and electroacupuncture group,20 cases in each group.The random digits table was used for grouping.The spinous processes and ertebral plates of T13-L1 of rabbits in sham-operated group were removed to expose 0.5 cm of endorachis in width without spinal cord injuries,while the models of acute spinal cord injury were built in rabbits in model group and electroacupuncture group by using improved Allens method and the models were identified by spinal cord MRI scanning.The rabbits in model group and sham-operated group were untreated,while the rabbits in electroacupuncture group were treated with electroacupuncture at two pairs of Point JIAJI(EX-B2)and upper and lower Point governor vessel(Point JINSUO and Point MINGMEN)at the injured segments of spinal cord,30 minutes at a time,once a day for consecutive 7 times.At 1,3 and 7 days after the beginning of electroacupuncture intervention,the neural function of hindlimbs of rabbits were evaluated by using improved Tarlov scoring method.After the end of intervention,10 rabbits were randomly selected from each group and were treated by perfusing their hearts with 4% paraformaldehyde.Then approximate 1 cm injured spinal cord were fetched out and sectioned for HE staining.The spinal cord tissue morphology were observed under the microscope and the proliferations of spinal cord cells and Nestin expression levels were detected by using immunohistochemical method.The sciatic nerves of the remaining 10 rabbits in each group were separated under anesthesia and 30% horseradish peroxidase(HRP)were injected into the sciatic nerves in dosage of 5 μL.Twenty-four hours later,the dorsal spinal cord tissues near the injuried spinal cord were fetched out and the numbers of red nucleus neuronal cells were marked by using HRP retrograde tracing technique.Results:The MRI signals of spinal cord in the transverse plane were normal and clear cerebrospinal fluids were found in sham-operated group; while enhanced MRI signals of spinal cord in transverse plane and compressed cerebrospinal fluids were found in model group and electroacupuncture group,and the injured segments of spinal cord presented with obvious high signal on T2-weighted MRI and low signal on T1-weighted MRI in the sagittal plane,which suggested that there were engorgement and edema arround the broken ends of spinal cord and the models were successfully built.There was statistical difference in Tarlov scores between different time points after electroacupuncture intervention,in other words,there was time effect(F=6.920,P=0.001).The Tarlov scores increased gradually in model group and electroacupuncture group after intervention.There was statistical difference in Tarlov scores between the 3 groups in general,in other words,there was group effect(F=426.78,P=0.000).At 1 day after the beginning of intervention,the Tarlov scores were higher in electroacupuncture group compared to model group(0.30+/-0.47 vs 0.20+/-0.41 points,q=7.010,P=0.486)and were lower in electroacupuncture group compared to sham-operated group(0.30+/-0.47 vs 4.70+/-0.47 points,q=30.843,P=0.000)and were lower in model group compared to sham-operated group(q=31.544,P=0.000).At 3 days after the beginning of intervention,the Tarlov scores were higher in electroacupuncture group compared to model group(1.55+/-0.60 vs 1.05+/-0.53 points,q=2.877,P=0.006)and were lower in electroacupuncture group compared to sham-operated group(1.55+/-0.60 vs 4.80+/-0.41 points,q=20.775,P=0.000),and the Tarlov scores were lower in model group compared to sham-operated group(q=23.651,P=0.000).At 7 days after the beginning of intervention,the Tarlov scores were higher in electroacupuncture group compared to model group(2.65+/-0.59 vs 1.40+/-0.50 points,q=8.004,P=0.000)and were lower in electroacupuncture group compared to sham-operated group(2.65+/-0.59 vs 4.85+/-0.41 points,q=14.081,P=0.000),and the Tarlov scores were lower in model group compared to sham-operated group(q=22.093,P=0.000).There was interaction between time factor and group factor(F=70.786,P=0.000).In sham-operated group,the shape and structure of spinal cord were complete and the neurons were uniformly distributed in normal structure; the cellular membrane,cellular nucleus and tissue spaces were normal and Nissl bodies were clear.In model group,the normal structure of spinal cord tissue disappeared,and hyperaemia and edema in the tissues and decrease of number of neurons were found; some Nissl body disappeared and there were big cavity in some areas of grey matter.In electroacupuncture group,the shape and structure of spinal cord were basically complete and the structure of neuron was basically normal; the vacuolar degeneration decreased and hyperaemia and edema in the tissues disappeared.At 7 days after the beginning of electroacupuncture intervention,there was statistical difference in the number of proliferative spinal cord cells under the optical microscope(×200)between the 3 groups(43.67+/-8.19,18.77+/-3.55,6.30+/-2.32,F=382.413,P=0.000); the number of proliferative spinal cord cells was more in electroacupuncture group compared to model group(q=20.889,P=0.000)and sham-operated group(q=31.348,P=0.000),and the number of proliferative spinal cord cells was more in model group compared to sham-operated group(q=10.459,P=0.000).At 7 days after the beginning of intervention,there was statistical difference in the number of Nestin positive expression cells of spinal cord under the optical microscope(×200)between the 3 groups(23.37+/-5.10,10.77+/-3.26,4.43+/-1.33,F=218.084,P=0.000); the number of Nestin positive expression cells was more in electroacupuncture group compared to model group(q=15.770,P=0.000)and sham-operated group(q=23.696,P=0.000),and the number of Nestin positive expression cells was more in model group compared to sham-operated group(q=7.926,P=0.000).At 7 days after the beginning of intervention,there was statistical difference in the number of red nucleus neuronal cells under the optical microscope(×200)between the 3 groups(513.40+/-46.79,417.90+/-43.03,578.40+/-43.91,F=32.760,P=0.000); the number of red nucleus neuronal cells was more in electroacupuncture group compared to model group(q=6.771,P=0.000)and was less compared to sham-operated group(q=4.608,P=0.000),and the number of red nucleus neuronal cells was less in model group compared to sham-operated group(q=11.379,P=0.000).Conclusion:Electroacupuncture at Point JIAJI(EX-B2)and Point governor vessel can obviously improve the neural function of hindlimbs in rabbits with acute spinal cord injury.It can promote the differentiation and proliferation of neural stem cells and inhibit the apoptosis of red nucleus neuronal cells,which may be the mechanisms of action for repairing the nervous tissues at the injuried segments and recirculating the neural pathway.

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備注/Memo

備注/Memo:
基金項(xiàng)目:浙江省杭州市科技發(fā)展計(jì)劃項(xiàng)目(20140733Q51); 浙江省杭州市蕭山區(qū)科技計(jì)劃項(xiàng)目(2013313)
通訊作者:陳榮良 E-mail:[email protected]
更新日期/Last Update: 2016-12-30