Effect of electroacupuncture Zhibian( BL54) and Shuidao( ST28) on motor function and urination function in rats with acute spinal cord injuries

目的:观察电针秩边穴与水道穴对大鼠急性脊髓损伤后运动和排尿功能的影响。方法:将48只Sd大鼠随机分为电针组和对照组,每组24只。采用改良AllEn’S重物打击法将所有大鼠制成急性脊髓损伤模型。造模后24 H,电针组大鼠在秩边穴和水道穴进行电针治疗,对照组大鼠在秩边穴和水道穴旁开0.5 CM处进行电针治疗。每天治疗1次,每次15 MIn。分别于实验干预1 d、3 d、7 d和14 d后采用bbb评分法评定2组大鼠的运动功能,同时于每次运动功能测试完成后,测定大鼠的膀胱容量和剩余尿量。结果:1运动功能。干预后不同时间bbb评分的差异有统计学意义,即存在时间效应[(0.50±0.53)分,(2.90±1.10)分,(7.50±1.27)分,(11.50±0.71)分;(0.60±0.52)分,(2.70±0.95)分,(4.60±0.70)分,(8.60±0.97)分;f=625.000,P=0.000]。2组大鼠bbb评分的组间差异总体上有统计学意义,即存在分组效应(f=29.866,P=0.000);干预1 d、3 d后,2组大鼠的bbb评分比较,差异均无统计学意义(T=0.429,P=0.678;T=0.435,P=0.674);干预7 d、14 d后,电针组大鼠的bbb评分均大于对照组(T=6.328,P=0.000;T=9.222,P=0.000)。时间因素和分组因素之间存在交互效应(f=24.939,P=0.000)。2排尿功能。干预后不同时间膀胱容量的差异有统计学意义,即存在时间效应[(1.10±0.05)M l,(1.37±0.05)M l,(1.89±0.09)M l,(2.31±0.19)M l;(1.10±0.06)M l,(1.36±0.05)M l,(1.67±0.08)M l,(1.88±0.08)M l;f=193.885,P=0.000]。2组大鼠膀胱容量的组间差异总体上有统计学意义,即存在分组效应(f=65.970,P=0.000);干预1 d、3 d后,2组大鼠的膀胱容量比较,差异均无统计学意义(T=0.177,P=0.868;T=0.366,P=0.733);干预7 d、14 d后,电针组大鼠的膀胱容量均大于对照组(T=4.186,P=0.014;T=4.647,P=0.009)。时间因素和分组因素之间存在交互效应(f=10.283,P=0.000)。干预后不同时间剩余尿量的差异有统计学意义,即存在时间效应[(2.00±0.20)M l,(1.88±0.15)M l,(1.49±0.07)M l,(0.98±0.11)M l;(1.99±0.19)M l,(1.89±0.17)M l,(1.73±0.12)M l,(1.29±0.07)M l;f=131.825,P=0.000]。2组大鼠剩余尿量的组间差异总体上有统计学意义,即存在分组效应(f=3.787,P=0.038);干预1 d、3 d后,2组大鼠的剩余尿量比较,差异均无统计学意义(T=0.016,P=0.988;T=0.039,P=0.971);干预7 d、14 d后,电针组大鼠的剩余尿量均小于对照组(T=3.791,P=0.019;T=5.334,P=0.006)。时间因素和分组因素之间存在交互效应(f=5.646,P=0.028)。结论:电针秩边穴与水道穴能改善大鼠急性脊髓损伤后的运动和排尿功能。Objective: To observe the effect of electroacupuncture Zhibian( BL54) and Shuidao( ST28) on motor function and urination function in rats with acute spinal cord injuries.Methods: Forty-eight SD rats were randomly divided into electroacupuncture group and control group,24 cases in each group.The acute spinal cord injuries models were built by modified Allen's method in all rats.The rats in the electroacupuncture group were treated with electroacupuncture at Zhibian( BL54) and Shuidao( ST28),while the others in the control group were treated with electroacupuncture at points 0.5 cm apart from Zhibian( BL54) and Shuidao( ST28) at 24 hours after the modeling,once a day for 15 minutes at a time.The motor function were evaluated by using BBB score at 1st,3rd,7th and 14 th days after experimental intervention.Meanwhile,the bladder capacity and residual urine volume of the rats were measured.Results: There was statistical difference in BBB scores between different time points after intervention,in other words,there was time effect( 0.50 + /- 0.53,2.90 + /- 1.10,7.50 + /-1.27,11.50 + /- 0.71 points; 0.60 + /- 0.52,2.70 + /- 0.95,4.60 + /- 0.70,8.60 + /- 0.97 points; F = 625.000,P = 0.000).In general,there was statistical difference in BBB scores between the 2 groups,in other words,there was grouping effect( F = 29.866,P = 0.000).There was no statistical difference in BBB scores between the 2 groups at 1 day and 3 days after intervention( t = 0.429,P = 0.678; t =0.435,P = 0.674),and the BBB scores of the electroacupuncture group were greater than those of control group at 7 and 14 days after intervention( t = 6.328,P = 0.000; t = 9.222,P = 0.000).There was interaction between time factor and grouping factor( F = 24.939,P =0.000).There was statistical difference in bladder capacity between different time points after intervention,in other words,there was time effect( 1.10 + /- 0.05,1.37 + /- 0.05,1.89 + /- 0.09,2.31 + /- 0.19 m L; 1.10 + /- 0.06,1.36 + /- 0.05,1.67 + /- 0.08,1.88 + /-0.08 m L; F = 193.885,P = 0.000).In general,there was statistical difference in bladder capacity between the 2 groups,in other words,there was grouping effect( F = 65.970,P = 0.000).There was no statistical difference in bladder capacity between the 2 groups at 1 day and 3 days after intervention( t = 0.177,P = 0.868; t = 0.366,P = 0.733),and the bladder capacity of the electroacupuncture group was larger than that of control group at 7 and 14 days after intervention( t = 4.186,P = 0.014; t = 4.647,P = 0.009).There was interaction between time factor and grouping factor( F = 10.283,P = 0.000).There was statistical difference in residual urine volume between different time points after intervention,in other words,there was time effect( 2.00 + /- 0.20,1.88 + /- 0.15,1.49 + /- 0.07,0.98 + /- 0.11 m L;1.99 + /- 0.19,1.89 + /- 0.17,1.73 + /- 0.12,1.29 + /- 0.07 m L; F = 131.825,P = 0.000).In general,there was statistical difference in residual urine volume between the 2 groups,in other words,there was grouping effect( F = 3.787,P = 0.038).There was no statistical difference in residual urine volume between the 2 groups at 1 day and 3 days after intervention( t = 0.016,P = 0.988; t = 0.039,P =0.971),and the residual urine volume of the electroacupuncture group was smaller than that of control group at 7 and 14 days after intervention( t = 3.791,P = 0.019; t = 5.334,P = 0.006).There was interaction between time factor and grouping factor( F = 5.646,P =0.028).Conclusion: The motor function and urination function of rats with acute spinal cord injuries can be improved by electroacupuncture Zhibian( BL54) and Shuidao( ST28).浙江省中医药科研计划项目(2008CB067

Mechanism Study of the Promoting Action of Elongated Needle Acupuncture on Functional Repair in Rats with Acute Spinal Cord Injury

目的 观察芒针对急性脊髓损伤炎症反应和神经细胞凋亡的影响,研究PI3K/Akt和MAPK/ERK信号转导途径是否参与芒针的神经保护作用,探讨芒针促脊髓损伤修复的具体作用机制.方法 将150只成年雄性SD大鼠随机分为A组、B组、C组、D组和E组,每组30只.采用改良Allen＇s法制作大鼠脊髓中度损伤模型,A组为假手术组,不予以损伤脊髓及芒针治疗;B组造模后不予以芒针治疗;C组造模后采用芒针治疗;D组造模前0.5 h鞘内注射LY294002,造模后采用芒针治疗;E组造模前0.5 h鞘内注射PD98059,造模后采用芒针治疗.分别采用BBB评分检测大鼠的自发活动;ELISA检测炎性因子TNF-α 、IL-6、IL-1β、NF-κB的含量;TUNEL检测细胞凋亡的程度;免疫组化检测Bcl-2和Bax阳性细胞水平;Western印迹分析p-Akt和p-ERK在脊髓组织的表达,RT-PCR分析Cyt-C和Caspase-3在体内的表达.相对的下行Akt和ERK信号通路通过LY294002和PD98059特异性抑制剂处理分析在体内的抑制作用.结果 炎症反应和PI3K/Akt和MAPK/ERK信号通路抑制的神经元凋亡参与了脊髓损伤大鼠模型的损害,芒针介导的神经保护作用与Bax蛋白阳性神经元数目的减少及Bcl-2蛋白阳性神经元数量的增加有关.芒针治疗能改善大鼠的运动功能,PI3K/Akt和MAPK/ERK信号通路中p-Akt和p-ERK的激活,通过下调Bax蛋白和Bcl-2表达上调,抑制了线粒体凋亡途径关键因子Cyt-C的表达.TUNEL法检测并抑制激活神经元凋亡的Caspase-3的级联.PI3K/Akt和MAPK/ERK信号通路特异性抑制剂LY294002和PD98059的应用抑制了p-Akt和p-ERK的表达.结论 芒针促脊髓损伤修复的神经保护作用可能与炎症反应和PI3K/Akt和MAPK/ERK信号通路的激活、通过下调Bax蛋白和Bcl-2表达上调以及抑制线粒体途径诱导的凋亡有关.Objective To observe the effect of elongated needle acupuncture on inflammatory reactions and apoptosis in acute spinal cord injury, investigate if PI3K/Akt and MAPK/ERK signal transduction pathways are involved in the neuroprotective effect of elongated needle acupuncture and explore the promoting action of elongated needle acupuncture on spinal cord injury repair.Method One hundred and fifty adult male SD rats were randomized into groups A, B, C, D and E, 30 each. A model of moderate spinal cord injury was made by modified Allen＇s method. Group A received a sham operation without spinal cord injury and no elongated needle acupuncture. Group B did not receive elongated needle acupuncture after model making. Group C received elongated needle acupuncture after model making. Group D received an intrathecal injection of LY294002 at 0.5 hour before model making and elongated needle acupuncture after model making. Group E received an intrathecal injection of PD98059 at 0.5 hour before model making and elongated needle acupuncture after model making. Rat spontaneous activity was examined using the BBB rating. Inflammatory cytokines TNF-α, IL-6, IL-1β and NF-κB contents were measured by ELISA. The degree of apoptosis was determined by TUNEL. Bcl-2- and Bax-positive cell levels were measured by an immunohistochemical method. Spinal p-Akt and p-ERK expressions were determined by Western blot. In vivo expressions of Cyt-C and Caspase-3 were determined by RT-PCR. The in vivo inhibitory effect on downstream Akt and ERK signaling pathways was investigated using specific inhibitors LY294002 and PD98059.Result Inflammatory reactions and neuronal apoptosis due to the inhibition of PI3K/Akt and MAPK/ERK signal pathways were involved in the damage in a rat model of spinal cord injury. The neuroprotective effect of elongated needle acupuncture was related to a decrease in the number of Bax protein-positive neurons and an increase in the number of Bcl-2 protein-positive neurons. Elongated needle acupuncture treatment improved r浙江省中医药科研计划项目（2008CB067

Effects of electro-acupuncture on neuronal apoptosis and associative function in rats with spinal cord injury

目的 :探讨电针对大鼠急性脊髓损伤后膀胱功能改善的影响及作用机制。方法 :取健康成年雄性SPf级Sd大鼠60只,体重220~250 g,适应性饲养1周后,将大鼠按照随机数字表法随机分为假手术组、模型组、电针组和电针对照组,各15只。假手术组不予任何刺激,模型组、电针组和电针对照组大鼠采用改良AllEnS法制作脊髓损伤中度损伤Sd大鼠模型,模型组不予治疗,电针组给予秩边与水道穴电针治疗,电针对照组给予秩边与水道穴旁开0.5寸电针治疗,频率2/100 Hz,电流1 M A,刺激15 MIn,电针左右隔次交替,每日1次,共7次;分别于术后1、7 d观察大鼠残余尿量、排尿量的变化;术后7 d处死大鼠取伤段脊髓观察各组大鼠凋亡情况,检测bCl-2、bAX、bAd含量的变化。结果:造模后3组大鼠均出现不同程度的膀胱功能障碍。术后7 d,电针组、电针对照组残余尿量较术后1 d明显降低(P<0.001),且电针组与电针对照组比较差异有统计学意义(P<0.01);电针组、电针对照组较模型组在术后7 d排尿量增加,且电针组与电针对照组比较差异有统计学意义(P<0.001);TunEl发现电针可以抑制脊髓神经细胞的凋亡,电针组、电针对照组与模型组相比在术后7 d脊髓神经细胞凋亡率显著增加(P<0.01,P<0.05),且电针组与电针对照组比较差异有统计学意义(P<0.05);与模型组相比,电针组、电针对照组bAX、bAd的阳性表达率降低(P<0.01,P<0.05),而bCl-2的阳性表达率升高(P<0.01);且电针组与电针对照组比较差异有统计学意义(P<0.05)。结论 :电针能明显促进急性脊髓损伤的修复,其机制可能为通过增加bCl-2、抑制bAX、bAd的表达,从而抑制脊髓神经元细胞的凋亡发生作用的。Objective:To explore the effect of electro acupuncture to improve the bladder function after acute spinal cord injury in rats and its possible mechanism.Methods:Sixty healthy adult male SD rats of SPF grade,with body weight of 220 to250 g,one week after feeding adaptation,were randomly divided into sham operation group,model group,electro acupuncture group,electro acupuncture control group with 15 rats in each group.Sham operation group underwent no stimulation,and the moderate damage model of spinal cord injury were made in other three groups according to modified Allens method.The model group were not treated,electro acupuncture group were treated with electro acupuncture on Zhibianxue and Shuidaoxue,and electro acupuncture control group were treated with electro acupuncture on 0.5 inch next to Zhibianxue and Shuidaoxue.The frequency of 2 / 100 Hz,current of 1 m A,stimulation time of 15 min,once a day,left and right alternately stimulate every time,for a total of 7 times.The changes of residual urine volume and urine output in rats at the 1st and the 7th days after operation were observed.And 7 d later,the rats were sacrificed and the injured spinal cord were taken out to observe the apoptosis,and to detect the changes of Bcl-2,Bax,Bad content.Results:After modeling,the rats of three groups showed different bladder dysfunction.In electro acupuncture group and electro acupuncture control group,the residual urine volume of the 7th day after operation was significant lower than the 1st day after operation(P<0.001),and there was statistically significant difference on the 7th day after operation between two groups(P<0.001).Compared with model group,the urine output of electro acupuncture group and electro acupuncture control group was significantly increased on the 7th day after operation,and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.001).Electro acupuncture can inhibit apoptosis of spinal cord neurons by TUNEL detection.Postoperative at 7 d,the rate of nerve cell apoptosis in electro acupuncture group and electro acupuncture control group was significant increased than model group(P<0.01,P<0.05),and there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.005).Compared with model group,the positive expression rate of Bax,Bad decreased(P<0.01,P<0.05),and Bcl-2 increased(P<0.01)in electro acupuncture group and electro acupuncture control group,there was significant difference between electro acupuncture group and electro acupuncture control group(P<0.01).Conclusion:Electro acupuncture can obviously promote the repair of acute spinal cord injury,its mechanism may be through increasing Bcl-2,inhibiting the expression of Bax,Bad,which inhibits the apoptosis of spinal cord neurons.浙江省中医药科研计划项目(编号:2008CB067)~

Effects of Elongated Needle with Paired Needling “Shuidao”(ST28) and “Zhibian”(BL54) on Neurological Function of Spinal Cord Injury Rats

目的观察芒针偶刺秩边与水道穴对大鼠脊髓损伤后神经功能的影响及可能作用机制。方法 30只SD大鼠随机分为假手术组、模型组、芒针治疗组各10只。模型组、芒针治疗组采用改良Allen's法制作脊髓损伤模型。芒针治疗组大鼠手术苏醒后即刻给予芒针秩边和水道穴,用偶刺法针刺大约2 cm深度,捻转1 min,并使同侧秩边、水道组成一回路连接于电针仪上,电针30 min,每日1次,假手术组和模型组正常饲养。5日后采用BBB评分法观察各组大鼠后肢运动功能情况,取脊髓组织行组织学观察、计算细胞凋亡指数、检测Bcl-2、Bax表达情况。结果与假手术组比较,模型组大鼠BBB运动功能评分明显降低,神经细胞凋亡指数升高,脊髓组织Bax、Bcl-2阳性细胞百分率升高(P<0.05或P<0.01)。芒针治疗组大鼠BBB运动功能评分较模型组明显升高、神经细胞凋亡指数降低(P<0.05或P<0.01);芒针治疗组脊髓组织Bax阳性细胞百分率显著低于模型组,Bcl-2阳性细胞百分率显著高于模型组(P<0.01)。结论芒针偶刺秩边与水道穴可能通过调控凋亡相关基因Bax、Bcl-2的表达,减少神经细胞凋亡,从而促进神经功能恢复。Objective To observe effects and possible mechanism of elongated needle with paired needling"Shuidao"( ST28) and"Zhibian"( BL54) on neurological function of spinal cord injury rats. Methods Thirty SD rats were randomly divided into control group,model group and treatment group,10 rats in each group. The model group and the treatment group were used improved Allen 's method to make models of spinal cord injury. In the treatment group,when rats revived after operation,the researchers immediately used elongated needle to needle"Shuidao"( ST28) and "Zhibian"( BL54) with method of paired needling to acupuncture the rats,about 2 cm depth and twirl for 1 min and made two ipsilateral acupoints to form a loop connected to electric acupuncture apparatus,electric acupuncture for 30 min,once per day. After 5 days the researchers used BBB scoring method to observe motor function of rats' hind limbs and took tissue of spinal cord to make histological observation,count apoptotic index and detect expression of the Bcl-2 and Bax. Results Compared with the control group,in the model group,BBB motor function score significantly decreased but nerve cell apoptotic index and percentage of positive cells about Bax and Bcl-2 of spinal cord tissue increased( P < 0. 05 or P < 0. 01). Compared with the model group,in the treatment group,BBB motor function score significantly increased but nerve cell apoptotic index decreased( P < 0. 05 or P < 0. 01); And percentage of positive cells about Bax of spinal cord tissue significantly decreased but percentage of positive cells about Bcl-2 significantly increased( P < 0. 01). Conclusion Elongated needle with paired needle"Shuidao"( ST28) and"Zhibian"( BL54) may promote neural functional to recover by regulating the expression of apoptotic gene that Bax and Bcl-2 to reduce apoptosis of nerve cells.浙江省中医药科研计划(2008CB067

Effects of elongated needle on the expression of TNF-α, IL-6β, IL-1β and NF-Kβ in rats with spinal cord injury

目的:应用改良AllEn’S造模法制备大鼠急性脊髓损伤(SCI)模型,观察芒针对大鼠急性脊髓损伤(ASCI)后脊髓组织中肿瘤坏死因子α(Tnf-α)、白介素-6(Il-6),白介素-1(Il-1β)和核转录因子kβ(nf-kβ)含量的影响。方法:将72只Sd大鼠随机分为两组:模型组36只和芒针组36只,采用改良AllEn’S打击法致伤模型组和芒针组大鼠T9~T11脊髓。芒针组采用芒针刺秩边与水道穴,1次/d,共治疗7d。分别于术后1d,3d,7d行bbb评分测定;术后1d,3d,7d取材,用EllSA法检测脊髓组织中Tnf-α、Il-6、Il-1β、nf-kβ的含量。结果:实验发现7d时芒针组bbb评分明显高于模型组,差异具有统计学意义(P<0.05);脊髓损伤后,模型组和芒针治疗组Tnf-α、Il-6、Il-1β、nf-kβ含量显著升高。经治疗,与模型组相比,芒针组受损脊髓组织内Tnf-α,Il-6、Il-1β、nf-kβ的表达均降低,且在1d,3d,7d的差异具有统计学意义(P<0.01)。结论:早期芒针治疗可显著抑制脊髓损伤炎性因子Tnf-α、Il-6、Il-1β、nf-kβ的表达,改善局部微环境,促进损伤后期大鼠运动功能的恢复。Objective: By SCI model, effects of elongated needle on TNF-α, IL-1β, IL-6 and NF-Kβ were observed.Methods: 72 SD rats are randomly divided into the model group(n=36) and the elongated needle treatment group(n=36).T9~T11 spinal cord injury model is created by Allen's method.The elongated needle group took Zhi Bian and Shuidao acupoint.BBB scores were invested on 1d, 3d, 7d after injury.The expression of TNF-α? IL-6? IL-1β and NF-K β in the injuryed spinal cords are examined by ELISA method at 1d, 3d, 7d after injury.Results: In 7 days, BBB scores in the treatment group was higher(P<0.05).The expression of TNF-α, IL-6? IL-1β and NF-Kβ in two groups were significantly increased after injuried.The expression of TNF-α, IL-6? IL-1β and NF-Kβ of elongated needle group was significantly lower than that in the model group; in 1d, 3d, 7d, the difference was statistically significant(P<0.01).Conclusion: Elongated needle treatment can effectively inhibit the expression of pro-inflammatory cytokine TNF-α, IL-6? IL-1β and NF-Kβ in rats of acute spinal cord injuries, improve the micro-environment, and promote functional recovery of acute spinal cord injury rats.浙江省中医药科研计划项目(2008CB067