电穿孔介导睫状神经营养因子基因转染延缓失神经肌萎缩

目的　探索一次性电穿孔介导睫状神经营养因子 (ciliaryneurotrophicfactor ,CNTF)转染延缓失神经骨骼肌萎缩的疗效。方法　制备 3 6只SD大鼠右下肢腓肠肌失神经支配模型 ,按手术先后顺序随机分成失神经对照组 (A组 )和CNTF基因转染组 (B组 ) ,每组 18只大鼠。于术后 2、4、8周测定肌湿重维持率、肌细胞截面积、肌肉蛋白含量、胶原纤维与肌细胞面积比和细胞凋亡数。结果　术后 2、4周B组的肌湿重维持率、肌细胞截面积和肌肉蛋白含量均明显高于A组 (P <0 .0 5 ) ,但胶原纤维与肌细胞面积比和细胞凋亡数明显低于A组 (P <0 .0 5 )。术后 8周 ,两组间各参数无明显区别 (P >0 .0 5 )。结论　一次性电穿孔介导CNTF基因转染可延缓失神经骨骼肌萎缩四周。     

缺血对失神经肌肉超微结构及酶组织化学的影响

目的 研究肢体缺血对失神经支配骨骼肌组织学、超微结构及Na、K—ATP酶和Ca—ATP酶活性的影响。方法 选用SD大鼠24只，建立双下肢失神经支配腓肠肌实验模型后，结扎右侧股动脉，观察肌细胞的组织学，超微结构及Na、K—ATP酶和Ca—ATP酶活性变化。结果 缺血侧肢体肌细胞直径及截面积较对照侧下降速度快25．48％及32．33％(t=2．94，P＜0．05；t＝2．88，P＜0．05)；缺血加速肌细胞线粒体，肌质网退变；Na、K—ATP酶活性术后2周实验侧较对照例增高59．68％，而术后4周缺血侧较对照侧下降17．45％(t＝2．24，P＜0．05)；Ca—ATP酶活性下降速度缺血侧较对照侧分别快26．42％及21．87％(t＝2．64、2．24，P＜0．05)。结论 缺血加速失神经支配骨骼肌萎缩的发生与发展，周围神经损伤合并肢体动脉损伤时，动脉尽可能予以全部修复。     

细胞外ATP防治失神经肌肉萎缩的实验研究

目的　探索细胞外ATP是否对失神经支配肌肉有保护作用。方法　SD大鼠 12只 ,在梨状肌下缘切断坐骨神经 ,制作腓肠肌失神经支配模型。左侧为实验组 ,术后于腓肠肌内注射ATP 0 .1mg/d ;右侧为对照组 ,腓肠肌内注射等量生理盐水。于术后 8、12周取 2组标本称肌湿重 ,检测运动终板、肌纤维横截面积及组织学变化。结果　实验侧的腓肠肌饱满有弹性、色泽好 ;对照侧肌纤维萎缩变细、色泽苍白。实验组运动终板边缘清晰 ,终板乙酰胆碱酯酶染色较深。比较两组的运动终板平均灰度值和平均光密度值 ,差异有显著性意义 (t =3 .0 5 7、4.13 8,P <0 .0 5 ) ,两组肌纤维横截面积相比 ,差异有显著性意义(t =4.191,P <0 .0 5 )。结论　ATP具有明显延缓失神经肌肉肌萎缩和减轻皮肤溃疡的作用     

大鼠失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化

目的 研究失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化规律.方法 实验以失神经支配腓肠肌为动物模型.选用体质量为200～250 g的雌性SD大鼠42只,随机分成2组,实验组36只,健康对照组6只.按术后取材时间的不同再将实验组分为6组,每组6只大鼠.分别运用Tunel法及免疫组织化学方法检测失神经后不同时间萎缩腓肠肌细胞凋亡及Bcl-2、Bax的表达变化,同时测量肌湿重比和肌纤维横截面积,透射电镜观察肌细胞的超微结构变化.结果 萎缩的腓肠肌湿重比在失神经4周内和第10周到第12周下降最快,而在第4周到10周和第12周到16周下降较慢,且变化差异无统计学意义(P＞0.05).Bcl-2仅在失神经早期表达增高,于第4周达高峰;而Bax分别于失神经第2周与第12周达高峰,增高幅度大于Bcl-2.Bcl-2/Bax的比值除对照组(1.522±0.215)和失神经第8周时(1.065±0.165)大于1外,其余各时间点均小于1.肌细胞凋亡率分别于失神经第4周和第12周达到高峰,其变化趋势与Bax相仿.结论 失神经腓肠肌萎缩过程呈现4个阶段;凋亡和凋亡相关基因Bax过量表达在失神经不可逆肌萎缩过程中发挥着重要的作用;靶向作用Bax基因,抑制Bax表达可能延缓失神经骨骼肌萎缩.     

外源性促红细胞生成素对失神经骨骼肌萎缩的影响

目的 探讨外源性促红细胞生成素(erythropoietin,EPO)对失神经骨骼肌萎缩的影响.方法 取雄性SD大鼠24只,体重200～220 g,于右侧梨状肌下缘切断坐骨神经,制备小腿三头肌失神经支配模型.模型制备后随机分为两组(n=12),EPO组:术后每天右小腿腓肠肌注射rhEPO(2 500 U/kg);对照组:注射等体积生理盐水.术后观察动物一般情况,于第2、4周检测肌湿重、肌肉蛋白含量,行HE及TUNEL染色,测量肌细胞直径、横切面积及细胞凋亡率,并测定肌肉Na+-K+-ATP酶和Ca2+-ATP酶活性.结果 术后两组动物右后肢均拖膝行走,切口无感染,动物均存活至实验完成,4周时对照组5只及EPO组2只大鼠发生足跟溃疡.术后2、4周EPO组肌湿重分别为(885.2.35)、(697.62±94.74)g,均明显重于对照组(760.63±109.05)、(458.71±58.76)g(P<0.01);肌肉蛋白含量分别为(77.37±5.24)、(66.37±4.87)mg/mL,明显高于对照组(65.39±4.97)、(54.62±6.32)mg/mL(P<0.01).术后2、4周EPO组肌纤维形态基本正常;对照组肌纤维萎缩变细,部分断裂,肌束问结缔组织增生较明显;EPO组肌细胞直径和横切面积明显大于对照组(P<0.01).术后2、4周,EPO组骨骼肌细胞凋亡数明显少于对照组,EPO组腓肠肌细胞凋亡率分别为11.80%±1.74%、28.47%±1.81%,明显低于对照组21.48%±2.21%、55.89%±2.88%(P<0.01).术后2、4周EPO组Na+-K+-ATP酶和Ca2+-ATP酶活性均高于对照组(P<0.01).结论 EPO具有明显延缓大鼠失神经骨骼肌萎缩的作用.     

大鼠失神经支配骨骼肌萎缩的实验研究

目的　观察大鼠失神经支配骨骼肌组织学、电生理及酶组织化学改变 ,探讨反映肌肉萎缩程度的指标。方法　选用SD大鼠 ,建立失神经支配腓肠肌模型 ,观察术后肌细胞直径及截面积 ,运动终板超微结构、纤颤电位波幅、Na 、K ATP酶及Ca2 ATP酶活性变化。结果　肌细胞直径及截面积随失神经支配时间延长呈进行性下降 ;运动终板 4周内改变不明显 ,16周后消失 ;纤颤电位波幅在 8周内维持在高水平 ,12周后呈进行性下降 ;Na 、K ATP酶活性随失神经支配时间延长呈进行性下降 ;Ca2 ATP酶活性在 8周内维持在较高水平 ,12周后明显下降。结论　肌细胞直径及截面积可作为反映肌肉萎缩的可靠形态学指标 ,纤颤电位波幅可作为潜在电生理指标 ;Na 、K ATP酶活性是可靠酶组织化学指标。     

活性氧、线粒体通透性转化孔介导肌细胞凋亡在失神经骨骼肌萎缩中的相关性

目的 观察活性氧(ROS)、线粒体通透性转化孔(MPTP)在失神经骨骼肌萎缩后的表达变化且与肌细胞凋亡的相关性,探讨ROS、MPTP参与失神经骨骼肌萎缩的具体分子机制.方法 将30只Vistar大鼠随机分为对照组、失神经2d组、失神经7d组、失神经14d组、失神经28d组,每组6只.制作坐骨神经切断后失神经支配的腓肠肌Vistar大鼠模型.应用流式细胞术(FCM)检测失神经支配后腓肠肌细胞ROS的含量,激光共聚焦显微镜检测MPTP的开放,脱氧核糖核苷酸转移酶介导的缺口末端标记(TUNEL)法检测肌细胞凋亡.结果 大鼠失神经支配后,肌细胞中的ROS、MPTP及凋亡率与正常对照组比较,表达随失神经支配时间的延长(<28d)而持续增加,且各组的表达均显著高于对照组(P<0.05),ROS的表达与MPTP的开放呈正相关(r=0.884,P<0.01),与肌细胞的凋亡率呈正相关(r=0.893,P<0.01),MPTP的开放与肌细胞凋亡率呈正相关(r=0.927,P<0.01)与肌细胞萎缩指标肌湿重比呈负相关(r=-0.907,P<0.01).结论 ROS、MPTP为调控失神经支配后骨骼肌萎缩的重要分子,其具体机制是通过线粒体介导的凋亡通路促进骨骼肌萎缩.

Abstract：

Objective To study the expression of reactive oxygen species (ROS) and mitochondrial permeability transition pore (MPTP) in denervated skeletal muscle atrophy and its correlation with cell apoptosis, and explore specific molecular mechanism in denervated skeletal muscle atrophy. MethodsThirty Vista rats were randomly divided into five group: control group, 2-days group, 7-days group, 14-days group, 28-days group. Standard model of denervated gastrocnemius muscle was established. The content of ROS and the opening of MPTP in the gastrocnemius were detected by flow cytometry (FCM) and fluorescence microscope respectively. The apoptotic cells in atrophic muscle were examined by TdT-mediated dUTP nick end labeling (TUNEL). Results As compared with the control group, the content of ROS, the opening of MPTP and the apoptosis of gastrocnemius were increased continuously (<28 days) in 2-days group, 7-days group, 14-days group, 28-days group (P<0.05). The content of ROS had a positive correlation with the opening of MPTP (r=0.884,P<0.01) and the apoptosis rate (r=0.893,P<0.01), and the opening of MPTP had a positive correlation with the apoptosis rate (r=0.927,P<0.01), but a negtive correlation with the ratio of muscle wet weight (r=-0.907,P<0.01). Conclusion ROS and MPTP are important elements in regulating skeletal muscle atrophy after denervation by the mitochondrial apoptosis pathway.     

肢体制动对失神经支配骨骼肌超微结构及酶组织化学影响

目的：研究肢体制动对失神经支配骨骼肌超微结构及酶组织化学影响。方法：用SD大鼠，建立左下肢失神经支配腓肠肌及右下肢制动加失神经支配腓肠肌的实验模型，观察肌细胞直径、截面积，超微结构及Na^ 、K^ —ATP酶和Ca^2 -ATP酶活性变化。结果：制动例肌细胞直径及截面积较对照例下降速度明显加快；肢体制动加速了肌细胞的线粒体，肌质网退变Na^ 、K^ —ATP酶活性制动例较对照例下降速度快18．24％；而Ca^2 -ATP酶活性在术后2周无明显差异，术后4周制动例较对照例下降速度快13．99％。结论：肢体制动加速了失神经支配骨骼肌萎缩的发生与发展。因此，临床上神经损伤合并有肌膜、骨折等复合损伤情况下，肢体制动范围及时间尽可能予以减少。     

细胞外三磷腺苷对失神经大鼠腓肠肌细胞中FoxO3a通路的影响

目的 探讨细胞外三磷腺苷(ATP)对失神经大鼠腓肠肌细胞中FoxO3a/MAFbx通路的影响.方法 雌性成年Wistar大鼠54只随机分为3组:失神经对照组,ATP治疗组,健康对照组.其中失神经对照组和ATP治疗组在右侧梨状肌下缘切断坐骨神经,制作失神经动物模型.术后ATP治疗组于右侧腓肠肌内注射ATP0.1mg/d,左侧腓肠肌内注射等量生理盐水;失神经对照组双下肢注射等量的生理盐水;健康对照组不做任何处理.在术后0、2、7、14、28d分别处死一组大鼠,取其两侧腓肠肌,称肌湿重,计算肌失重比(右侧/左侧),采用实时荧光定量PCR和Western Blot检测大鼠腓肠肌FoxO3a和MAFbx的mRNA和蛋白表达水平以及253位磷酸化的FoxO3a(p-FoxO3a)蛋白表达.结果 ATP治疗组肌湿重比高于失神经对照组,差异有统计学意义(P＜0.01);ATP治疗组FoxO3a的mRNA和蛋白表达略低于失神经对照组,差异无统计学意义(P＞0.05);MAFbx的mRNA和蛋白表达低于失神经对照组,差异有统计学意义(P＜0.05);p-FoxO3a的蛋白表达量较对照组明显增加,不同时段差异均有统计学意义(P＜0.05).结论 细胞外ATP可能通过磷酸化FoxO3a进而抑制MAFbx蛋白的表达来延缓大鼠骨骼肌失神经萎缩.     

不同时间神经修复后失神经肌肉及运动终板变化的实验研究

目的 研究失神经支配肌肉不同时间神经修复后,肌肉及运动终板变化.探讨神经修复的最佳时机.方法 建立失神经支配腓肠肌实验模型,以损伤后不同的神经修复时间(0、2、4、6、8、10周)随机分为6组,每组6只.右后肢为实验侧,左后肢不作任何处理为对照侧(对照组).神经修复手术后第6周取材,测定各项检测指标.结果 肌肉失神经支配0～4周组行神经修复后肌肉湿重呈下降趋势,但各组间比较差异无统计学意义(P>0.05);4周后神经修复组肌肉湿重下降尤为明显,维持于一定水平,肌细胞直径及截面积呈持续性下降.2周内神经修复组,运动终板降钙素基因相关肽(calcitonin gene-related peptide,CGRP)灰度值与对照组比较差异无统计学意义(P>0.05);4,6周组,CGRP灰度值明显低于2周内神经修复组(P<0.05);8周后进行神经修复其灰度值进一步下降,与6周内神经修复组比较差异有统计学意义(P<0.05).超微结构的变化趋势与透射电镜观察基本一致.结论 实验提示神经损伤后2～4周内修复效果较好,6周后修复萎缩肌肉逆转可能性降低,但其各项指标仍能维持在一定水平,有指征尝试手术修复.     

电刺激对失神经支配骨骼肌萎缩的影响

目的 研究电刺激对失神经支配骨骼肌萎缩的影响。方法 选用SD大鼠16只，建立双侧下肢失神经支配胙肠肌的实验模型，电刺激右侧胙肠肌为实验侧，左侧不作电刺激为对照侧，术后2、4周分别观察肌肉的组织学，超微结构，纤颤电位波幅及Na^ -K^ -ATP酶和Ca^2 -ATP酶活性变化。结果 实验侧术后2、4周肢体肌细胞直径及截面积较对照侧下降速度明显减慢；电刺激能延缓肌细胞的线粒体，肌质网退变，但对肌肉纤颤电位波幅无明显影响；实验侧术后2、4周Na^ -K^ -ATP酶活性下降速度比对照侧分别慢15．59％和27．38％；实验侧术后2、4周Ca^2 -ATP酶活性下降速度较对照侧分别慢4．83％和21．64％。结论 电刺激对失神经支配骨骼肌的组织学、电生理及酶组织化学的各项指标均有保护作用，是延缓肌肉萎缩的有效方法。     

失神经纤颤电位及正尖波波幅与神经修复后肌肉功能恢复关系的实验研究

目的 研究大鼠腓肠肌失神经支配后纤颤电位与正尖波波幅的变化,并探讨其与在损伤后不同时期修复断伤胫神经后腓肠肌功能恢复间的相关性。方法 雄性SD大鼠54只,按手术先后顺序随机分为8组。第1组12只大鼠,于切断其右侧胫神经后即刻、48h、72h、1周、2周、4周、8周、12周、16周,测定右侧腓肠肌的纤颤电位及正尖波。第2－7组（每组6只大鼠）,分别在胫神经切断后1、2、4、8、12、16周时修复神经,各组于修复后12周取腓肠肌,测定肌湿重及肌纤维直径及截面积。第8组（6只大鼠）作为正常对照组。结果 第1组的正尖波先于纤颤电位出现,纤颤电位的波幅在术后1周最高,在神经切断后4周内维持在较高水平;术后8周起春波幅呈进行性,16周时全部消失。正尖波于神经切断后478h开始出现,术后8周其波幅最大,术后16周时半数大鼠的正尖波消失。于神经断伤后8周内修复后胫神经,恢复的肌肉功能与正常对照组相比,差异无显著性意义（t＝1．952,P＞0．05）;神经断伤后12周以后修复组恢复的肌肉功能明显变差,与正常对照组相比差异有显著性意义（t＝3．127,P＜0．05）。结论 失神经支配腓肠肌纤颤电位下正尖波的波幅。与神经修复后功能恢复程度间有密切的相关性,可作为大鼠失神经骨骼肌萎缩是可逆的一个量化指标。     

失神经支配骨骼肌退变组织形态学及电生理实验研究

目的研究大鼠失神经支配骨骼肌组织形态学及其电生理变化,为临床寻找检测肌肉萎缩程度的方法及更好防治肌肉萎缩提供理论基础。方法选用成年ＳＤ大鼠,切断胫神经建立失神经支配腓肠肌实验模型。测定萎缩肌肉湿重、肌细胞直径及截面积,观察骨骼肌运动终板及肌肉纤颤电位波幅与频数变化。结果４周内肌肉湿重下降最快,以后逐渐减慢并维持于一定水平,而肌细胞直径及截面积呈持续性下降;在４周内运动终板改变不明显,６周后退变逐渐加重,１６周后消失;２周时肌肉纤颤电位波幅最大,１２周后进行性下降,２０周时有半数以上大鼠出现电静息,频数与波幅值变化规律相一致。结论肌肉湿重、肌细胞直径及截面积可作为反映肌肉萎缩的组织形态学指标,而后两者更为可靠;肌肉纤颤电位波幅与频数可作为电生理指标,周围神经损伤后修复手术越早越好,应力争在运动终板消失前进行。     

An experimental study on denervated muscle atrophy--effect of electrostimulation and comparison with immobilization muscle atrophy

The efficacy of causing muscle atrophy was compared among denervation, arthrodesis and tenotomy in rat anterior tibial muscle. Reduction of wet weight was most pronounced in denervated muscle and least in arthrodesed muscle. Histochemical investigation by ATPase stain revealed that atrophy of Type 1 and Type 2 fiber was more severe in denervated muscle group than in the other two groups. Type 2 fiber atrophy was dominant in denervated muscle and in arthrodesed muscle. Type 1 fiber atrophy was dominant in tenotomized muscle. The effect of electrostimulation on denervated muscle was investigated. Electrostimulation significantly reduced the degree of denervation atrophy. Four weeks after severance of peroneal nerve, tibial nerve-crossing was done and electrostimulation was continued for eight weeks. Recovery of wet weight of re-innervated muscle with electrostimulation was significantly better than that without electrostimulation. Electrostimulation applied to denervated muscle reduced the progress of atrophy and improved the recovery after nerve repair.     

Experimental study on free muscle transplantation--histological changes and functional recovery of transplanted muscles in relation to the duration of ischemia and denervation

The purpose of this study was to clarify the influence of temporary ischemia and denervation on the characteristics of transplanted muscles. Materials and methods: The rectus femoris brevis muscle of white rabbit was used. Under an operating microscope, this muscle was mobilized preserving its feeding vessels, and the innervating nerve was severed at 1 cm proximal to the entrance of the muscle. Six groups were made as follows. Group D: The severed nerve was left as it was. Group S: The nerve was sutured immediately after severance. No treatment was made on the feeding vessels. Group SI: The artery and vein were clamped for 1, 2, 3 and 4 hours (group SI-1, 2, 3 and 4) after nerve repair. The proximal and distal tendon were reattached to their original place in every group. Examination I: Transplanted muscles were examined histologically and histochemically from 48 hours to 6 months. Motor end-plate was one of the targets of observation. Results: Main patho-histological changes observed on the muscle and motor end-plates at one week were thought due to denervation, and the longer ischemic time caused more severe change in the group SI-3 and 4. Phagocytosis, destruction of muscle fibers and edema in the interstitial tissue were seen in these long ischemic time groups. A few motor end-plates in the group SI-3 and many of them in the group SI-4 were atrophic and their acetylcholinesterase (AChE) activity was low. Reduced nicotinamide-adenine dinucleotide-tetrazolium reductase (NADH-TR) and phosphorylase activity were reduced in all groups at one week. At 3 weeks, muscle atrophy was pronounced and was found earlier in type II fibers than in type I as a result of denervation. Pathological changes such as elongation, atrophy or segmentation and low AChE activity were observed in the motor end-plates of all groups at 3 and 6 weeks. These findings were more pronounced in the group SI-3 and 4. In all groups at 6 weeks, phosphorylase activity was found to be generally low but NADH-TR reaction stained a few regenerated muscle fibers. Although muscle atrophy was still observed at 9 weeks, recovery in size and shape and histochemical reaction was seen in all groups at this stage. These findings became more manifest at 15 weeks and almost normal appearance of muscle fibers was observed at 6 months. Interstitial connective tissue, however, increased, and necrotized muscle fibers were seen extensively in the group SI-3 and 4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental study of denervated muscle atrophy following severance of posterior rami of the lumbar spinal nerves.

The morphologic changes in denervation atrophy of paravertebral muscles after severance of the posterior rami in cats were investigated, using histochemical methods and electromyography. Using a paraspinal approach, three branches of the posterior rami on the left side were cut under microscopy at one, two, or three levels (L2 approximately L4). Muscle atrophy was evaluated, using the percent wet weight and the percent diameter of muscle fibers as parameters. Myosine ATPase stain was used to observe reinnervation. Four weeks after surgery, the range and severity of muscle atrophy increased proportionally to the number of posterior rami severed. Muscle atrophy was revealed at one or two levels caudal to the injured nerve level. At 12 and 24 weeks, muscle atrophy recovered gradually. In more than two-level injury groups, however, recovery of percent wet weight reached up to 80% even after 24 weeks, despite the fact of reinnervation demonstrated in some parts of the denervated muscles.     

不同时间神经修复后失神经肌肉及运动终板变化的实验研究

目的 研究失神经支配肌肉不同时间神经修复后,肌肉及运动终板变化.探讨神经修复的最佳时机.方法 建立失神经支配腓肠肌实验模型,以损伤后不同的神经修复时间(0、2、4、6、8、10周)随机分为6组,每组6只.右后肢为实验侧,左后肢不作任何处理为对照侧(对照组).神经修复手术后第6周取材,测定各项检测指标.结果 肌肉失神经支配0～4周组行神经修复后肌肉湿重呈下降趋势,但各组间比较差异无统计学意义(P>0.05);4周后神经修复组肌肉湿重下降尤为明显,维持于一定水平,肌细胞直径及截面积呈持续性下降.2周内神经修复组,运动终板降钙素基因相关肽(calcitonin gene-related peptide,CGRP)灰度值与对照组比较差异无统计学意义(P>0.05);4,6周组,CGRP灰度值明显低于2周内神经修复组(P<0.05);8周后进行神经修复其灰度值进一步下降,与6周内神经修复组比较差异有统计学意义(P<0.05).超微结构的变化趋势与透射电镜观察基本一致.结论 实验提示神经损伤后2～4周内修复效果较好,6周后修复萎缩肌肉逆转可能性降低,但其各项指标仍能维持在一定水平,有指征尝试手术修复.