aFGF预防失神经支配运动终板退变及肌萎缩的实验研究
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摘要
研究背景
很多研究发现肌细胞正常结构与功能维持需靠一些神经营养因子的作用，运动神经损伤后，骨骼肌失神经支配后最大变化便是这种神经营养因子丢失，同时骨骼肌废用，最终导致肌肉形态结构和生理生化等方面改变，组织形态学上表现为运动终板退变、肌细胞浆丢失及直径减小，并随时间延长而加重。目前，运动神经损伤后功能的重建主要还是集中在恢复神经的连续性上，忽视了对其靶器官的保护，由于神经漫长的再生过程、再生过程中神经出现再退变、损伤部位的阻挡因素、修复后肢体固定等原因，即使及时修复神经，肌肉失神经支配时间很长，神经对肌肉的营养作用丧失，运动终板逐渐退变，给神经肌肉接头的重建带来极大的困难，临床常见肌肉呈念珠状变性、横纹消失，肌肉萎缩，时间过长肌肉坏死、消失；而且，肌肉失用、萎缩亦会影响运动终板的形态，二者之间形成恶性循环，成为目前严重阻碍运动神经修复后功能恢复的最重要因素之一。因此，如何预防运动终板退变及肌萎缩已成为当前临床运动神经损伤后功能重建过程中急需解决的难题。
迄今对运动终板退变的预防研究甚少，治疗方法不多。临床常用的方法是提倡功能锻炼，但其只能避免肌肉废用不能提供神经营养、且外科修复病例早期常需固定肢体而无法开展，作用有限。曾经有学者提出电刺激可以替代失神经肌肉的兴奋性，并提出了实验鼠模拟正常运动终板兴奋的电刺激模式，取得了一定的效果，但电刺激作用不能持久，并有诸多不便。近年在药物实验研究方面取得了一些成果：有作者报道大鼠腹腔注射地塞米松可以减缓减轻非离断性神经损伤支配运动终板的退变；口服氨哮素可以防止失神经支配运动终板的退变，防止肌肉萎缩，其作用可能是通过某种途径使肌卫星细胞产生一种胰岛素样生长因子(IGF)的营养物质模仿神经对肌肉的营养作用；此外，神经断端间应用神经生长因子(NGF)及肝细胞生长因子(HGF)可减轻运动终板的退变；1996年，Margaret等报道在Ⅱ期修复神经的同时将酸性成纤维细胞生长因子(aFGF)纤维蛋白凝胶植入其支配的萎缩肌腹，初步研究可以诱导运动终板的再生，但对不修复神经者无明显作用，其结果仅仅说明aFGF的作用是在修复神经的前提下，没有说明能否保护失神经支配的运动终板，也没有探讨其对功能恢复效果的作用。
aFGF具有广泛的生物学效应，如促进细胞增殖与分化，促进组织修复，也是一种形态发生因子；其靶细胞有成肌细胞、成纤维细胞、血管内皮细胞等；在重建神经肌肉接头时能激活静止的未分化神经靶细胞形成基底膜，有促进血管生成改善微循环，对多种组织有体内营养作用。成纤维细胞有促进肌细胞基底膜形成的作用，而骨胳肌基底膜是神经肌肉接头形成的重要条件，因此，aFGF促进成纤维细胞的分化对神经肌肉接头的形成具有重要作用；很多研究表明肌肉萎缩的原因主要是肌膜受到不同程度的脂类过氧化作用，这一因素也将累及运动终板，因此，促进基底膜的形成有助于补充受损的膜性结构。亦有研究表明aFGF能调节细胞内钙离子的流出与流入过程，有利于促进黄嘌呤脱氢酶转变为黄嘌呤氧化酶，从而有力于自由基的清除而保护膜性结构如运动终板。此外，对成肌细胞的促进分化可以形成肌细胞并分化成肌管，有助于防止肌肉萎缩；作用于血管内皮细胞有助于改善血循环。
aFGF对运动神经损伤后运动终板退变及肌萎缩的预防是否有作用尚无报道，本研究试图探讨aFGF对失神经支配运动终板退变及肌萎缩的预防作用及其作用机理。
研究目的1、建立为失神经支配运动终板及肌肉提供替代性神经营养的aFGF缓释系统。
2、建立aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的动物模型。
3、探讨aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的形态学及机能学效果。
4、初步探讨aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的作用机理。
材料和方法
1、纤维蛋白凝胶复合aFGF缓释系统的制备：将冻干人纤维蛋白原及抑肽酶直接溶于注射用水，其浓度分别为60mg．ml~(-1)，3000 KIU．ml~(-1)，得溶液Ⅰ；凝血酶用40mmol．L~(-1)CaCL_2溶液配成250 U．ml~(-1)，为溶液Ⅱ。每支aFGF(50μg／支)和0.5ml溶液Ⅰ混合，使aFGF充分混悬后加等体积溶液Ⅱ，在模具中制成形状为圆柱形、半透明、乳白色复合物。将等体积溶液Ⅰ与溶液Ⅱ直接混合制备纤维蛋白凝胶。
2、制做动物模型及分组：155只成年纯系Sprague-Dawley大鼠在全麻(10％水合氯醛腹腔注射)下切断右侧腓总神经胫前肌分支(距神经入肌点约1cm)，随机分为5组：修复神经植入aFGF组(NR+aFGFgel)：缝合神经外膜+胫前肌植入aFGF纤维蛋白凝胶(aFGF 25μg／只)；修复神经植入凝胶组(NR+gel)：缝合神经外膜+胫前肌植入纤维蛋白凝胶；未修复神经植入aFGF组(aFGFgel)：胫前肌植入aFGF纤维蛋白凝胶(aFGF 25μg／只)；单纯修复神经组(NR)：缝合神经外膜；未修复神经未植入组(Blank)：神经肌肉不做处理。NR+gel、NR、Blank为三个对照组。术后观察6周。
3、形态学研究：采用肉眼形态学检查观察肌肉的外观形态，测量胫前肌湿重，采用氯化金法染色显示运动终板，采用超薄切片、铅铀染色、透射电镜观察运动终板超微结构，统计各组运动终板均数及其退变率并进行统计学分析。观察aFGF纤维蛋白凝胶在体内的降解及异常反应。
4、机能学研究：采用Karnovsky-Roots亚铁氰化铜法显示运动终板乙酰胆碱酯酶(AchE)，观察各组运动终板AchE变化情况。退变终板计数：每例随机观察30个运动终板，计数Ache染色显示活性减弱的运动终板数量并进行统计学分析。图像分析：染色切片在400X光镜下经摄像头摄入，利用计算机图像分析软件计算运动终板Ache平均染色面积及灰度值，每例随机测10个运动终板，取均数。采用诱发电位肌电图仪进行神经低频重复电刺激检测：刺激坐骨神经，记录胫前肌肌电波波幅，在脉冲电流刺激频率为3～5Hz的条件下连续刺激9次，观察有无衰减，以第五个肌电波波幅比第一个降低10％以上为阳性，统计各组第五次刺激肌电波波幅衰减率均数。
5、机理研究：采用超薄切片、铅铀染色、透射电镜观察各组运动终板附近胫前肌肌卫星细胞的形态，每例随机观察20个超薄切片视野，统计肌卫星细胞数量并进行统计学分析。采用免疫组织化学染色检查aFGF受体(aFGFR)表达部位；统计每例aFGFR阳性血管表达率；图像分析仪测量毛细血管壁内aFGFR平均灰度。
6、试验结果统计学分析：采用SPSS13.0软件包(第一军医大学生物统计学教研室)对结果进行统计学分析(计量资料采用One-WayANOVA进行分析，多重比较采用LSD法；计数资料采用R×C表资料的x~2检验进行分析)，差异显著性水准置于0.05。
结果
1、肉眼形态学检查：NR+aFGFgel组：自身对照可见两侧胫前肌对称且无明显肌萎缩，肌张力正常；NR+gel组及NR组表现相近：右侧胫前肌明显萎缩，肌张力下降；aFGFgel组：右侧胫前肌略有萎缩，但不如NR+gel组及NR组明显，肌张力消失；Blank组：右侧胫前肌明显萎缩，肌张力消失。NR+aFGFgel组、MR+gel组、aFGFgel组纤维蛋白凝胶植入局部组织无异常反应。
2、胫前肌湿重：NR+aFGFgel组胫前肌湿重为1.26±0.10g，aFGFgel组胫前肌湿重为1.20±0.12g，两组胫前肌湿重高于三个对照组，统计学分析有显著性意义。
3、终板染色检查：NR+aFGFgel组可见运动终板结构完整、形态正常；NR+gel组及NR组表现相近：失神经肌纤维上的终板形态不规则，轴索终末分支减少而杂乱，终板呈细条状或点状；aFgFgel组：未见轴突，运动终板形态大致正常，但区域缩小。Blank组：未见轴突，运动终板数量明显减少，形态不规则。
4、电镜检查：NR+aFGFgel、aFGFgel组运动终板的数量较多，容易辨认，结构基本正常，运动终板退变例数少。三个对照组运动终板数量减少，结构退变，运动终板退变例数多。NR+aFGFgel组运动终板数量为12.00±2.24，aFGFgel组运动终板数量为11.40±1.14，两组运动终板数量高于三个对照组，统计学分析有显著性意义。NR+aFGFgel组运动终板退变率为26.67，aFGFgel组运动终板退变率为33．33，两组运动终板退变率低于三个对照组，统计学分析有显著性意义。
5、神经低频重复电刺激检查：NR+aFGFgel组所有大鼠神经低频重复电刺激时没有出现大的肌电波波幅衰减反应，衰减率为5.70±3.13％，没有出现阳性情况；NR+gel、NR组大鼠低频电刺激时出现较大的肌电波波幅衰减反应，阳性率均为90％。NR+aFGFgel组衰减率低于对照组，统计学分析有显著性意义。
6、运动终板AchE活性：NR+aFGFgel、aFGFgel组运动终板染色深，显示AchE活性强，结构接近正常，NR+aFGFgel组AchE染色面积为1693.98±168.16μm~2，灰度为24.81±1.44，aFGFgel组AchE染色面积为1520.60±303.00μm~2，灰度为24.93±1.54，AchE活性高于对照组，统计学分析有显著性意义；NR+aFGFgel组运动终板退变率为20.42％，aFGFgel组运动终板退变率为24.17％，运动终板退变率低于对照组，统计学分析有显著性意义。
7、肌卫星细胞观察：NR+aFGFgel、aFGFgel组肌卫星细胞体积增大，数量多。NR+aFGFgel组MSC数量为11.00±3.00，aFGFgel组MSC数量为10.40±1.14，MSC数量高于对照组，统计学分析有显著意义。
8、免疫组织化学检查：aFGFR阳性部位主要定位在毛细血管壁内，NR+aFGFgel、aFGFgel组显示毛细血管aFGFR表达密度高，毛细血管密集，对照组毛细血管aFGFR表达密度低，毛细血管稀少。NR+aFGFgel组毛细血管aFGFR表达阳性率为87.50％，aFGFgel组毛细血管aFGFR表达阳性率85.00％，两组aFGFR表达阳性率高于对照组，统计学分析有显著性意义。NR+aFGFgel组毛细血管壁aFGFR表达灰度为25.92±1.55，aFGFgel组毛细血管壁aFGFR表达灰度为26.85±1.23，两组毛细血管壁aFGFR表达量高于对照组，统计学分析有显著性意义。
结论
1、实验证实aFGF纤维蛋白凝胶可做为失神经支配运动终板及肌肉提供替代性神经营养的药物缓释系统，未见异常反应。
2、联合神经修复及应用aFGF纤维蛋白凝胶可明显减轻大鼠运动神经损伤后运动终板退变，减轻肌萎缩。
3、即使神经未及时修复，应用aFGF纤维蛋白凝胶可有效保护大鼠运动神经损伤后所支配的运动终板、减轻肌萎缩。
4、神经重复电刺激检查神经肌肉问传导功能表明：运动神经损伤后联合神经修复及应用aFGF纤维蛋白凝胶保护运动终板可有效改善神经肌肉接头间传导。
5、酶组织化学检查运动终板Ache活性表明：运动神经损伤后应用aFGF纤维蛋白凝胶保护运动终板可有效维持运动终板Ache活性，有利于神经递质的正常代谢，有利于神经冲动的正常传递。
6、应用aFGF纤维蛋白凝胶处理组MSC数量高于对照组，说明aFGF纤维蛋白凝胶预防运动终板退变及肌萎缩的作用与aFGF促MSC增殖作用有关。
7、应用aFGF纤维蛋白凝胶处理组运动终板附近分布了较多毛细血管，微循环良好；而未经aFGF纤维蛋白凝胶处理的运动终板附近毛细血管稀疏，微循环较差；说明aFGF具有明显的促血管形成及改善微循环作用。
8、应用aFGF纤维蛋白凝胶处理组运动终板附近毛细血管aFGFR表达阳性率及表达量高于未经aFGF纤维蛋白凝胶处理组，进一步说明aFGF纤维蛋白凝胶预防运动终板退变及肌萎缩的途径在于其作用于毛细血管，改善运动终板附近微循环，弥补失神经支配肌的神经营养作用。
9、本实验通过肌卫星细胞及运动终板附近微循环的形态观察提示给运动终板及肌肉人工提供“替代性”神经营养是防止失神经支配运动终板退变及肌萎缩的有效途径。
Backgrounds
Many researches show that the normal architecture of muscle and the maintenance of its function depend on the contributions of some neurotrophic factors. When motor nerve is damaged, the greatest change of denervated skeletal muscle is the loss of such kind of factors. Meanwhile, skeletal muscle is disused, which finally results in the change of muscles in morphosis and physiological biochemistry. What displays in histomorphology is the cataplasia of end plate, the loss of the muscle cytoplasm and the diminutus of anteroposterior diameter. Now the re-establishment of motor nerve function after its injury centers on the recovery of the succession of nerve while ignores the protection of its target organs. Such factors as the lengthy reactivation process of nerve with re-cataplasia of nerve during this process, the baffling effect of injury region, the immobilization of limbs after its reparation, even if the nerve is promptly repaired, the process that muscles with denervation lasts for a long time, and the trophic action of nerve lost and gradual cataplasia of end plate bring great difficulty to the re-establishment of myoneural junction. In clinic, the features that muscle presents intestini degeneration, transverse striation abolishment, myatrophy, the death and disappearance of muscle are found. Then, the apraxia and atrophy of muscle have influence on the morphology of end plate, which will lead to an infernal circle between them. This is one of the most important factors nowadays which hinder the recovery of function after nerve repair. As a result, how to prevent the cataplasia of end plate and myatrophy becomes an urgent and tough problem nowadays which awaits to be solved urgently in clinic once motor nerve is damaged.
However, there are few researches and treatments on how to obviate the cataplasia of end plate. The commonly used treatment in clinic is functional exercise, which can only avoid the disuse of muscle but can not offer neurotrophy. Particularly, in the morning of surgery recovery, the limbs are always fixed which limits the effect of such kind of treatment.
Certain scholar once introduced that the excitability of denervated muscles can be replaced by electrical stimulation and a modality of electrical stimulation that using an experimental rat to imitate the excitability of normal end plate was established. And some achievements were obtained. However, the effect of electrical stimulation can not last for a long period and there were many drawbacks. There are also some achievements in the empirical study of drug on the treatment of this aspect in recent years. It is reported that the experiment on rats indicates that Dexamethasone through intraperitoneal injection can alleviate and lessen the consequence that non-mutilation nerve injury leads to the cataplasia of end plate. Clenbuterol by mouth can avoid the cataplasia of denervated end plate and muscular atrophy. Through a certain way, muscle satellite cell can generate such nutritive materials as insulin-like growth factor (IGF), which can imitate the trophic action of nerve to muscles. In addition, NGF and HGF can lessen the cataplasia of end plate. In 1996, initial researches indicated, which was presented by Margaret, that in theⅡstage, when nerves were repaired, once carrier of fibrin gel of acid fibroblast growth factor (aFGF) was imbedded into atrophic muscle belly, it could induce the regeneration of end plate but there was no obvious contribution to those whose nerves were not repaired. Such kind of research showed that the contribution of aFGF could be displayed on the condition that the nerve was repaired and whether it could protect denervated end plate was not still be proved. The contribution that aFGF did to the recovery of function was not indicated as well.
AFGF can generate extensive biological effects such as enhancing cell multiplication and differentiation and promoting tissue repair. It is also a kind of morphogenetic factor whose target cell includes sarcoblast, collagenoblast, vascular endothelial cell. Angiogenesis can ameliorate microcirculation and can do great endotrophic action to many architectures. Collagenoblast can promote the formation of muscle cell's basal lamina, while basal lamina of skeletal muscle is one of the significant conditions in the formation of myoneural junction. As a result, aFGF plays an important role in promoting the differentiation of collagenoblast which does contribution to the formation of myoneural junction. Meanwhile many researches indicate that the principal cause of muscular atrophy is tunica muscularis suffers lipide peroxidation which also has effect on end plate. As a result, to enhance the formation of basal lamina will also enhance the replenishment of those impaired hymeno-structures. Other research indicates that aFGF can accommodate the affluence and effluence of Ca2+ in intra-cellular, which will enhance the transformation of xanthine oxidase from xanthine dehydrogenase and then eliminate free radical. Accordingly, those hymeno-structures such as end plate can be protected through the elimination of free radical. To hasten myoblastic differentiation will form muscle cell and differentiate myotube and thereby prevent muscle from myatrophy. AFGF can improve the blood circulation through the action of vascular endothelial cell.
There is still no report shows that aFGF can prevent the cataplasia of end plate and myatrophy after motor nerve injury. This study was undertaken to probe the effect on the prevention of the cataplasia of denervated end plate and myatrophy by aFGF and its mechanism of action.
Objectives
1. To establish aFGF slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy.
2. To establish the animal model of preventing cataplasia of denervated end plate and myatrophy with fibrin gel of aFGF.
3. To approach the morphological and functional effect that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.
4. To approach initially the mechanism of action that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.
Materials and Methods
1. The preparation of aFGF slow- releasing system: liquorⅠ: cryodesiccant human fibrinogen and aprotinin were dissolved into aqua pro injectione directly with concentration as 60mg.ml~(-1) and 3000 KIU.ml~(-1) respectively; liquorⅡ: 40mmol.L~(-1)CaCL_2 zymoplasm with concentration as 250 U.ml~(-1). Admixed aFGF 50μg with 0.5ml liquorⅠand add it with equal volumn liquorⅡby mixing thoroughly. Put this admixture into tooting and got a cylindrical, semitransparent ivory compound. Fibrin gel was prepared by admixed liquorⅠwith equal volumn liquorⅡ.
2. Establishing and grouping animal models: 155 adult inbred line Sprague-Dawley rats were selected and abscised their right tibial muscle branch of the common peroneal nerves (the distance between abscising point and the point where nerve entering into muscle is 1cm) with complete anesthesia which was through 10% chloral hydrate through intraperitoneal injection. The models were randomly divided into five groups. Group NR+aFGFgel: those that epineuriums were sutured and carriers of Fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR+gel: those that epineuriums were sutured and carriers of fibrin gel were imbedded in tibial muscles. Group aFGFgel: those that epineuriums were not sutured and carriers of fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR: those that only epineurium were sutured. Group Blank: those that epineuriums were not sutured and no implant was imbedded in tibial muscles. Group NR+gel, Group NR and Group Bland were control groups. All rats were observed for 6 weeks postoperation.
3. Morphological research: Observe the appearance of the muscle through morphological method with naked eyes and measure humid weight of tibial muscle. Staining with auric chloride was deployed to display end plate. Ultrathin sections were stained with uranium and lead and the ultrastructure of end plate was observed by transmission electron microscope. Calculate end plates and the percentage of cataplasia of denervated end plate in five groups and analyze it through statistical method. Observe the degradation of fibrin gel of aFGF and those possible adverse effects.
4. Functional research: Karnovsky-Roots copper-ferrocyanide was employed to display AchE of end plate and observe the change of AchE in end plate of each group. The count of cataplasia of end plate: take randomly 30 end plates in each group and count the quantity of end plates which were with subdued activity through staining with AchE and the statistical analysis was conducted. Image analysis: 10 end plates of each staining section were intaken randomly through PC camera with 400X microscope. Image analyzer was employed to observe the staining area of AchE and gradation. And statistical analysis on average results was conducted. Machine of evoked potential electromyogram was deployed to detect and stimulate ischiadic nerve through low frequency RNS and myoelectric wave amplitude of tibial muscle was recorded. Stimulate it repetitively for 9 times with the frequency from 3 to 5Hz pulsating current and observe whether there is decrement or not. Masculine was based on the fact that if the fifth myoelectric wave amplitude is lower 10% than that of the first one. Statistical analysis on the average decrement rate of the fifth myoelectric wave amplitude was conducted.
5. Mechanism research: Observe the morphology of muscle satellite cell of tibial muscle around end plate in each group through transmission electron microscope. Observe randomly 20 ultrathin sections randomly and count the quantity of muscle satellite cells and statistical analysis about it was conducted. Observe the masculine expressing area of aFGF receptor (aFGFR) through immunohistochemiscal staining. Blood vessel masculine expressing of aFGFR was counted and average aFGFR gradation of capillary vessel was measured with image analyzer.
6. Statistical analysis of the data was performed using the SPSS 13.0 software package(Department of Biostatistics, FIMMU). The measurement data were analyzed by one-way ANOVA and multiple comparison was performed by LSD. The numeration data were analyzed by Chi-square. The significant level of difference is 0.05.
Results
1. Morphological observation with naked eyes: group NR+aFGFgel: through own control, it could be observed that there was symmetry between bilateral tibial muscles and normal muscular tension while no manifest muscular atrophy in this group. There were similar performances in group NR+gel and NR: there was manifest muscular atrophy in the right tibial muscles and descending muscular tensions. Group aFGFgel: there was slight muscular atrophy in the right tibial muscle but not so manifest as that in group NR+gel and NR. There was no muscular tension. Group Blank: There was manifest muscular atrophy in the right tibial muscle and no muscular tension in this group. There was no abnormal reaction in which fibrin gel was emdedded in group NR+aFGFgel, group NR+gel and group aFGFgel.
2. The humid weight of tibial muscle of group NR+aFGFgel was 1.26+0.10g, and that of group aFGFgel was 1.20±0.12 g. The humid weight of tibial muscle of group NR+aFGFgel and group aFGFgel was heavier than that of control groups. And statistical analysis on this showed predominant significance.
3. Chromoscopy of end plate: majority end plates in group NR+aFGFgel were with integrated structure and normal morphology. There were similar performances in group NR+gel and group NR: the morphology of end plates in denervated muscle fiber was anomalistic. The branched ending of axon was decreasing and tangling as well. End plate was in the form of striga or punctiform. Group aFGFgel: no axon was observed, the morphology of majority end plates was basically normal, only the end plates region reduced. Group Blank: no axon was observed, the quantity of end plate decreased manifestly and the morphology of end plate was anomalistic.
4. Electron microscope observation: there were great amount of end plates in group NR+aFGFgel and aFGFgel and their architecture was fundamentally normal. Few cataplasia of end plates were observed. The quantity of end plate in control groups decreased obviously and the architecture of end plates was cataplasia. The amount of end plates of group NR+aFGFgel was 12.00±2.24, and that of group aFGFgel was 11.40±1.14. The amount of end plates of group NR+aFGFgel and group aFGFgel was higher than that of control groups, and statistical analysis on this showed predominant significance. The cataplasia percentage of end plates of group NR+aFGFgel was 26.67% and that of group aFGFgel was 33.33%. The cataplasia percentage of end plates of group NR+aFGFgel and group aFGFgel was lower than that of control groups, and statistical analysis on this showed predominant significance.
5. RNS detecting results: There was no obvious decrement of myoelectricity wave in group NR+aFGFgel when there was low frequency RNS. The decrement rate was lower than 10% and no masculine sample was observed. While there was obvious decrement of myoelectricity wave in group NR+gel and group NR when there was low frequency RNS. The masculine rate was 90% in these two groups. The decrement rate of group NR+aFGFgel was 5.70±3.13%, which was lower than that of control groups by statistical analysis.
6. AchE activity of end plate: end plates in group NR+aFGFgel and aFGFgel were stained thickly, which indicates that the acitivity of AchE was strong. The architecture of end plates was normal. Ache staining area of the end plates in group NR+aFGFgel was 1693.98±168.161μm~2, and the gradation was 24.81±1.44. AchE staining area of the end plates in group aFGFgel was 1520.60±303.00μm~2, and the gradation was 24.93±1.54. The acitivity of AchE was stronger than that of control groups by statistical analysis. The rate of cataplasia of end plate in group NR+aFGFgel was 20.42% and that in group aFGFgel was 24.17%,which was lower than that of control groups by statistical analysis.
7. Observation of MSC: the volume and the amount of MSC in group NR+aFGFgel and group aFGFgel increased. The amount of MSC in group NR+aFGFgel was 11.00±3.00 and that in group aFGFgel was 10.40±1.14, which was higher than that of control groups by statistical analysis.
8. Immunohistochemical observation: masculine expressing area of aFGFR mainly existed in capillary vessel wall. Observation showed that high density of expressing area of aFGFR was in group NR+aFGFgel and group aFGFgel. Meanwhile there were intensive capillary vessels in these two groups. Observation also showed that low density of masculine expressing area of aFGFR in capillary vessel was in control groups. And there were rare capillary vessels in control groups. The masculine percentage of aFGFR expression of capillary vessels of group NR+aFGFgel was 87.50%, that of group aFGFgel was 85.00%, which was higher than that of control groups by statistical analysis. The aFGFR gradation of capillary wall was 25.92±1.55 in group NR+aFGFgel, that was 26.85±1.23 in group aFGFgel, which indicated that the expressing amount of aFGFR in capillary wall was higher than that of control groups by statistical analysis.
Conclusions
1. Experiment confirmed that fibrin gel of aFGF could be deployed as the drug slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy and there was no abnormal reaction.
2. Combining nerve repair and the protection for end plate with fibrin gel of aFGF could manifestly prevent end plate from cataplasia and avoid myatrophy in rats.
3. Even if the nerves were not repaired, fibrin gel of aFGF could still protect end plate effectively and avoid myatrophy.
4. RNS detection for conduction of neuromuscular junction indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively improve the conduction of neuromuscular junction.
5. Observation of the activity of AchE through enzymohistochemical method indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively recover the activity of AchE of end plate and enhance normal metabolism of neurotransmitter and transmission of nerve impulse.
6. Those groups with fibrin gel of aFGF were with much more muscle satellite cells than those control groups without fibrin gel of aFGF. It indicated that the function of preventing end plate from cataplasia and avoiding myatrophy by fibrin gel of aFGF correlated with the proliferation of muscle satellite cell.
7. For those groups with fibrin gel of aFGF, there were more blood vessels with abundant transportation of blood around end plates. While for those without fibrin gel of aFGF, there were less blood vessels and bad microcirculation. This indicated that aFGF could enhance the formation of blood vessel manifestly and improve microcirculation.
8. For those groups with fibrin gel of aFGF, the percentage of masculine expressing of aFGFR in capillary vessel and the amount of expressing of aFGFR were much higher than that of those groups without fibrin gel of aFGF. This further indicated that fibrin gel of aFGF prevented end plate from cataplasia and avoided myatrophy correlated to its action to capillary vessel. Through improving the function of microcirculation around end plate, the neural trophic action of denervated muscles was redeemed.
9. Through observing the morphous of muscle satellite cells and the microcirculation around end plate, this experiment deemed that one effective way to prevent devervated end plate from cataplasia and myatrophy was to provide end plate and muscles with substituting neurotrophy.
很多研究发现肌细胞正常结构与功能维持需靠一些神经营养因子的作用，运动神经损伤后，骨骼肌失神经支配后最大变化便是这种神经营养因子丢失，同时骨骼肌废用，最终导致肌肉形态结构和生理生化等方面改变，组织形态学上表现为运动终板退变、肌细胞浆丢失及直径减小，并随时间延长而加重。目前，运动神经损伤后功能的重建主要还是集中在恢复神经的连续性上，忽视了对其靶器官的保护，由于神经漫长的再生过程、再生过程中神经出现再退变、损伤部位的阻挡因素、修复后肢体固定等原因，即使及时修复神经，肌肉失神经支配时间很长，神经对肌肉的营养作用丧失，运动终板逐渐退变，给神经肌肉接头的重建带来极大的困难，临床常见肌肉呈念珠状变性、横纹消失，肌肉萎缩，时间过长肌肉坏死、消失；而且，肌肉失用、萎缩亦会影响运动终板的形态，二者之间形成恶性循环，成为目前严重阻碍运动神经修复后功能恢复的最重要因素之一。因此，如何预防运动终板退变及肌萎缩已成为当前临床运动神经损伤后功能重建过程中急需解决的难题。
迄今对运动终板退变的预防研究甚少，治疗方法不多。临床常用的方法是提倡功能锻炼，但其只能避免肌肉废用不能提供神经营养、且外科修复病例早期常需固定肢体而无法开展，作用有限。曾经有学者提出电刺激可以替代失神经肌肉的兴奋性，并提出了实验鼠模拟正常运动终板兴奋的电刺激模式，取得了一定的效果，但电刺激作用不能持久，并有诸多不便。近年在药物实验研究方面取得了一些成果：有作者报道大鼠腹腔注射地塞米松可以减缓减轻非离断性神经损伤支配运动终板的退变；口服氨哮素可以防止失神经支配运动终板的退变，防止肌肉萎缩，其作用可能是通过某种途径使肌卫星细胞产生一种胰岛素样生长因子(IGF)的营养物质模仿神经对肌肉的营养作用；此外，神经断端间应用神经生长因子(NGF)及肝细胞生长因子(HGF)可减轻运动终板的退变；1996年，Margaret等报道在Ⅱ期修复神经的同时将酸性成纤维细胞生长因子(aFGF)纤维蛋白凝胶植入其支配的萎缩肌腹，初步研究可以诱导运动终板的再生，但对不修复神经者无明显作用，其结果仅仅说明aFGF的作用是在修复神经的前提下，没有说明能否保护失神经支配的运动终板，也没有探讨其对功能恢复效果的作用。
aFGF具有广泛的生物学效应，如促进细胞增殖与分化，促进组织修复，也是一种形态发生因子；其靶细胞有成肌细胞、成纤维细胞、血管内皮细胞等；在重建神经肌肉接头时能激活静止的未分化神经靶细胞形成基底膜，有促进血管生成改善微循环，对多种组织有体内营养作用。成纤维细胞有促进肌细胞基底膜形成的作用，而骨胳肌基底膜是神经肌肉接头形成的重要条件，因此，aFGF促进成纤维细胞的分化对神经肌肉接头的形成具有重要作用；很多研究表明肌肉萎缩的原因主要是肌膜受到不同程度的脂类过氧化作用，这一因素也将累及运动终板，因此，促进基底膜的形成有助于补充受损的膜性结构。亦有研究表明aFGF能调节细胞内钙离子的流出与流入过程，有利于促进黄嘌呤脱氢酶转变为黄嘌呤氧化酶，从而有力于自由基的清除而保护膜性结构如运动终板。此外，对成肌细胞的促进分化可以形成肌细胞并分化成肌管，有助于防止肌肉萎缩；作用于血管内皮细胞有助于改善血循环。
aFGF对运动神经损伤后运动终板退变及肌萎缩的预防是否有作用尚无报道，本研究试图探讨aFGF对失神经支配运动终板退变及肌萎缩的预防作用及其作用机理。
研究目的1、建立为失神经支配运动终板及肌肉提供替代性神经营养的aFGF缓释系统。
2、建立aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的动物模型。
3、探讨aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的形态学及机能学效果。
4、初步探讨aFGF纤维蛋白凝胶预防失神经支配运动终板退变及肌萎缩的作用机理。
材料和方法
1、纤维蛋白凝胶复合aFGF缓释系统的制备：将冻干人纤维蛋白原及抑肽酶直接溶于注射用水，其浓度分别为60mg．ml~(-1)，3000 KIU．ml~(-1)，得溶液Ⅰ；凝血酶用40mmol．L~(-1)CaCL_2溶液配成250 U．ml~(-1)，为溶液Ⅱ。每支aFGF(50μg／支)和0.5ml溶液Ⅰ混合，使aFGF充分混悬后加等体积溶液Ⅱ，在模具中制成形状为圆柱形、半透明、乳白色复合物。将等体积溶液Ⅰ与溶液Ⅱ直接混合制备纤维蛋白凝胶。
2、制做动物模型及分组：155只成年纯系Sprague-Dawley大鼠在全麻(10％水合氯醛腹腔注射)下切断右侧腓总神经胫前肌分支(距神经入肌点约1cm)，随机分为5组：修复神经植入aFGF组(NR+aFGFgel)：缝合神经外膜+胫前肌植入aFGF纤维蛋白凝胶(aFGF 25μg／只)；修复神经植入凝胶组(NR+gel)：缝合神经外膜+胫前肌植入纤维蛋白凝胶；未修复神经植入aFGF组(aFGFgel)：胫前肌植入aFGF纤维蛋白凝胶(aFGF 25μg／只)；单纯修复神经组(NR)：缝合神经外膜；未修复神经未植入组(Blank)：神经肌肉不做处理。NR+gel、NR、Blank为三个对照组。术后观察6周。
3、形态学研究：采用肉眼形态学检查观察肌肉的外观形态，测量胫前肌湿重，采用氯化金法染色显示运动终板，采用超薄切片、铅铀染色、透射电镜观察运动终板超微结构，统计各组运动终板均数及其退变率并进行统计学分析。观察aFGF纤维蛋白凝胶在体内的降解及异常反应。
4、机能学研究：采用Karnovsky-Roots亚铁氰化铜法显示运动终板乙酰胆碱酯酶(AchE)，观察各组运动终板AchE变化情况。退变终板计数：每例随机观察30个运动终板，计数Ache染色显示活性减弱的运动终板数量并进行统计学分析。图像分析：染色切片在400X光镜下经摄像头摄入，利用计算机图像分析软件计算运动终板Ache平均染色面积及灰度值，每例随机测10个运动终板，取均数。采用诱发电位肌电图仪进行神经低频重复电刺激检测：刺激坐骨神经，记录胫前肌肌电波波幅，在脉冲电流刺激频率为3～5Hz的条件下连续刺激9次，观察有无衰减，以第五个肌电波波幅比第一个降低10％以上为阳性，统计各组第五次刺激肌电波波幅衰减率均数。
5、机理研究：采用超薄切片、铅铀染色、透射电镜观察各组运动终板附近胫前肌肌卫星细胞的形态，每例随机观察20个超薄切片视野，统计肌卫星细胞数量并进行统计学分析。采用免疫组织化学染色检查aFGF受体(aFGFR)表达部位；统计每例aFGFR阳性血管表达率；图像分析仪测量毛细血管壁内aFGFR平均灰度。
6、试验结果统计学分析：采用SPSS13.0软件包(第一军医大学生物统计学教研室)对结果进行统计学分析(计量资料采用One-WayANOVA进行分析，多重比较采用LSD法；计数资料采用R×C表资料的x~2检验进行分析)，差异显著性水准置于0.05。
结果
1、肉眼形态学检查：NR+aFGFgel组：自身对照可见两侧胫前肌对称且无明显肌萎缩，肌张力正常；NR+gel组及NR组表现相近：右侧胫前肌明显萎缩，肌张力下降；aFGFgel组：右侧胫前肌略有萎缩，但不如NR+gel组及NR组明显，肌张力消失；Blank组：右侧胫前肌明显萎缩，肌张力消失。NR+aFGFgel组、MR+gel组、aFGFgel组纤维蛋白凝胶植入局部组织无异常反应。
2、胫前肌湿重：NR+aFGFgel组胫前肌湿重为1.26±0.10g，aFGFgel组胫前肌湿重为1.20±0.12g，两组胫前肌湿重高于三个对照组，统计学分析有显著性意义。
3、终板染色检查：NR+aFGFgel组可见运动终板结构完整、形态正常；NR+gel组及NR组表现相近：失神经肌纤维上的终板形态不规则，轴索终末分支减少而杂乱，终板呈细条状或点状；aFgFgel组：未见轴突，运动终板形态大致正常，但区域缩小。Blank组：未见轴突，运动终板数量明显减少，形态不规则。
4、电镜检查：NR+aFGFgel、aFGFgel组运动终板的数量较多，容易辨认，结构基本正常，运动终板退变例数少。三个对照组运动终板数量减少，结构退变，运动终板退变例数多。NR+aFGFgel组运动终板数量为12.00±2.24，aFGFgel组运动终板数量为11.40±1.14，两组运动终板数量高于三个对照组，统计学分析有显著性意义。NR+aFGFgel组运动终板退变率为26.67，aFGFgel组运动终板退变率为33．33，两组运动终板退变率低于三个对照组，统计学分析有显著性意义。
5、神经低频重复电刺激检查：NR+aFGFgel组所有大鼠神经低频重复电刺激时没有出现大的肌电波波幅衰减反应，衰减率为5.70±3.13％，没有出现阳性情况；NR+gel、NR组大鼠低频电刺激时出现较大的肌电波波幅衰减反应，阳性率均为90％。NR+aFGFgel组衰减率低于对照组，统计学分析有显著性意义。
6、运动终板AchE活性：NR+aFGFgel、aFGFgel组运动终板染色深，显示AchE活性强，结构接近正常，NR+aFGFgel组AchE染色面积为1693.98±168.16μm~2，灰度为24.81±1.44，aFGFgel组AchE染色面积为1520.60±303.00μm~2，灰度为24.93±1.54，AchE活性高于对照组，统计学分析有显著性意义；NR+aFGFgel组运动终板退变率为20.42％，aFGFgel组运动终板退变率为24.17％，运动终板退变率低于对照组，统计学分析有显著性意义。
7、肌卫星细胞观察：NR+aFGFgel、aFGFgel组肌卫星细胞体积增大，数量多。NR+aFGFgel组MSC数量为11.00±3.00，aFGFgel组MSC数量为10.40±1.14，MSC数量高于对照组，统计学分析有显著意义。
8、免疫组织化学检查：aFGFR阳性部位主要定位在毛细血管壁内，NR+aFGFgel、aFGFgel组显示毛细血管aFGFR表达密度高，毛细血管密集，对照组毛细血管aFGFR表达密度低，毛细血管稀少。NR+aFGFgel组毛细血管aFGFR表达阳性率为87.50％，aFGFgel组毛细血管aFGFR表达阳性率85.00％，两组aFGFR表达阳性率高于对照组，统计学分析有显著性意义。NR+aFGFgel组毛细血管壁aFGFR表达灰度为25.92±1.55，aFGFgel组毛细血管壁aFGFR表达灰度为26.85±1.23，两组毛细血管壁aFGFR表达量高于对照组，统计学分析有显著性意义。
结论
1、实验证实aFGF纤维蛋白凝胶可做为失神经支配运动终板及肌肉提供替代性神经营养的药物缓释系统，未见异常反应。
2、联合神经修复及应用aFGF纤维蛋白凝胶可明显减轻大鼠运动神经损伤后运动终板退变，减轻肌萎缩。
3、即使神经未及时修复，应用aFGF纤维蛋白凝胶可有效保护大鼠运动神经损伤后所支配的运动终板、减轻肌萎缩。
4、神经重复电刺激检查神经肌肉问传导功能表明：运动神经损伤后联合神经修复及应用aFGF纤维蛋白凝胶保护运动终板可有效改善神经肌肉接头间传导。
5、酶组织化学检查运动终板Ache活性表明：运动神经损伤后应用aFGF纤维蛋白凝胶保护运动终板可有效维持运动终板Ache活性，有利于神经递质的正常代谢，有利于神经冲动的正常传递。
6、应用aFGF纤维蛋白凝胶处理组MSC数量高于对照组，说明aFGF纤维蛋白凝胶预防运动终板退变及肌萎缩的作用与aFGF促MSC增殖作用有关。
7、应用aFGF纤维蛋白凝胶处理组运动终板附近分布了较多毛细血管，微循环良好；而未经aFGF纤维蛋白凝胶处理的运动终板附近毛细血管稀疏，微循环较差；说明aFGF具有明显的促血管形成及改善微循环作用。
8、应用aFGF纤维蛋白凝胶处理组运动终板附近毛细血管aFGFR表达阳性率及表达量高于未经aFGF纤维蛋白凝胶处理组，进一步说明aFGF纤维蛋白凝胶预防运动终板退变及肌萎缩的途径在于其作用于毛细血管，改善运动终板附近微循环，弥补失神经支配肌的神经营养作用。
9、本实验通过肌卫星细胞及运动终板附近微循环的形态观察提示给运动终板及肌肉人工提供“替代性”神经营养是防止失神经支配运动终板退变及肌萎缩的有效途径。
Backgrounds
Many researches show that the normal architecture of muscle and the maintenance of its function depend on the contributions of some neurotrophic factors. When motor nerve is damaged, the greatest change of denervated skeletal muscle is the loss of such kind of factors. Meanwhile, skeletal muscle is disused, which finally results in the change of muscles in morphosis and physiological biochemistry. What displays in histomorphology is the cataplasia of end plate, the loss of the muscle cytoplasm and the diminutus of anteroposterior diameter. Now the re-establishment of motor nerve function after its injury centers on the recovery of the succession of nerve while ignores the protection of its target organs. Such factors as the lengthy reactivation process of nerve with re-cataplasia of nerve during this process, the baffling effect of injury region, the immobilization of limbs after its reparation, even if the nerve is promptly repaired, the process that muscles with denervation lasts for a long time, and the trophic action of nerve lost and gradual cataplasia of end plate bring great difficulty to the re-establishment of myoneural junction. In clinic, the features that muscle presents intestini degeneration, transverse striation abolishment, myatrophy, the death and disappearance of muscle are found. Then, the apraxia and atrophy of muscle have influence on the morphology of end plate, which will lead to an infernal circle between them. This is one of the most important factors nowadays which hinder the recovery of function after nerve repair. As a result, how to prevent the cataplasia of end plate and myatrophy becomes an urgent and tough problem nowadays which awaits to be solved urgently in clinic once motor nerve is damaged.
However, there are few researches and treatments on how to obviate the cataplasia of end plate. The commonly used treatment in clinic is functional exercise, which can only avoid the disuse of muscle but can not offer neurotrophy. Particularly, in the morning of surgery recovery, the limbs are always fixed which limits the effect of such kind of treatment.
Certain scholar once introduced that the excitability of denervated muscles can be replaced by electrical stimulation and a modality of electrical stimulation that using an experimental rat to imitate the excitability of normal end plate was established. And some achievements were obtained. However, the effect of electrical stimulation can not last for a long period and there were many drawbacks. There are also some achievements in the empirical study of drug on the treatment of this aspect in recent years. It is reported that the experiment on rats indicates that Dexamethasone through intraperitoneal injection can alleviate and lessen the consequence that non-mutilation nerve injury leads to the cataplasia of end plate. Clenbuterol by mouth can avoid the cataplasia of denervated end plate and muscular atrophy. Through a certain way, muscle satellite cell can generate such nutritive materials as insulin-like growth factor (IGF), which can imitate the trophic action of nerve to muscles. In addition, NGF and HGF can lessen the cataplasia of end plate. In 1996, initial researches indicated, which was presented by Margaret, that in theⅡstage, when nerves were repaired, once carrier of fibrin gel of acid fibroblast growth factor (aFGF) was imbedded into atrophic muscle belly, it could induce the regeneration of end plate but there was no obvious contribution to those whose nerves were not repaired. Such kind of research showed that the contribution of aFGF could be displayed on the condition that the nerve was repaired and whether it could protect denervated end plate was not still be proved. The contribution that aFGF did to the recovery of function was not indicated as well.
AFGF can generate extensive biological effects such as enhancing cell multiplication and differentiation and promoting tissue repair. It is also a kind of morphogenetic factor whose target cell includes sarcoblast, collagenoblast, vascular endothelial cell. Angiogenesis can ameliorate microcirculation and can do great endotrophic action to many architectures. Collagenoblast can promote the formation of muscle cell's basal lamina, while basal lamina of skeletal muscle is one of the significant conditions in the formation of myoneural junction. As a result, aFGF plays an important role in promoting the differentiation of collagenoblast which does contribution to the formation of myoneural junction. Meanwhile many researches indicate that the principal cause of muscular atrophy is tunica muscularis suffers lipide peroxidation which also has effect on end plate. As a result, to enhance the formation of basal lamina will also enhance the replenishment of those impaired hymeno-structures. Other research indicates that aFGF can accommodate the affluence and effluence of Ca2+ in intra-cellular, which will enhance the transformation of xanthine oxidase from xanthine dehydrogenase and then eliminate free radical. Accordingly, those hymeno-structures such as end plate can be protected through the elimination of free radical. To hasten myoblastic differentiation will form muscle cell and differentiate myotube and thereby prevent muscle from myatrophy. AFGF can improve the blood circulation through the action of vascular endothelial cell.
There is still no report shows that aFGF can prevent the cataplasia of end plate and myatrophy after motor nerve injury. This study was undertaken to probe the effect on the prevention of the cataplasia of denervated end plate and myatrophy by aFGF and its mechanism of action.
Objectives
1. To establish aFGF slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy.
2. To establish the animal model of preventing cataplasia of denervated end plate and myatrophy with fibrin gel of aFGF.
3. To approach the morphological and functional effect that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.
4. To approach initially the mechanism of action that how fibrin gel of aFGF prevents cataplasia of denervated end plate and myatrophy.
Materials and Methods
1. The preparation of aFGF slow- releasing system: liquorⅠ: cryodesiccant human fibrinogen and aprotinin were dissolved into aqua pro injectione directly with concentration as 60mg.ml~(-1) and 3000 KIU.ml~(-1) respectively; liquorⅡ: 40mmol.L~(-1)CaCL_2 zymoplasm with concentration as 250 U.ml~(-1). Admixed aFGF 50μg with 0.5ml liquorⅠand add it with equal volumn liquorⅡby mixing thoroughly. Put this admixture into tooting and got a cylindrical, semitransparent ivory compound. Fibrin gel was prepared by admixed liquorⅠwith equal volumn liquorⅡ.
2. Establishing and grouping animal models: 155 adult inbred line Sprague-Dawley rats were selected and abscised their right tibial muscle branch of the common peroneal nerves (the distance between abscising point and the point where nerve entering into muscle is 1cm) with complete anesthesia which was through 10% chloral hydrate through intraperitoneal injection. The models were randomly divided into five groups. Group NR+aFGFgel: those that epineuriums were sutured and carriers of Fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR+gel: those that epineuriums were sutured and carriers of fibrin gel were imbedded in tibial muscles. Group aFGFgel: those that epineuriums were not sutured and carriers of fibrin gel of aFGF (25μg aFGF per rat)were imbedded in tibial muscles. Group NR: those that only epineurium were sutured. Group Blank: those that epineuriums were not sutured and no implant was imbedded in tibial muscles. Group NR+gel, Group NR and Group Bland were control groups. All rats were observed for 6 weeks postoperation.
3. Morphological research: Observe the appearance of the muscle through morphological method with naked eyes and measure humid weight of tibial muscle. Staining with auric chloride was deployed to display end plate. Ultrathin sections were stained with uranium and lead and the ultrastructure of end plate was observed by transmission electron microscope. Calculate end plates and the percentage of cataplasia of denervated end plate in five groups and analyze it through statistical method. Observe the degradation of fibrin gel of aFGF and those possible adverse effects.
4. Functional research: Karnovsky-Roots copper-ferrocyanide was employed to display AchE of end plate and observe the change of AchE in end plate of each group. The count of cataplasia of end plate: take randomly 30 end plates in each group and count the quantity of end plates which were with subdued activity through staining with AchE and the statistical analysis was conducted. Image analysis: 10 end plates of each staining section were intaken randomly through PC camera with 400X microscope. Image analyzer was employed to observe the staining area of AchE and gradation. And statistical analysis on average results was conducted. Machine of evoked potential electromyogram was deployed to detect and stimulate ischiadic nerve through low frequency RNS and myoelectric wave amplitude of tibial muscle was recorded. Stimulate it repetitively for 9 times with the frequency from 3 to 5Hz pulsating current and observe whether there is decrement or not. Masculine was based on the fact that if the fifth myoelectric wave amplitude is lower 10% than that of the first one. Statistical analysis on the average decrement rate of the fifth myoelectric wave amplitude was conducted.
5. Mechanism research: Observe the morphology of muscle satellite cell of tibial muscle around end plate in each group through transmission electron microscope. Observe randomly 20 ultrathin sections randomly and count the quantity of muscle satellite cells and statistical analysis about it was conducted. Observe the masculine expressing area of aFGF receptor (aFGFR) through immunohistochemiscal staining. Blood vessel masculine expressing of aFGFR was counted and average aFGFR gradation of capillary vessel was measured with image analyzer.
6. Statistical analysis of the data was performed using the SPSS 13.0 software package(Department of Biostatistics, FIMMU). The measurement data were analyzed by one-way ANOVA and multiple comparison was performed by LSD. The numeration data were analyzed by Chi-square. The significant level of difference is 0.05.
Results
1. Morphological observation with naked eyes: group NR+aFGFgel: through own control, it could be observed that there was symmetry between bilateral tibial muscles and normal muscular tension while no manifest muscular atrophy in this group. There were similar performances in group NR+gel and NR: there was manifest muscular atrophy in the right tibial muscles and descending muscular tensions. Group aFGFgel: there was slight muscular atrophy in the right tibial muscle but not so manifest as that in group NR+gel and NR. There was no muscular tension. Group Blank: There was manifest muscular atrophy in the right tibial muscle and no muscular tension in this group. There was no abnormal reaction in which fibrin gel was emdedded in group NR+aFGFgel, group NR+gel and group aFGFgel.
2. The humid weight of tibial muscle of group NR+aFGFgel was 1.26+0.10g, and that of group aFGFgel was 1.20±0.12 g. The humid weight of tibial muscle of group NR+aFGFgel and group aFGFgel was heavier than that of control groups. And statistical analysis on this showed predominant significance.
3. Chromoscopy of end plate: majority end plates in group NR+aFGFgel were with integrated structure and normal morphology. There were similar performances in group NR+gel and group NR: the morphology of end plates in denervated muscle fiber was anomalistic. The branched ending of axon was decreasing and tangling as well. End plate was in the form of striga or punctiform. Group aFGFgel: no axon was observed, the morphology of majority end plates was basically normal, only the end plates region reduced. Group Blank: no axon was observed, the quantity of end plate decreased manifestly and the morphology of end plate was anomalistic.
4. Electron microscope observation: there were great amount of end plates in group NR+aFGFgel and aFGFgel and their architecture was fundamentally normal. Few cataplasia of end plates were observed. The quantity of end plate in control groups decreased obviously and the architecture of end plates was cataplasia. The amount of end plates of group NR+aFGFgel was 12.00±2.24, and that of group aFGFgel was 11.40±1.14. The amount of end plates of group NR+aFGFgel and group aFGFgel was higher than that of control groups, and statistical analysis on this showed predominant significance. The cataplasia percentage of end plates of group NR+aFGFgel was 26.67% and that of group aFGFgel was 33.33%. The cataplasia percentage of end plates of group NR+aFGFgel and group aFGFgel was lower than that of control groups, and statistical analysis on this showed predominant significance.
5. RNS detecting results: There was no obvious decrement of myoelectricity wave in group NR+aFGFgel when there was low frequency RNS. The decrement rate was lower than 10% and no masculine sample was observed. While there was obvious decrement of myoelectricity wave in group NR+gel and group NR when there was low frequency RNS. The masculine rate was 90% in these two groups. The decrement rate of group NR+aFGFgel was 5.70±3.13%, which was lower than that of control groups by statistical analysis.
6. AchE activity of end plate: end plates in group NR+aFGFgel and aFGFgel were stained thickly, which indicates that the acitivity of AchE was strong. The architecture of end plates was normal. Ache staining area of the end plates in group NR+aFGFgel was 1693.98±168.161μm~2, and the gradation was 24.81±1.44. AchE staining area of the end plates in group aFGFgel was 1520.60±303.00μm~2, and the gradation was 24.93±1.54. The acitivity of AchE was stronger than that of control groups by statistical analysis. The rate of cataplasia of end plate in group NR+aFGFgel was 20.42% and that in group aFGFgel was 24.17%,which was lower than that of control groups by statistical analysis.
7. Observation of MSC: the volume and the amount of MSC in group NR+aFGFgel and group aFGFgel increased. The amount of MSC in group NR+aFGFgel was 11.00±3.00 and that in group aFGFgel was 10.40±1.14, which was higher than that of control groups by statistical analysis.
8. Immunohistochemical observation: masculine expressing area of aFGFR mainly existed in capillary vessel wall. Observation showed that high density of expressing area of aFGFR was in group NR+aFGFgel and group aFGFgel. Meanwhile there were intensive capillary vessels in these two groups. Observation also showed that low density of masculine expressing area of aFGFR in capillary vessel was in control groups. And there were rare capillary vessels in control groups. The masculine percentage of aFGFR expression of capillary vessels of group NR+aFGFgel was 87.50%, that of group aFGFgel was 85.00%, which was higher than that of control groups by statistical analysis. The aFGFR gradation of capillary wall was 25.92±1.55 in group NR+aFGFgel, that was 26.85±1.23 in group aFGFgel, which indicated that the expressing amount of aFGFR in capillary wall was higher than that of control groups by statistical analysis.
Conclusions
1. Experiment confirmed that fibrin gel of aFGF could be deployed as the drug slow-releasing system which providing denervated end plate and muscle with substitution of neurotrophy and there was no abnormal reaction.
2. Combining nerve repair and the protection for end plate with fibrin gel of aFGF could manifestly prevent end plate from cataplasia and avoid myatrophy in rats.
3. Even if the nerves were not repaired, fibrin gel of aFGF could still protect end plate effectively and avoid myatrophy.
4. RNS detection for conduction of neuromuscular junction indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively improve the conduction of neuromuscular junction.
5. Observation of the activity of AchE through enzymohistochemical method indicated that when there was motor nerve injury, combining nerve repair and the protection for end plate with fibrin gel of aFGF could effectively recover the activity of AchE of end plate and enhance normal metabolism of neurotransmitter and transmission of nerve impulse.
6. Those groups with fibrin gel of aFGF were with much more muscle satellite cells than those control groups without fibrin gel of aFGF. It indicated that the function of preventing end plate from cataplasia and avoiding myatrophy by fibrin gel of aFGF correlated with the proliferation of muscle satellite cell.
7. For those groups with fibrin gel of aFGF, there were more blood vessels with abundant transportation of blood around end plates. While for those without fibrin gel of aFGF, there were less blood vessels and bad microcirculation. This indicated that aFGF could enhance the formation of blood vessel manifestly and improve microcirculation.
8. For those groups with fibrin gel of aFGF, the percentage of masculine expressing of aFGFR in capillary vessel and the amount of expressing of aFGFR were much higher than that of those groups without fibrin gel of aFGF. This further indicated that fibrin gel of aFGF prevented end plate from cataplasia and avoided myatrophy correlated to its action to capillary vessel. Through improving the function of microcirculation around end plate, the neural trophic action of denervated muscles was redeemed.
9. Through observing the morphous of muscle satellite cells and the microcirculation around end plate, this experiment deemed that one effective way to prevent devervated end plate from cataplasia and myatrophy was to provide end plate and muscles with substituting neurotrophy.
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