电刺激治疗股二头肌急性拉伤的模拟实验研究
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摘要
实验目的:
1.模拟运动状态下股二头肌的急性拉伤,建立一种反映运动实践特点的股二头肌急性拉伤动物模型,从而使进一步深入的研究成为可能;
2.通过对股二头肌急性拉伤后力学以及组织学的观察,明确其结构、功能和病理学等方面的特点,为下一步实验研究打下基础;
3.采用低强度20Hz和80Hz电刺激治疗股二头肌急性拉伤,观察其对于肌肉损伤部位结构重塑的影响;
4.采用低强度20Hz和80Hz电刺激治疗股二头肌急性拉伤,观察电刺激治疗对于成肌细胞分化、融合以及肌纤维形成的MyoD和Myogenin基因表达调控的影响。为阐明电刺激治疗肌肉拉伤的机制寻找的突破口,从而为肌肉拉伤的临床治疗提供科学的参数与依据,优化电刺激治疗方案。
实验方法:
雄性SD大鼠45只,随机分为正常对照组、D0组、D3组、D7组、D7-20Hz组、D7-80Hz组、D14组、D14-20Hz组和D14-80Hz组。除正常对照组外,其余各组建立股二头肌急性拉伤动物模型。麻醉大鼠,采用生理刺激仪针电刺激固定侧肢体的坐骨神经,使股二头肌发生强直收缩,同时牵拉膝关节迅速做角速度为9600·s-1的伸膝运动,股二头肌做离心收缩,造成一侧肢体股二头肌急性拉伤。拉伤前后做超声诊断。动物造模完成后,D7-20Hz组、D7-80Hz组、D14-20Hz组和D14-80Hz于动物造模后第5天开始电刺激,每日两次,每次30分钟,相隔4小时,其余组继续喂养。各组分别在相应的时间进行力矩测试,待实验结束后处死大鼠,分离股二头肌,固定保存以备用,进行病理组织、免疫组化观察。
实验结果:
1.肌肉拉伤部位病理改变光镜观察结果:肌肉拉伤当天肌纤维坏死,肌肉组织结构紊乱,肌纤维水肿变性,肌肉间隙大小不一,部分肌纤维间隙明显加大,并可见血管扩张充血。其中股二头肌损伤程度较之半膜肌与半腱肌严重。拉伤后第3天病理改变光镜观察可见炎性反应明显。拉伤后第7天病理改变光镜观察可见,肌纤维出现修复,但是不成熟,肌纤维排列不严整,大小不一。经过20Hz电刺激治疗后,新生的肌纤维更成熟,成簇排列,结构更严整,边界更清晰。80Hz电刺激治疗的效果不明显。各组于拉伤后第14天肌纤维基本修复。
2.肌肉拉伤后超声图像:大鼠肌肉拉伤后,半腱肌与半膜肌肌肉损伤较轻微,无超声影像的改变;股二头肌肌肉拉伤较前两者严重,表现为受累肌肉的局部回声减低,部分撕裂声像图显示肌纤维部分或完全不连续,裂口间出现血肿,肌肉拉伤位置处于远端肌腱结合部靠近肌肉端。各组于拉伤当日4小时后,经肌肉诊断超声横断面测量损伤所占横径比例处于5%-50%,属于二度损伤,各组之间比较差异不显著(P>0.05)。
3.关节最大等长力矩测定:正常对照组关节最大等长力矩值为0.337±0.025(Nm),大鼠肌肉拉伤后不同时刻关节最大等长力矩指标的变化,肌肉拉伤后即刻关节最大等长力矩下降为正常的73.05±5.37%,与正常对照组比较存在显著性差异(P<0.05);第三天为79.79±6.95%;第七天为89.84±4.75%;第14天基本恢复到正常。
4.电刺激治疗后关节最大等长力矩测定: D7-20Hz组关节最大等长力矩值为0.312±0.043(Nm),D7-80Hz组关节最大等长力矩值为0.302±0.033(Nm),与D7组比较不存在显著性差异(P>0.05)。D14-20Hz组关节最大等长力矩值为0.335±0.028(Nm),D14-80Hz组关节最大等长力矩值为0.334±0.060(Nm),与D14以及受伤前力矩比较比较不存在显著性差异(P>0.05)。
5.力矩-角度关系测定结果:正常对照组最优角度出现在130.000±3.540,肌肉拉伤后即刻力矩-角度关系指标测定结果显示关节最大等长力矩出现右侧偏移,最优角度为144.500±3.710,与受伤前比较存在显著性差异(P<0.01);大鼠肌肉拉伤后第14天力矩-角度关系指标测定结果显示,D14组关节最大等长力矩出现左侧偏移,最优角度出现在120.000±3.530,与受伤前比较存在显著性差异(P<0.05)。大鼠肌肉拉伤后经电刺激治疗,力矩-角度关系指标测定结果显示关节最优角度出现左侧偏移, D14-20Hz和D14-80Hz最优角度分别出现在125.000±3.530以及121.000±4.180,其中D14-20Hz组偏移较小,与D14组比较存在显著性差异(P<0.05)。
6. MyoD与Myogenin蛋白表达测定结果:肌肉拉伤后第7天MyoD与Myogenin蛋白表达, D7组、D7-20Hz组以及D7-80Hz组明显增强,与正常对照组比较差异显著(P<0.01),其中D7-20Hz组增强更明显,与D7组比较存在显著性差异(P<0.01),但是D7-80Hz组与D7组比较不存在显著性差异(P>0.05)。肌肉拉伤后第14天MyoD与Myogenin蛋白表达观察结果,其中肌肉拉伤后第14天各组MyoD蛋白表达,D14组以及D14-80Hz组减弱,与正常对照组比较无差异(P>0.05),但是D14-20Hz组依旧比正常对照组增强明显(P<0.05)。肌肉拉伤后第14天Myogenin蛋白表达,D14-20Hz组Myogenin蛋白表达依旧强于D14动物模型组(P<0.05)。
实验结论:
1.本实验研究成功地建立了股二头肌急性拉伤动物模型,并具有接近运动实践的特点;
2.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,可以有效地促进损伤后肌肉结构的重塑,对于损伤后肌肉组织功能结构的恢复非常有利;
3.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,并不能在两周内明显提高关节最大等长力矩,但是可以很好地优化关节力矩-角度的关系;
4.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,促进了MyoD与Myogenin的基因表达,并观察到损伤股二头肌结构重塑和力矩-角度关系的优化,这表明采用低强度20Hz的电刺激治疗通过对MyoD与Myogenin的基因表达调控的影响,从而促进损伤股二头肌组织的修复;
5.股二头肌急性拉伤后采用低强度80Hz时,其对受损伤肌肉在形态结构和功能上的恢复没有明显促进作用,并且对于MyoD与Myogenin的基因表达调控没有显著影响。
Objectives:
1. To create an animal model that simulates exercise-induced acute biceps femoris strain, in order to provide basis for the further study on muscle structure, functions and pathology.
2. To observe the mechanics and histology, so as to identify the features of structure, function and pathology after biceps femoris strain.
3. To observe the effect of low-intensity electrical stimulation at different frequencies (20 Hz and 80 Hz) on the rebuilding of muscle structure.
4. To observe the effect of low-intensity electrical stimulation at different frequencies (20 Hz and 80 Hz) on the gene regulatory of MyoD and Myogenin on proliferation, termination and new fibers rebuilding after acute biceps femoris strain, in order to find a new way for analysis the manchnism of muscular strain. Therefore, it will be helpful to choose better protocol of electrical therapy.
Methods:
Forty-five male SD rats were randomly divided into control group, D0 group, D3 group, D7 group, D7-20Hz group, D7-80Hz group, D14 group, D14-20Hz group and D14-80Hz group. All male SD rats except ones of control group were anesthetized. One limb was chosen at random to create a partial biceps femoris stretch injury.The biceps femoris was stimulated to tetany by activating the sciatic nerve via subcutaneous needle electrodes, which were placed over the sciatic nerve in the region of the ischial tuberosity. The limb was moved from knee flexion 700 to full knee extension at 9600?s-1 angular velocity and then returned to the starting position.After torque test and the observation of ultrasound, the biceps femoris was harvested for procedure of histology. After injury, no interventions were given to D0 group, D3 group, D7 group and D14 group. The electrical protocol was made in D7-20Hz group, D7-80Hz group, D14-20Hz group and D14-80Hz group. The biceps femoris was stimulated using a modified stimulator. Square wave stimuli at 20Hz\80Hz were applied twice a day for 30 minutes each time with a 4 hour interval. After experiment, we had an observation on torque, ultrasound diagnosis, HE staining, western-blotting and immuno-staining.
Results:
1. After injury, histology showed fiber necrosis, and hemorrhage. HE staining revealed structural disorder, edema, and increased gap between muscle fibers.The injuries of biceps were more severe than injury of Semitendinosus and Semimembrinosus. At 3 days post-injury, there was intense inflammatory cell proliferation. At 7 days, injured muscle fibers recovered but did not mature. Given electrical therapy in D7-20Hz group, the new fibers became more mature, neat formation and clear boundary.However, low-intensity 80Hz electrical stimulation show no significant effects. At 14 days, injured muscle fibers became mature and recovered.
2. The images of Semitendinosus and Semimembrinosus are lack of any ultrasonic lesion. Injured Biceps correspond to lesions involving from 5 to 50% of the muscle volume or cross-sectional diameter, which belongs toⅡdegree injury. No significant difference between them(P>0.05).
3. The isometric maximal knee torque of control group was 0.337±0.025Nm. The isometric maximal torque showed a significant drop by 73.05±5.37% of control muscle at day 0(P<0.05), 79.79±6.95% at Day 3 post-injury, 89.84±4.75% at Day 7 post-injury. At Day 14, the isometric maximal torque recovered to normal level.
4. The isometric maximal knee torque of D7-20Hz group and D7-80Hz group respectively were 0.312±0.043Nm and 0.302±0.033Nm. Compared to the isometric maximal torque of D7 animal model group, no significant difference existed(P>0.05). The isometric maximal knee torque of D14-20Hz group and D14-80Hz group respectively were 0.335±0.028Nm and 0.334±0.060Nm. Compared to the isometric maximal knee torque of D7 animal model group, no significant difference existed(P>0.05).
5. The optimal angle of control group was 130.000±3.540. It showed a significant right shift to 144.500±3.710 at day 0 post-injury. Compared to the optimal angle of control group,there was significant difference(P<0.05). At day 14 post-injury, it showed a significant left shift to 120.000±3.530 compared to the optimal angle of control group (P<0.05). After electrical stimulation, the optimal angle of D14-20Hz group and D14-80Hz group were 125.000±3.530 and 121.000±4.180. A significant difference existed between D14 group and D14-20Hz group(P<0.05).
6. At 7 days post-injury,the protein expression of MyoD and Myogenin increased in D7group、D7-20Hz group and D7-80Hz group. Compared to control group, there was a significant difference (P<0.01). Between D7 group and D7-20Hz group, there was also significant difference(P<0.01).At 14 day after injury, the protein expression of MyoD decreased in D14 group and D14-80Hz group. Compared to control group, there was no significant difference (P>0.05). However, the protein expression of MyoD was still significant higher in D14-20Hz group compared to D14 animal model group (P<0.05). The protein expression of Myogenin was still higher in D14-20Hz. Compared to D14 group, there was a significant difference (P<0.05).
Conclusions:
1. The animal model, which was created in this experiment, simulated a sports injury well.
2. The low intensity 20Hz electrical stimulation was useful for muscular rebuilding of the injured biceps femoris in morphological structure and function.
3. The low intensity the 20Hz electrical stimulation could not increase the maximal isometric toque but was able to improve the torque-angle relationship in injured biceps femoris during 2 weeks post-injury.
4. The low-intensity 20Hz electrical stimulation could improve the gene regulatory of MyoD and Myogenin as well as muscular rebuilding, which proved the low-intensity 20Hz electrical stimulation was helpful for the rapair of injured biceps femoris through the intervention on gene regulatory of MyoD and Myogenin.
5. The low-intensity 80Hz electrical stimulation had no effects on the gene regulatory of MyoD and Myogenin as well as muscular rebuilding.
1.模拟运动状态下股二头肌的急性拉伤,建立一种反映运动实践特点的股二头肌急性拉伤动物模型,从而使进一步深入的研究成为可能;
2.通过对股二头肌急性拉伤后力学以及组织学的观察,明确其结构、功能和病理学等方面的特点,为下一步实验研究打下基础;
3.采用低强度20Hz和80Hz电刺激治疗股二头肌急性拉伤,观察其对于肌肉损伤部位结构重塑的影响;
4.采用低强度20Hz和80Hz电刺激治疗股二头肌急性拉伤,观察电刺激治疗对于成肌细胞分化、融合以及肌纤维形成的MyoD和Myogenin基因表达调控的影响。为阐明电刺激治疗肌肉拉伤的机制寻找的突破口,从而为肌肉拉伤的临床治疗提供科学的参数与依据,优化电刺激治疗方案。
实验方法:
雄性SD大鼠45只,随机分为正常对照组、D0组、D3组、D7组、D7-20Hz组、D7-80Hz组、D14组、D14-20Hz组和D14-80Hz组。除正常对照组外,其余各组建立股二头肌急性拉伤动物模型。麻醉大鼠,采用生理刺激仪针电刺激固定侧肢体的坐骨神经,使股二头肌发生强直收缩,同时牵拉膝关节迅速做角速度为9600·s-1的伸膝运动,股二头肌做离心收缩,造成一侧肢体股二头肌急性拉伤。拉伤前后做超声诊断。动物造模完成后,D7-20Hz组、D7-80Hz组、D14-20Hz组和D14-80Hz于动物造模后第5天开始电刺激,每日两次,每次30分钟,相隔4小时,其余组继续喂养。各组分别在相应的时间进行力矩测试,待实验结束后处死大鼠,分离股二头肌,固定保存以备用,进行病理组织、免疫组化观察。
实验结果:
1.肌肉拉伤部位病理改变光镜观察结果:肌肉拉伤当天肌纤维坏死,肌肉组织结构紊乱,肌纤维水肿变性,肌肉间隙大小不一,部分肌纤维间隙明显加大,并可见血管扩张充血。其中股二头肌损伤程度较之半膜肌与半腱肌严重。拉伤后第3天病理改变光镜观察可见炎性反应明显。拉伤后第7天病理改变光镜观察可见,肌纤维出现修复,但是不成熟,肌纤维排列不严整,大小不一。经过20Hz电刺激治疗后,新生的肌纤维更成熟,成簇排列,结构更严整,边界更清晰。80Hz电刺激治疗的效果不明显。各组于拉伤后第14天肌纤维基本修复。
2.肌肉拉伤后超声图像:大鼠肌肉拉伤后,半腱肌与半膜肌肌肉损伤较轻微,无超声影像的改变;股二头肌肌肉拉伤较前两者严重,表现为受累肌肉的局部回声减低,部分撕裂声像图显示肌纤维部分或完全不连续,裂口间出现血肿,肌肉拉伤位置处于远端肌腱结合部靠近肌肉端。各组于拉伤当日4小时后,经肌肉诊断超声横断面测量损伤所占横径比例处于5%-50%,属于二度损伤,各组之间比较差异不显著(P>0.05)。
3.关节最大等长力矩测定:正常对照组关节最大等长力矩值为0.337±0.025(Nm),大鼠肌肉拉伤后不同时刻关节最大等长力矩指标的变化,肌肉拉伤后即刻关节最大等长力矩下降为正常的73.05±5.37%,与正常对照组比较存在显著性差异(P<0.05);第三天为79.79±6.95%;第七天为89.84±4.75%;第14天基本恢复到正常。
4.电刺激治疗后关节最大等长力矩测定: D7-20Hz组关节最大等长力矩值为0.312±0.043(Nm),D7-80Hz组关节最大等长力矩值为0.302±0.033(Nm),与D7组比较不存在显著性差异(P>0.05)。D14-20Hz组关节最大等长力矩值为0.335±0.028(Nm),D14-80Hz组关节最大等长力矩值为0.334±0.060(Nm),与D14以及受伤前力矩比较比较不存在显著性差异(P>0.05)。
5.力矩-角度关系测定结果:正常对照组最优角度出现在130.000±3.540,肌肉拉伤后即刻力矩-角度关系指标测定结果显示关节最大等长力矩出现右侧偏移,最优角度为144.500±3.710,与受伤前比较存在显著性差异(P<0.01);大鼠肌肉拉伤后第14天力矩-角度关系指标测定结果显示,D14组关节最大等长力矩出现左侧偏移,最优角度出现在120.000±3.530,与受伤前比较存在显著性差异(P<0.05)。大鼠肌肉拉伤后经电刺激治疗,力矩-角度关系指标测定结果显示关节最优角度出现左侧偏移, D14-20Hz和D14-80Hz最优角度分别出现在125.000±3.530以及121.000±4.180,其中D14-20Hz组偏移较小,与D14组比较存在显著性差异(P<0.05)。
6. MyoD与Myogenin蛋白表达测定结果:肌肉拉伤后第7天MyoD与Myogenin蛋白表达, D7组、D7-20Hz组以及D7-80Hz组明显增强,与正常对照组比较差异显著(P<0.01),其中D7-20Hz组增强更明显,与D7组比较存在显著性差异(P<0.01),但是D7-80Hz组与D7组比较不存在显著性差异(P>0.05)。肌肉拉伤后第14天MyoD与Myogenin蛋白表达观察结果,其中肌肉拉伤后第14天各组MyoD蛋白表达,D14组以及D14-80Hz组减弱,与正常对照组比较无差异(P>0.05),但是D14-20Hz组依旧比正常对照组增强明显(P<0.05)。肌肉拉伤后第14天Myogenin蛋白表达,D14-20Hz组Myogenin蛋白表达依旧强于D14动物模型组(P<0.05)。
实验结论:
1.本实验研究成功地建立了股二头肌急性拉伤动物模型,并具有接近运动实践的特点;
2.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,可以有效地促进损伤后肌肉结构的重塑,对于损伤后肌肉组织功能结构的恢复非常有利;
3.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,并不能在两周内明显提高关节最大等长力矩,但是可以很好地优化关节力矩-角度的关系;
4.股二头肌急性拉伤后采用低强度20Hz的电刺激治疗,促进了MyoD与Myogenin的基因表达,并观察到损伤股二头肌结构重塑和力矩-角度关系的优化,这表明采用低强度20Hz的电刺激治疗通过对MyoD与Myogenin的基因表达调控的影响,从而促进损伤股二头肌组织的修复;
5.股二头肌急性拉伤后采用低强度80Hz时,其对受损伤肌肉在形态结构和功能上的恢复没有明显促进作用,并且对于MyoD与Myogenin的基因表达调控没有显著影响。
Objectives:
1. To create an animal model that simulates exercise-induced acute biceps femoris strain, in order to provide basis for the further study on muscle structure, functions and pathology.
2. To observe the mechanics and histology, so as to identify the features of structure, function and pathology after biceps femoris strain.
3. To observe the effect of low-intensity electrical stimulation at different frequencies (20 Hz and 80 Hz) on the rebuilding of muscle structure.
4. To observe the effect of low-intensity electrical stimulation at different frequencies (20 Hz and 80 Hz) on the gene regulatory of MyoD and Myogenin on proliferation, termination and new fibers rebuilding after acute biceps femoris strain, in order to find a new way for analysis the manchnism of muscular strain. Therefore, it will be helpful to choose better protocol of electrical therapy.
Methods:
Forty-five male SD rats were randomly divided into control group, D0 group, D3 group, D7 group, D7-20Hz group, D7-80Hz group, D14 group, D14-20Hz group and D14-80Hz group. All male SD rats except ones of control group were anesthetized. One limb was chosen at random to create a partial biceps femoris stretch injury.The biceps femoris was stimulated to tetany by activating the sciatic nerve via subcutaneous needle electrodes, which were placed over the sciatic nerve in the region of the ischial tuberosity. The limb was moved from knee flexion 700 to full knee extension at 9600?s-1 angular velocity and then returned to the starting position.After torque test and the observation of ultrasound, the biceps femoris was harvested for procedure of histology. After injury, no interventions were given to D0 group, D3 group, D7 group and D14 group. The electrical protocol was made in D7-20Hz group, D7-80Hz group, D14-20Hz group and D14-80Hz group. The biceps femoris was stimulated using a modified stimulator. Square wave stimuli at 20Hz\80Hz were applied twice a day for 30 minutes each time with a 4 hour interval. After experiment, we had an observation on torque, ultrasound diagnosis, HE staining, western-blotting and immuno-staining.
Results:
1. After injury, histology showed fiber necrosis, and hemorrhage. HE staining revealed structural disorder, edema, and increased gap between muscle fibers.The injuries of biceps were more severe than injury of Semitendinosus and Semimembrinosus. At 3 days post-injury, there was intense inflammatory cell proliferation. At 7 days, injured muscle fibers recovered but did not mature. Given electrical therapy in D7-20Hz group, the new fibers became more mature, neat formation and clear boundary.However, low-intensity 80Hz electrical stimulation show no significant effects. At 14 days, injured muscle fibers became mature and recovered.
2. The images of Semitendinosus and Semimembrinosus are lack of any ultrasonic lesion. Injured Biceps correspond to lesions involving from 5 to 50% of the muscle volume or cross-sectional diameter, which belongs toⅡdegree injury. No significant difference between them(P>0.05).
3. The isometric maximal knee torque of control group was 0.337±0.025Nm. The isometric maximal torque showed a significant drop by 73.05±5.37% of control muscle at day 0(P<0.05), 79.79±6.95% at Day 3 post-injury, 89.84±4.75% at Day 7 post-injury. At Day 14, the isometric maximal torque recovered to normal level.
4. The isometric maximal knee torque of D7-20Hz group and D7-80Hz group respectively were 0.312±0.043Nm and 0.302±0.033Nm. Compared to the isometric maximal torque of D7 animal model group, no significant difference existed(P>0.05). The isometric maximal knee torque of D14-20Hz group and D14-80Hz group respectively were 0.335±0.028Nm and 0.334±0.060Nm. Compared to the isometric maximal knee torque of D7 animal model group, no significant difference existed(P>0.05).
5. The optimal angle of control group was 130.000±3.540. It showed a significant right shift to 144.500±3.710 at day 0 post-injury. Compared to the optimal angle of control group,there was significant difference(P<0.05). At day 14 post-injury, it showed a significant left shift to 120.000±3.530 compared to the optimal angle of control group (P<0.05). After electrical stimulation, the optimal angle of D14-20Hz group and D14-80Hz group were 125.000±3.530 and 121.000±4.180. A significant difference existed between D14 group and D14-20Hz group(P<0.05).
6. At 7 days post-injury,the protein expression of MyoD and Myogenin increased in D7group、D7-20Hz group and D7-80Hz group. Compared to control group, there was a significant difference (P<0.01). Between D7 group and D7-20Hz group, there was also significant difference(P<0.01).At 14 day after injury, the protein expression of MyoD decreased in D14 group and D14-80Hz group. Compared to control group, there was no significant difference (P>0.05). However, the protein expression of MyoD was still significant higher in D14-20Hz group compared to D14 animal model group (P<0.05). The protein expression of Myogenin was still higher in D14-20Hz. Compared to D14 group, there was a significant difference (P<0.05).
Conclusions:
1. The animal model, which was created in this experiment, simulated a sports injury well.
2. The low intensity 20Hz electrical stimulation was useful for muscular rebuilding of the injured biceps femoris in morphological structure and function.
3. The low intensity the 20Hz electrical stimulation could not increase the maximal isometric toque but was able to improve the torque-angle relationship in injured biceps femoris during 2 weeks post-injury.
4. The low-intensity 20Hz electrical stimulation could improve the gene regulatory of MyoD and Myogenin as well as muscular rebuilding, which proved the low-intensity 20Hz electrical stimulation was helpful for the rapair of injured biceps femoris through the intervention on gene regulatory of MyoD and Myogenin.
5. The low-intensity 80Hz electrical stimulation had no effects on the gene regulatory of MyoD and Myogenin as well as muscular rebuilding.
引文
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