大鼠腰5神经根牵拉模型建立和机械牵拉对神经根功能和形态学影响

发布时间：2018-05-17 08:38

本文选题：脊神经根 + 大鼠　；参考：《山东大学》2008年博士论文

【摘要】： 目的:神经根牵拉引起的神经功能异常在临床工作中十分常见,如腰椎间盘突出症、臂丛神经损伤等。关于机械牵拉作用对神经组织的研究主要集中在外周神经,以至于很多关于神经根方面的观点都是通过外周神经的研究推论得来。外周神经和神经根在解剖结构和生物力学性质上都有明显不同,所以牵拉神经根出现的功能学和形态学变化也必然与外周神经不同。但是,目前直接利用神经根研究机械牵拉影响还比较少,特别是用不同的程度和速度牵拉神经根研究其出现病理生理学变化还未见报道。因此我们利用生物力学的一些研究方法建立大鼠L5神经根机械牵拉模型,设定不同的程度和速度进行牵拉。然后通过神经电生理的方法研究牵拉后神经根神经功能的变化,并且在完成测试后对神经根进行组织学研究,了解牵拉后神经根的病理变化以及与神经功能改变之间的关系。方法:手术暴露大鼠L5神经根,将神经背根近端切断并连接于Endura-Tec-3200材料测试仪,Wintest软件控制牵拉的长度和速度。高速相机记录神经根标记各点间的运动,记录的图像通过ReadCam软件传输到电脑。Image Express软件分析神经根上标记各点在图像中的位移,计算出牵拉神经根实际牵拉程度。神经根牵拉前后10分钟内,进行神经电生理测试。在暴露的同侧坐骨神经下方放置刺激电极,L5神经背根下放置两个记录电极。分别以0-3V刺激激活神经复合动作电位。获得的信号记录到EGAA系统进行分析。利用神经传导速度、复合神经动作电位波幅的峰值、动作电位曲线下的面积三个指标评价神经根牵拉前后以及不同的牵拉速度和程度对神经功能的影响。电生理实验结束后取L5神经背根进行形态学观察。银浸染色用来观察神经纤维撕裂和纤维之间的空隙。HE染色观察神经根内血管的变化(血管破裂,出血)。另外利用β-APP免疫组织化学染色的方法来评价神经轴突轴浆运输损伤。建立评分系统并分别对三种染色方法进行评分。统计学方法:单因素的方差分析和独立样本的t检验用来分析不同牵拉程度和速度对神经功能和形态学的影响。Logistic回归分析出现神经传导完全阻断的牵拉阈值。同时线性回归分析出现的功能学和形态学变化与牵拉程度、速度之间是否存在线形关系。P＜0.05认为有显著性差异。结果:1.神经电生理结果显示:(1)神经传导速度的降低率随牵拉程度和速度的增加而增加。牵拉程度R＜10%,三种速度(0.01mm/sec,1mm/sec和15mm/sec)神经传导速度降低率分别为42.8±11.5%、46±29.2%、77.1±21.5%;牵拉程度R10-20%时三种速度(同上)牵拉的降低率为77.2±19.8%、94.5±13.5%、95.2±10.8%;牵拉程度为R＞20%时三种速度牵拉均引起神经传导的完全阻断,这时认为神经传导降低率为100%。线性回归分析显示在0.01mm/sec,牵拉程度增加和传导速度减少呈线性关系(R~2=0.714)。(2)Logistic回归分析神经传导功能完全阻断,当R＜20%时与牵拉速度有关。当速度分别为0.01m/sec,1mm/sec和15mm/sec时CV完全阻断(50%occurrence)的牵拉程度分别为16%,10%,9%。(3)随着牵拉程度的增加复合动作电位(CAP)波幅峰值的降低率也逐渐增加,牵拉程度R＜10%,三种速度(0.01mm/sec,1 mm/sec和15mm/sec)CAP波幅峰值的降低率分别为35.8±18.7%、36.5±32.5%、85.6±28.4%;牵拉程度R10-20%时三种速度(同上)牵拉的降低率为66.2±40%、90.3±23.7%、93.5±20.6%;牵拉程度为R＞20%时三种速度牵拉CAP波幅峰值降低率为100%。直线回归分析显示在速度0.01mm/sec时牵拉程度和峰值降低率有线性关系(R~2=0.633)。(4)CAP曲线下面积(AUC)表示当神经受到电刺激后产生动作电位的轴突的数量。AUC的降低率也显示同样的变化。牵拉程度R＜10%,三种速度(0.01mm/sec,1mm/sec,15mm/sec)AUC降低率分别为30.5±5.4%、69±18.6%、81.6±25.2%;牵拉程度R10-20%时三种速度(同上)牵拉的降低率为83.2±16%、98.9±2.6%、98.5±4.7%;牵拉程度为R＞20%时三种速度牵拉AUC均为降低率为100%。直线回归分析显示在速度0.01mm/sec时牵拉程度增加和AUC减低率之间存在线性关系(R~2=0.738)。 2.组织学结果显示:(1)βAPP免疫组织化学。随着牵拉程度和速度增加,βAPP染色阳性率也逐渐增加。正常对照组未发现阳性结果,假手术组染色阳性低于5%。所有被牵拉过的神经根染色均发现βAPP聚集,提示在部分或全部高倍视野下有轴突损伤。牵拉程度R＜10%,三种速度(0.01 mm/sec,1 mm/sec,15mm/sec)βAPP染色的阳性率分别为8.6±4.0%、10.1±6.0%、20.8±4.7%。牵拉程度R10-20%时三种速度(同上)BAPP染色的阳性率分别为30.8±15.7%、47.6±4.1%、51±30%。牵拉程度为R＞20%时三种速度βAPP染色的阳性率分别为61±14.2%、73.4±11.2%、73.7±19.6%。直线回归分析显示βAPP染色阳性率和在三种不同速度时与牵拉程度均存在线性关系(0.01mm/sec,R~2=0.708,1mm/sec,R~2=0.912,15mm/sec,R~2=0.719)。(2)HE染色。HE染色用来观察神经根内是否有血管破裂,提示神经根内是否发生病理性出血。对照组观察到少量的血管破裂但发生率低于5%。假手术组观察到血管破裂,发生率为25%。牵拉程度R＜10%,三种速度(0.01mm/sec,1 m/sec,15 mm/sec)血管破裂率分别为32.6±12.8%、38.2±10.5%、36.6±5.9%;牵拉程度R10-20%时三种速度血管破裂率为52.4±10.6%、61.4±4.7%、62.4±6.5%;牵拉程度为R＞20%时三种速度血管破裂率为57.7±21.9%、79.3±15.3%、89.7±6.9%。直线回归分析发现拉伸程度和血管破裂率之间在速度15mm/sec(R~2=0.7738)和1mm/sec(R~2=0.7692)时存在线性关系。(3)银浸染色观察到正常对照组形态保持完整,神经纤维之间发现有空隙但是发生率低于10%。假手术组形态基本保持完整,偶尔发现有神经纤维断裂和空隙,但发生率分别低于15%和3%。牵拉程度R＜10%,三种速度(0.01mm/sec,1mm/sec,15mm/sec)染色正常率为47.3±25.4%、46.8±26.1%、45.6±18.9%;牵拉程度R10-20%时三种速度染色正常率为40.3±7.7%、29.8±37.2%、17.9±18.4%;牵拉程度为R＞20%时三种速度染色正常率为29.7±14.9%、29.6±16.6%、3.8±5.2%。直线回归分析在速度为0.01mm/sec和1 mm/sec时,牵拉程度和出现空隙之间无线形关系。在速度15mm/sec时有线形关系(R~2=0.488)。神经纤维撕裂和牵拉程度,在速度0.01mm/sec(R~2=0.6108)和15mm/sec(R~2=0.6531)时存在线形关系。结论:建立的大鼠L5神经根牵拉模型可以有效研究神经根在牵拉作用下的损伤机制。特别是在牵拉过程中利用高速相机采集图像可以分析不同时间牵拉程度和力量的变化有助于进行神经根生物力学的分析。利用传导速度,CAP波幅峰值和曲线下面积三个指标分析神经功能增加了神经电生理试验的准确性。实验结果证实神经根牵拉损伤除了与牵拉程度有关外,还与牵拉时的速度有关系。随着牵拉程度和速度的增加,神经功能丧失也逐渐增加。与神经功能研究类似,牵拉后神经根的形态学变化与牵拉速度程度都有关系。说明神经功能丧失在一定程度上是由于神经根形态结构变化引起,但是可能还有其它的机制也起作用,如离子通道和神经受体系统。实验中实验组与假手术组比较差异并不十分明显,因此在下一步关于神经根的病理学研究中还需要进一步探讨。另外我们发现了牵拉后神经根呈现弥散性损伤,该模型造成的轴突损伤与观察到的人脑损伤出现的形态学变化很相似。因此,我们建立的神经根牵拉模型也是研究中枢系统轴突损伤较好的体内研究模型。
[Abstract]:Objective: neural dysfunction caused by nerve root traction is very common in clinical work, such as lumbar intervertebral disc herniation, brachial plexus injury, and so on. The study of nerve tissue is mainly focused on peripheral nerve, so that many points about nerve root are deduced from peripheral nerve. There are obvious differences in the anatomical structure and the biomechanical properties of the peripheral nerve and the nerve root, so the changes of the functional and morphological changes of the traction nerve root are also different from the peripheral nerve. However, the influence of the mechanical traction on the direct use of the nerve root is still less, especially with the different degree and speed of the traction nerve root. The changes in pathophysiology have not been reported. Therefore, we use some methods of biomechanics to establish the mechanical pull model of L5 nerve root in rats, set different degrees and speed to pull. Then, the nerve root nerve function changes after traction are studied by the method of neurophysiology, and the nerve root after the test is completed. Histological study was carried out to understand the relationship between the pathological changes of the nerve roots after traction and the changes of nerve function.
Methods: the L5 nerve root was exposed in the operation. The proximal end of the dorsal root of the nerve was severed and connected to the Endura-Tec-3200 material tester. The length and speed of the traction were controlled by the Wintest software. The motion of the nerve root markers was recorded by the high-speed camera. The recorded images were transferred to the computer.Image Express software to mark the nerve root by ReadCam software. The actual traction of the traction nerve root was calculated with the displacement in the image. The nerve electrophysiological test was performed within 10 minutes of the nerve root before and after traction. The stimulation electrodes were placed under the exposed sciatic nerve and two recording electrodes were placed under the dorsal root of the L5 nerve. The signals obtained by stimulating the active nerve compound action potential were recorded with 0-3V. The EGAA system was analyzed. The nerve conduction velocity, the peak value of the compound nerve action potential wave amplitude and the area under the action potential curve were used to evaluate the effect of the nerve roots before and after traction and the different pulling speed and degree on the nerve function. After the electrophysiological experiment, the dorsal root of the nerve was taken to observe the morphological observation of the dorsal root of the L5 nerve. The silver immersion staining was used. To observe the changes in nerve root blood vessels (vascular rupture, bleeding) by.HE staining of nerve fibers and fiber laceration. In addition, beta -APP immunohistochemical staining was used to evaluate axonal axonal transport damage. Score system was established and three staining methods were scored. Statistical method: single factor prescription Difference analysis and t test of independent samples were used to analyze the effects of different stretch and speed on neural function and morphology..Logistic regression analysis showed the traction threshold of complete blocking of nerve conduction. At the same time, the linear regression analysis showed that there was a linear relationship between the functional and morphological changes and the degree of traction, and the linear relationship between the velocity and the speed was.P < 0.05. There is a significant difference.
Results: 1. the neurophysiological results showed that (1) the reduction rate of nerve conduction velocity increased with the increase of traction degree and speed. The degree of traction was R < 10%, three kinds of velocity (0.01mm/sec, 1mm/sec and 15mm/sec) were 42.8 + 11.5%, 46 + 29.2%, 77.1 + 21.5%, respectively, and traction at R10-20%. The reduction rate was 77.2 + 19.8%, 94.5 + 13.5%, 95.2 + 10.8%, and the stretch degree was R > 20% when three kinds of speed dragging all caused the complete block of nerve conduction. At this time, the reduction rate of nerve conduction was 100%. linear regression analysis showed in 0.01mm/sec, the degree of traction increased and the decrease of conduction velocity was linear (R~2=0.714). (2) Logistic regression analysis of God The conduction function was completely blocked, when the R < 20% was related to the pull speed. When the speed was 0.01m/sec, 1mm/sec and 15mm/sec were respectively, CV completely blocked (50%occurrence) was 16%, 10%, 9%. (3) increased with the degree of traction, and the decrease rate of the amplitude of CAP wave amplitude increased gradually, and the degree of traction was R < 10%, three speed. The reduction rate of the peak amplitude of CAP amplitude (0.01mm/sec, 1 mm/sec and 15mm/sec) was 35.8 + 18.7%, 36.5 + 32.5%, 85.6 + 28.4%, and the reduction rate of the stretch was 66.2 + 40%, 90.3 + 23.7%, 93.5 + 18.7% when the degree of traction was R10-20%, and the decrease rate of CAP wave amplitude was 100%. linear regression analysis when the stretch degree was R > 100%.. There is a linear relationship between the stretch degree and the peak reduction rate at the speed of 0.01mm/sec (R~2=0.633). (4) the area under the CAP curve (AUC) indicates that the reduction rate of the number of.AUC of the axon of the action potential when the nerve is stimulated by electrical stimulation also shows the same change. The stretch degree is R < 10%, and the three kinds of velocity (0.01mm/sec, 1mm/sec, 15mm/sec) AUC reduction rate The difference is 30.5 + 5.4%, 69 + 18.6%, 81.6 + 25.2%, and the reduction rate of three kinds of pullup is 83.2 + 16%, 98.9 + 2.6%, 98.5 + 4.7% when the stretch degree is R10-20%, and the reduction rate of AUC is 100%. linear regression analysis when the stretch degree is R > 69. Sexual relations (R~2=0.738).
2. histological results showed: (1) beta APP immuno histochemistry. With the increase of traction and speed, the positive rate of beta APP staining increased gradually. No positive results were found in the normal control group. The positive staining of the sham operation group was lower than that of the 5%.. The degree of traction was R < 10%, the positive rates of three kinds of velocity (0.01 mm/sec, 1 mm/sec, 15mm/sec) were 8.6 + 4%, 10.1 + 6%, and 20.8 + 4.7%. traction R10-20%, respectively, and the positive rates of three kinds of BAPP staining were 30.8 + 15.7%, 47.6 + 0.01, respectively. No 61 + 14.2%, 73.4 + 11.2%, 73.7 + 19.6%. linear regression analysis showed that there was a linear relationship between the positive rate of beta APP staining and the degree of traction at three different speeds (0.01mm/sec, R~2=0.708,1mm/sec, R~2=0.912,15mm/sec, R~2=0.719). (2) HE staining.HE staining was used to observe whether there was a vascular rupture within the nerve root, indicating whether the nerve roots were within the nerve root. In the control group, the control group observed a small amount of vascular rupture but the incidence of vascular rupture was lower than that of 5%. sham operation group. The incidence of 25%. traction was R < 10%, and the rate of vascular rupture was 32.6 + 12.8%, 38.2 + 10.5%, 36.6 + 5.9%, respectively, at the three kinds of speed (0.01mm/sec, 1 m/sec, 15 mm/sec), and the rupture of blood vessels when the traction degree was R10-20%. The rate was 52.4 + 10.6%, 61.4 + 4.7%, 62.4 + 6.5%, and the rate of vascular rupture was 57.7 + 21.9% at R > 20%, 79.3 + 15.3%, and 89.7 + 6.9%. linear regression analysis showed that there was a linear relationship between the tensile degree and the rupture rate of blood vessels at the speed of 15mm/sec (R~2=0.7738) and 1mm/sec (R~2= 0.7692). The shape of the group remained intact, but the incidence of the nerve fibers was found to be empty but the incidence of the group was less than that of the 10%. sham operation group. Occasionally, the nerve fiber fracture and the gap were found, but the incidence was lower than 15% and 3%. R < 10% respectively. The three kinds of speed (0.01mm/sec, 1mm/ sec, 15mm/sec) were 47.3 + 25.4%, 46.8 + 26.1%, 45.. 6 + 18.9%; the normal rate of three kinds of velocity dyeing at R10-20% was 40.3 + 7.7%, 29.8 + 37.2% and 17.9 + 18.4%, and the normal rate of three velocity dyeing was 29.7 + 14.9% and 17.9 + 18.4% when the traction degree was R > 20%, and when the velocity was 0.01mm/sec and mm/sec, the relationship between the stretch degree and the gap appeared. There is linear relationship (R~2=0.488) at 5mm/sec. The degree of tear and traction of nerve fibers is in line relationship at speed 0.01mm/sec (R~2=0.6108) and 15mm/sec (R~2=0.6531).
Conclusion: the established rat L5 nerve root traction model can effectively study the damage mechanism of nerve root under traction, especially in the process of pulling the image by high-speed camera to analyze the change of traction and strength at different time. It is helpful for the analysis of nerve root biomechanics. Using the conduction velocity and the peak amplitude of CAP amplitude, the peak value of nerve root can be analyzed. The accuracy of neurophysiological tests was increased by analyzing the three indexes of the area under the curve. The results showed that the nerve root traction injury was related to the pulling speed except the degree of traction, and the loss of nerve function increased with the increase of traction degree and speed. The morphological changes of the posterior nerve root are related to the degree of traction speed. It shows that the loss of nerve function is caused by the changes of the morphological structure of the nerve root to a certain extent, but there may be other mechanisms, such as the ion channel and the nervous receptor system. The experimental group is not very different from the sham operation group. Therefore, further discussion is needed in the next histopathological study of nerve roots. In addition, we found that the nerve root causes diffuse damage after traction, and the axon damage caused by this model is similar to the observed morphological changes in the human brain damage. Therefore, the nerve root traction model we established is also the central system. In vivo research model of axon injury.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R-332

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