本文選題：脊神經根 + 大鼠�。� 參考：《山東大學》2008年博士論文

【摘要】： 目的:神經根牽拉引起的神經功能異常在臨床工作中十分常見,如腰椎間盤突出癥、臂叢神經損傷等。關于機械牽拉作用對神經組織的研究主要集中在外周神經,以至于很多關于神經根方面的觀點都是通過外周神經的研究推論得來。外周神經和神經根在解剖結構和生物力學性質上都有明顯不同,所以牽拉神經根出現的功能學和形態(tài)學變化也必然與外周神經不同。但是,目前直接利用神經根研究機械牽拉影響還比較少,特別是用不同的程度和速度牽拉神經根研究其出現病理生理學變化還未見報道。因此我們利用生物力學的一些研究方法建立大鼠L5神經根機械牽拉模型,設定不同的程度和速度進行牽拉。然后通過神經電生理的方法研究牽拉后神經根神經功能的變化,并且在完成測試后對神經根進行組織學研究,了解牽拉后神經根的病理變化以及與神經功能改變之間的關系。 方法:手術暴露大鼠L5神經根,將神經背根近端切斷并連接于Endura-Tec-3200材料測試儀,Wintest軟件控制牽拉的長度和速度。高速相機記錄神經根標記各點間的運動,記錄的圖像通過ReadCam軟件傳輸到電腦。Image Express軟件分析神經根上標記各點在圖像中的位移,計算出牽拉神經根實際牽拉程度。神經根牽拉前后10分鐘內,進行神經電生理測試。在暴露的同側坐骨神經下方放置刺激電極,L5神經背根下放置兩個記錄電極。分別以0-3V刺激激活神經復合動作電位。獲得的信號記錄到EGAA系統(tǒng)進行分析。利用神經傳導速度、復合神經動作電位波幅的峰值、動作電位曲線下的面積三個指標評價神經根牽拉前后以及不同的牽拉速度和程度對神經功能的影響。電生理實驗結束后取L5神經背根進行形態(tài)學觀察。銀浸染色用來觀察神經纖維撕裂和纖維之間的空隙。HE染色觀察神經根內血管的變化(血管破裂,出血)。另外利用β-APP免疫組織化學染色的方法來評價神經軸突軸漿運輸損傷。建立評分系統(tǒng)并分別對三種染色方法進行評分。統(tǒng)計學方法:單因素的方差分析和獨立樣本的t檢驗用來分析不同牽拉程度和速度對神經功能和形態(tài)學的影響。Logistic回歸分析出現神經傳導完全阻斷的牽拉閾值。同時線性回歸分析出現的功能學和形態(tài)學變化與牽拉程度、速度之間是否存在線形關系。P＜0.05認為有顯著性差異。 結果:1.神經電生理結果顯示:(1)神經傳導速度的降低率隨牽拉程度和速度的增加而增加。牽拉程度R＜10%,三種速度(0.01mm/sec,1mm/sec和15mm/sec)神經傳導速度降低率分別為42.8±11.5%、46±29.2%、77.1±21.5%;牽拉程度R10-20%時三種速度(同上)牽拉的降低率為77.2±19.8%、94.5±13.5%、95.2±10.8%;牽拉程度為R＞20%時三種速度牽拉均引起神經傳導的完全阻斷,這時認為神經傳導降低率為100%。線性回歸分析顯示在0.01mm/sec,牽拉程度增加和傳導速度減少呈線性關系(R~2=0.714)。(2)Logistic回歸分析神經傳導功能完全阻斷,當R＜20%時與牽拉速度有關。當速度分別為0.01m/sec,1mm/sec和15mm/sec時CV完全阻斷(50%occurrence)的牽拉程度分別為16%,10%,9%。(3)隨著牽拉程度的增加復合動作電位(CAP)波幅峰值的降低率也逐漸增加,牽拉程度R＜10%,三種速度(0.01mm/sec,1 mm/sec和15mm/sec)CAP波幅峰值的降低率分別為35.8±18.7%、36.5±32.5%、85.6±28.4%;牽拉程度R10-20%時三種速度(同上)牽拉的降低率為66.2±40%、90.3±23.7%、93.5±20.6%;牽拉程度為R＞20%時三種速度牽拉CAP波幅峰值降低率為100%。直線回歸分析顯示在速度0.01mm/sec時牽拉程度和峰值降低率有線性關系(R~2=0.633)。(4)CAP曲線下面積(AUC)表示當神經受到電刺激后產生動作電位的軸突的數量。AUC的降低率也顯示同樣的變化。牽拉程度R＜10%,三種速度(0.01mm/sec,1mm/sec,15mm/sec)AUC降低率分別為30.5±5.4%、69±18.6%、81.6±25.2%;牽拉程度R10-20%時三種速度(同上)牽拉的降低率為83.2±16%、98.9±2.6%、98.5±4.7%;牽拉程度為R＞20%時三種速度牽拉AUC均為降低率為100%。直線回歸分析顯示在速度0.01mm/sec時牽拉程度增加和AUC減低率之間存在線性關系(R~2=0.738)。 2.組織學結果顯示:(1)βAPP免疫組織化學。隨著牽拉程度和速度增加,βAPP染色陽性率也逐漸增加。正常對照組未發(fā)現陽性結果,假手術組染色陽性低于5%。所有被牽拉過的神經根染色均發(fā)現βAPP聚集,提示在部分或全部高倍視野下有軸突損傷。牽拉程度R＜10%,三種速度(0.01 mm/sec,1 mm/sec,15mm/sec)βAPP染色的陽性率分別為8.6±4.0%、10.1±6.0%、20.8±4.7%。牽拉程度R10-20%時三種速度(同上)BAPP染色的陽性率分別為30.8±15.7%、47.6±4.1%、51±30%。牽拉程度為R＞20%時三種速度βAPP染色的陽性率分別為61±14.2%、73.4±11.2%、73.7±19.6%。直線回歸分析顯示βAPP染色陽性率和在三種不同速度時與牽拉程度均存在線性關系(0.01mm/sec,R~2=0.708,1mm/sec,R~2=0.912,15mm/sec,R~2=0.719)。(2)HE染色。HE染色用來觀察神經根內是否有血管破裂,提示神經根內是否發(fā)生病理性出血。對照組觀察到少量的血管破裂但發(fā)生率低于5%。假手術組觀察到血管破裂,發(fā)生率為25%。牽拉程度R＜10%,三種速度(0.01mm/sec,1 m/sec,15 mm/sec)血管破裂率分別為32.6±12.8%、38.2±10.5%、36.6±5.9%;牽拉程度R10-20%時三種速度血管破裂率為52.4±10.6%、61.4±4.7%、62.4±6.5%;牽拉程度為R＞20%時三種速度血管破裂率為57.7±21.9%、79.3±15.3%、89.7±6.9%。直線回歸分析發(fā)現拉伸程度和血管破裂率之間在速度15mm/sec(R~2=0.7738)和1mm/sec(R~2=0.7692)時存在線性關系。(3)銀浸染色觀察到正常對照組形態(tài)保持完整,神經纖維之間發(fā)現有空隙但是發(fā)生率低于10%。假手術組形態(tài)基本保持完整,偶爾發(fā)現有神經纖維斷裂和空隙,但發(fā)生率分別低于15%和3%。牽拉程度R＜10%,三種速度(0.01mm/sec,1mm/sec,15mm/sec)染色正常率為47.3±25.4%、46.8±26.1%、45.6±18.9%;牽拉程度R10-20%時三種速度染色正常率為40.3±7.7%、29.8±37.2%、17.9±18.4%;牽拉程度為R＞20%時三種速度染色正常率為29.7±14.9%、29.6±16.6%、3.8±5.2%。直線回歸分析在速度為0.01mm/sec和1 mm/sec時,牽拉程度和出現空隙之間無線形關系。在速度15mm/sec時有線形關系(R~2=0.488)。神經纖維撕裂和牽拉程度,在速度0.01mm/sec(R~2=0.6108)和15mm/sec(R~2=0.6531)時存在線形關系。 結論:建立的大鼠L5神經根牽拉模型可以有效研究神經根在牽拉作用下的損傷機制。特別是在牽拉過程中利用高速相機采集圖像可以分析不同時間牽拉程度和力量的變化有助于進行神經根生物力學的分析。利用傳導速度,CAP波幅峰值和曲線下面積三個指標分析神經功能增加了神經電生理試驗的準確性。實驗結果證實神經根牽拉損傷除了與牽拉程度有關外,還與牽拉時的速度有關系。隨著牽拉程度和速度的增加,神經功能喪失也逐漸增加。與神經功能研究類似,牽拉后神經根的形態(tài)學變化與牽拉速度程度都有關系。說明神經功能喪失在一定程度上是由于神經根形態(tài)結構變化引起,但是可能還有其它的機制也起作用,如離子通道和神經受體系統(tǒng)。實驗中實驗組與假手術組比較差異并不十分明顯,因此在下一步關于神經根的病理學研究中還需要進一步探討。另外我們發(fā)現了牽拉后神經根呈現彌散性損傷,該模型造成的軸突損傷與觀察到的人腦損傷出現的形態(tài)學變化很相似。因此,我們建立的神經根牽拉模型也是研究中樞系統(tǒng)軸突損傷較好的體內研究模型。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R-332

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