面神经缺损修复以及神经元凋亡及其调控的实验研究
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摘要
目的:面神经损伤后神经元凋亡及基因调控,与面神经缺损的修复是面神经损伤目前研究的二大热点。本研究探讨利用神经再生室以及组织工程技术修复兔面神经缺损的可行性及优越性,寻找一种新的,简单、有效修复长段神经缺损的手术方法。同时研究大鼠面神经总干压榨伤及总干切断伤后,其面神经元的形态学改变及凋亡相关基因caspase 3、caspase 8、cyto-c、bcl-2和p53的表达变化及其相互关系,并探讨面神经损伤后胶质细胞源性神经营养因子(GDNF)和米诺环素对其神经元的保护及其功能恢复的作用。
方法:本研究分二大部分四个实验,第一部分为再生室及组织工程修复兔面神经缺损的实验研究。手术制作兔面神经下颊支15mm缺损模型,分别用不同方法修复:几丁聚糖—胶原再生室双桥接小间隙组、双桥接无间隙组、翻转静脉复合异种去细胞神经基质桥接修复组以及原位神经吻合组。术后通过大体观察、神经电生理检测、组织学观察以及图像分析来评价神经再生恢复情况。第二部分为面神经损伤后诱导面神经元凋亡及相关基因表达的实验研究。通过制作大鼠面神经总干切断伤、不同损伤程度的总干压榨伤、切断伤局部使用GDNF及总干压榨伤和总干切断伤分别口服米诺环素组。通过大体观察了解不同程度压榨伤神经功能恢复情况,组织学观察及透射电镜观测术后各期实验组与正常对照组面神经元的形态学变化。免疫组织化学法、图像分析技术检测实验组及正常对照组面神经元内bcl-2、p53、caspase 3、caspase 8和cyto-c的基因表达变化。
结果:在实验时间段内,(1)几丁聚糖—胶原再生室吸收明显且无异物反应,能抑制神经纤维瘤形成,可为神经再生提供良好的微环境;(2)双桥接小间隙与双桥接无间隙组均能有效修复长段面神经缺损,其神经传导速度与原位神经吻合组无差异;(3)双桥接修复组轴突再生率与原位吻合组无差别,但再生轴突成熟度和有髓神经面积恢复率略低于原位吻合组;(4)翻转静脉复合异种去细胞神经基质桥接修复面神经缺损取得了与自体神经移植几乎相同的修复效果。异种去细胞神经基质组织相容性良好,无明显排异反应:(5)不同损伤程度面神经总干压榨伤后,其神经元反应不同,严重压榨伤可引起神经元死亡;(6)神经总干切断伤会引起神经元死亡,于术后21-28d时达到死亡高峰,神经元数目明显少于正常对照组;(7)面神经损伤后各蛋白表达与神经损伤形式有关。面神经干切断21d时BCL-2/P53比值达最低,切断侧神经元死亡率与BCL-2/P53的比值有明显相关性。Caspase 8,Cyto-c蛋白表达与Caspase 3蛋白表达相关;(8)局部应用GDNF对面神经切断伤后面运动神经元有明显的保护作用;(9)口服米诺环素对压榨伤后的面神经有明显的恢复作用,对总干切断伤后的面神经无恢复作用,但对总干切断伤后的面神经元有显著的保护作用。
结论:(1)几丁聚糖—胶原再生室生物相容性良好,促神经再生作用肯定,适于体内植入修复神经缺损;(2)双桥接技术修复长段神经缺损方法简单、效果肯定,双桥接无间隙组和双桥接小间隙组修复兔的面神经缺损效果无差别;(3)异种神经去细胞处理后可成为低抗原性的神经支架桥接体。异种神经去细胞基质复合翻转静脉修复兔面神经缺损效果肯定,无排异反应发生,其修复效果与自体神经移植相同;(4)面神经元是否发生凋亡以及凋亡数目与其神经干受损伤形式有关,严重压榨伤会引起面神经元死亡,而切断伤明显会引起面神元死亡;(5)损伤后Caspase3、8和Cyto-c蛋白表达增高与面神经损伤形式有关,Cyto-c表达与Caspase 3表达相关。BCL-2、P53蛋白参与面神经总干切断后诱导其神经元凋亡过程的调控,BCL-2/P53的比值变化决定面神经元的凋亡;(6)GDNF局部应用对切断伤后两周内的面运动神经元有明显的保护作用;(7)早期连续口服米诺环素有助于单纯压榨伤的面神经功能恢复,且对面神经切断后的神经元有显著保护作用。
Objective: To evaluate the feasibility and the superiority of repairing the longer facial nerve defects in rabbits by regenerating chambers and compound bridge grafts which made of acellular rat sciatic nerves and autogenous inside-out veins, and to find a simple and effective method for treatment of long nerve defects, which could be widely performed by surgeons. Meanwhile, to investigate the morphology of facial motoneurons and the expression of caspase 3、caspase 8、cyto-c、Bcl-2 and p53 following the facial nerve crush or transection at the level of the stylomastoid foramen in SD rats. At the same time, to assess the effect of Minocycline and GDNF in the function recovery of the facial nerve and the protection to its motoneurons after facial nerve injury.
Methods: The study included two parts: The first part is an Experimental Study of Repairing Facial Nerve Defect by Regenerating Chambers and Tissue engineering in Rabbits and the second part is a Study on Facial Motoneurons Death and its Regulatory Gene Expression Following Facial Nerve Injury.
In the first study, a 15mm nerve defect was created at the marginal mandibular branch of the facial nerve in rabbits, and then the defects were treated by different methods. Including, (1)chitolsan-collagen chambers which included double-bridging with little gap, (2)double-bridging without gap, and (3)combined bridge grafting which was made of acellular rat sciatic nerves by chemical extraction and autogenous inside-out vein, (4)nerve autografts and (5)normal control.
In the second study, the facial nerve trunks were transected distally or crushed with different severities in the SD rats, the GDNF was applied locally for the transection injury group and the Minocycline was taken orally for the both groups. Gross observation, electrophysiologic evaluation, histologic morphometrical analysis and transmission electron microscope were performed. Moreover, the expression of Caspase 3、Caspase 8、Cyto-c、Bcl-2 and P53 protein in facial motoneurons was analyzed by immunohistochemical staining.
Results: (1) chitosan-collagen chambers were obviously degraded in 12 weeks postoperatively and there was no foreign body reaction over all experimental periods. What's more, they also restrained the formation of neuroma and provided a good microcircumambience for nerve regeneration. (2)The nerve regeneration was good in double-bridging groups. There was no significant difference between the double-bridging and nerve autografts groups, but they all did not recover to the level before the surgery. (3)There was no significant difference between double-bridging groups and nerve autografts group according to the rate of regeneration of myelinated axon, the double-bridging groups were slightly lower than the nerve autografts group in the recovery rate and the maturity degree of myelinated axon. (4)The compound bridge did not show severe inflammatory reaction and provided a better microenvironment for nerve regeneration. (5)The result of facial nerve repairing was the same between the combined bridge and nerve autografts groups. (6)Different facial nerve crushes caused different results in the death of facial motoneurons; Severe nerve crushes resulted in death of facial motoneurons. (7) The loss of motoneurons was peaked in 21-28d after injuries and was mainly through by apoptotic pathway. The number of motoneurons loss in the transection group was larger than the crush group. (8) The Minocycline could promote the rapid recovery of crushed facial nerve. In addition, the protective effect of the Minocycline and GDNF for the facial motoneurons was detected in transection group. (9)The expression of Bcl-2 and P53 following facial nerve transaction was significantly higher than those of normal control group. (10)Caspase 3、8 protein and Cyto-c protein expressions were observed in wide spread areas of normal rat facial nucleus. Cells of the transection group stained more intense than that of crush group. Expressions of Cyto-c protein were correlated with expression of Caspase 3 protein.
Conclusion: (1) The Double-bridging technique is proved as a simple and effective method for treatment of the long nerve defect. (2) The Chitosan-collage chamber shows the excellent biocompatibility in repairing of the nerve defects. There was no significant difference between the double-bridging with little gap group and double-bridging without gap group. (3)After acellularization by chemical extraction, the acellular heterogeneous nerve matrix has become a low antigenicity xenograft; the compound bridge provides a better microenvironment for nerveregeneration without severe inflammatory reaction. (4)There is a correlation between the facial motoneurons apoptosis and the patterns of facial nerve injuries. A severe nerve crush or a nerve transaction can cause the death of motoneurons. (5) The Minocycline can promote the rapid recovery of the crushed facial nerve; Both GDNF and Minocycline obviously prevent facial motoneurons from dying after transaction. (6)Bcl-2 and P53 genes might play a great role in the process of facial motoneurons apoptosis following facial nerve transection, the ratio of Bcl-2 / P53 may control facial motoneurons apoptosis. (7)The expressions of Caspase 3、8 and Cyto-c proteins were related with facial nerve injury severities. Cyto-c proteins' expressions were correlated with caspase 3 protein's expression.
方法:本研究分二大部分四个实验,第一部分为再生室及组织工程修复兔面神经缺损的实验研究。手术制作兔面神经下颊支15mm缺损模型,分别用不同方法修复:几丁聚糖—胶原再生室双桥接小间隙组、双桥接无间隙组、翻转静脉复合异种去细胞神经基质桥接修复组以及原位神经吻合组。术后通过大体观察、神经电生理检测、组织学观察以及图像分析来评价神经再生恢复情况。第二部分为面神经损伤后诱导面神经元凋亡及相关基因表达的实验研究。通过制作大鼠面神经总干切断伤、不同损伤程度的总干压榨伤、切断伤局部使用GDNF及总干压榨伤和总干切断伤分别口服米诺环素组。通过大体观察了解不同程度压榨伤神经功能恢复情况,组织学观察及透射电镜观测术后各期实验组与正常对照组面神经元的形态学变化。免疫组织化学法、图像分析技术检测实验组及正常对照组面神经元内bcl-2、p53、caspase 3、caspase 8和cyto-c的基因表达变化。
结果:在实验时间段内,(1)几丁聚糖—胶原再生室吸收明显且无异物反应,能抑制神经纤维瘤形成,可为神经再生提供良好的微环境;(2)双桥接小间隙与双桥接无间隙组均能有效修复长段面神经缺损,其神经传导速度与原位神经吻合组无差异;(3)双桥接修复组轴突再生率与原位吻合组无差别,但再生轴突成熟度和有髓神经面积恢复率略低于原位吻合组;(4)翻转静脉复合异种去细胞神经基质桥接修复面神经缺损取得了与自体神经移植几乎相同的修复效果。异种去细胞神经基质组织相容性良好,无明显排异反应:(5)不同损伤程度面神经总干压榨伤后,其神经元反应不同,严重压榨伤可引起神经元死亡;(6)神经总干切断伤会引起神经元死亡,于术后21-28d时达到死亡高峰,神经元数目明显少于正常对照组;(7)面神经损伤后各蛋白表达与神经损伤形式有关。面神经干切断21d时BCL-2/P53比值达最低,切断侧神经元死亡率与BCL-2/P53的比值有明显相关性。Caspase 8,Cyto-c蛋白表达与Caspase 3蛋白表达相关;(8)局部应用GDNF对面神经切断伤后面运动神经元有明显的保护作用;(9)口服米诺环素对压榨伤后的面神经有明显的恢复作用,对总干切断伤后的面神经无恢复作用,但对总干切断伤后的面神经元有显著的保护作用。
结论:(1)几丁聚糖—胶原再生室生物相容性良好,促神经再生作用肯定,适于体内植入修复神经缺损;(2)双桥接技术修复长段神经缺损方法简单、效果肯定,双桥接无间隙组和双桥接小间隙组修复兔的面神经缺损效果无差别;(3)异种神经去细胞处理后可成为低抗原性的神经支架桥接体。异种神经去细胞基质复合翻转静脉修复兔面神经缺损效果肯定,无排异反应发生,其修复效果与自体神经移植相同;(4)面神经元是否发生凋亡以及凋亡数目与其神经干受损伤形式有关,严重压榨伤会引起面神经元死亡,而切断伤明显会引起面神元死亡;(5)损伤后Caspase3、8和Cyto-c蛋白表达增高与面神经损伤形式有关,Cyto-c表达与Caspase 3表达相关。BCL-2、P53蛋白参与面神经总干切断后诱导其神经元凋亡过程的调控,BCL-2/P53的比值变化决定面神经元的凋亡;(6)GDNF局部应用对切断伤后两周内的面运动神经元有明显的保护作用;(7)早期连续口服米诺环素有助于单纯压榨伤的面神经功能恢复,且对面神经切断后的神经元有显著保护作用。
Objective: To evaluate the feasibility and the superiority of repairing the longer facial nerve defects in rabbits by regenerating chambers and compound bridge grafts which made of acellular rat sciatic nerves and autogenous inside-out veins, and to find a simple and effective method for treatment of long nerve defects, which could be widely performed by surgeons. Meanwhile, to investigate the morphology of facial motoneurons and the expression of caspase 3、caspase 8、cyto-c、Bcl-2 and p53 following the facial nerve crush or transection at the level of the stylomastoid foramen in SD rats. At the same time, to assess the effect of Minocycline and GDNF in the function recovery of the facial nerve and the protection to its motoneurons after facial nerve injury.
Methods: The study included two parts: The first part is an Experimental Study of Repairing Facial Nerve Defect by Regenerating Chambers and Tissue engineering in Rabbits and the second part is a Study on Facial Motoneurons Death and its Regulatory Gene Expression Following Facial Nerve Injury.
In the first study, a 15mm nerve defect was created at the marginal mandibular branch of the facial nerve in rabbits, and then the defects were treated by different methods. Including, (1)chitolsan-collagen chambers which included double-bridging with little gap, (2)double-bridging without gap, and (3)combined bridge grafting which was made of acellular rat sciatic nerves by chemical extraction and autogenous inside-out vein, (4)nerve autografts and (5)normal control.
In the second study, the facial nerve trunks were transected distally or crushed with different severities in the SD rats, the GDNF was applied locally for the transection injury group and the Minocycline was taken orally for the both groups. Gross observation, electrophysiologic evaluation, histologic morphometrical analysis and transmission electron microscope were performed. Moreover, the expression of Caspase 3、Caspase 8、Cyto-c、Bcl-2 and P53 protein in facial motoneurons was analyzed by immunohistochemical staining.
Results: (1) chitosan-collagen chambers were obviously degraded in 12 weeks postoperatively and there was no foreign body reaction over all experimental periods. What's more, they also restrained the formation of neuroma and provided a good microcircumambience for nerve regeneration. (2)The nerve regeneration was good in double-bridging groups. There was no significant difference between the double-bridging and nerve autografts groups, but they all did not recover to the level before the surgery. (3)There was no significant difference between double-bridging groups and nerve autografts group according to the rate of regeneration of myelinated axon, the double-bridging groups were slightly lower than the nerve autografts group in the recovery rate and the maturity degree of myelinated axon. (4)The compound bridge did not show severe inflammatory reaction and provided a better microenvironment for nerve regeneration. (5)The result of facial nerve repairing was the same between the combined bridge and nerve autografts groups. (6)Different facial nerve crushes caused different results in the death of facial motoneurons; Severe nerve crushes resulted in death of facial motoneurons. (7) The loss of motoneurons was peaked in 21-28d after injuries and was mainly through by apoptotic pathway. The number of motoneurons loss in the transection group was larger than the crush group. (8) The Minocycline could promote the rapid recovery of crushed facial nerve. In addition, the protective effect of the Minocycline and GDNF for the facial motoneurons was detected in transection group. (9)The expression of Bcl-2 and P53 following facial nerve transaction was significantly higher than those of normal control group. (10)Caspase 3、8 protein and Cyto-c protein expressions were observed in wide spread areas of normal rat facial nucleus. Cells of the transection group stained more intense than that of crush group. Expressions of Cyto-c protein were correlated with expression of Caspase 3 protein.
Conclusion: (1) The Double-bridging technique is proved as a simple and effective method for treatment of the long nerve defect. (2) The Chitosan-collage chamber shows the excellent biocompatibility in repairing of the nerve defects. There was no significant difference between the double-bridging with little gap group and double-bridging without gap group. (3)After acellularization by chemical extraction, the acellular heterogeneous nerve matrix has become a low antigenicity xenograft; the compound bridge provides a better microenvironment for nerveregeneration without severe inflammatory reaction. (4)There is a correlation between the facial motoneurons apoptosis and the patterns of facial nerve injuries. A severe nerve crush or a nerve transaction can cause the death of motoneurons. (5) The Minocycline can promote the rapid recovery of the crushed facial nerve; Both GDNF and Minocycline obviously prevent facial motoneurons from dying after transaction. (6)Bcl-2 and P53 genes might play a great role in the process of facial motoneurons apoptosis following facial nerve transection, the ratio of Bcl-2 / P53 may control facial motoneurons apoptosis. (7)The expressions of Caspase 3、8 and Cyto-c proteins were related with facial nerve injury severities. Cyto-c proteins' expressions were correlated with caspase 3 protein's expression.
引文
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