应用荧光剂Neuro-DiI（DiO）示踪周围神经再生及端侧吻合神经来源的实验研究
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摘要
神经再生的客观指标包括三个方面:一是神经轴突连续性的恢复,二是神经电生理的恢复,三是神经功能的恢复。临床上需要通过一种检测方法能在活体微创、直观、客观、尽早地显示周围神经损伤及再生的情况,为指导临床的诊断及治疗提供准确客观的依据。神经轴突连续性的恢复是神经电生理及功能恢复的基础和前提,所以神经形态学的检测与神经电生理及行为学相比有不可比拟的优越性。但常规的神经形态学检测需要特异的免疫组化反应通过组织学切片来观察。我们的目的是利用能在周围神经中运输的示踪剂标记神经并反应神经轴浆流的情况,且能通过相应的仪器在活体动物的体外直接检测到神经形态变化。现今发现的能在体外检测到的示踪剂只有两大类,一种是放射性核素,一种是荧光剂。放射性核素与氨基酸结合后可以随着轴浆流转运,直观地观测神经形态的改变,但不能标记神经。荧光剂的检测主要是通过荧光显微镜发出激发波后接受荧光剂的反射而产生图像,利用图像分析系统在电脑上直接成像。羰化青类荧光剂有高度的亲脂性,本实验利用Neuro-DiI或DiO注射到周围神经干内,观察荧光剂对神经纤维的标记及在周围神经中运输的情况,从而评价神经损伤及再生的程度;在高倍及低倍荧光镜下观察示踪剂在正常神经、损伤神经及再生神经的标记及运输情况,研究Neuro-DiI及Neuro-DiO在周围神经中运输的机制、特性及代谢情况;并利用不同颜色的羰化青类示踪剂直观观察不同损伤类型、不同修复方式神经再生情况、再生神经纤维的来源;同时直观地评价神经再生促进因素(促红细胞生成素)对周围神经再生的影响,并与传统的神经行为学、电生理、组织学的检测加以对比,为临床上能够早期准确地判断神经损伤及再生的程度提供可靠的理论和实验依据。
Objectives:The goal of the present work was to develop minimally invasive,direct,objective,imaging techniques to monitor local injury and regeneration of peripheral nerves as ealier as possible and to provide the accurate proof for directing the next diagnosis and treatment associated with that nerve. At present there are two kinds of nerve tracing that can be detect in vitro.one is radionuclide,the other is fluorescer , radionuclides can not detect the morphology changes of nerve .The way of detection of fluorescer is by imaging excitation wave invented by fluorescence microscope to produce image that is analysed by computer. a lipophilic, fluorescence probe (DiI and DiO) would move along the nerve’s membrane following a direct injection into the sciatic nerve and to produce the nerve image directly.
Methods: The sciatic nerve model was applied to Sprague–Dawley rats to detect the tracer transportation in normal ,injury and regeneration nerves and to develop the mechanism and character of tracer transportation .two different color fluorescence probes were applied to detect the scource of regeneration of end-to- side anastomosis and to evaluate the influence of EPO on peripheral nerve regeneration . conventional behavioral testing, histological staining and Electrophysiology are also detected as the method in contrast at the same time.
Results: Neuro-DiI and Neuro-DiO for have the axonal specificity in peripheral nerve that provide the effective developing method in peripheral injury and regeneration in non-destructive manner.nerve tracer can reflect the immediate axoplasma flow to provide the direct nerve imaging index.
Conclusions:The tracer can persist 3 months in vivo Without interfering the nerve regerneration and fluorescence alternation ,this character can provide the nerve regeneration index directly. If Electrophysiology are detected at the same time ,we can get the accurate and complete information for nerve injury and regeneration .
Objectives:The goal of the present work was to develop minimally invasive,direct,objective,imaging techniques to monitor local injury and regeneration of peripheral nerves as ealier as possible and to provide the accurate proof for directing the next diagnosis and treatment associated with that nerve. At present there are two kinds of nerve tracing that can be detect in vitro.one is radionuclide,the other is fluorescer , radionuclides can not detect the morphology changes of nerve .The way of detection of fluorescer is by imaging excitation wave invented by fluorescence microscope to produce image that is analysed by computer. a lipophilic, fluorescence probe (DiI and DiO) would move along the nerve’s membrane following a direct injection into the sciatic nerve and to produce the nerve image directly.
Methods: The sciatic nerve model was applied to Sprague–Dawley rats to detect the tracer transportation in normal ,injury and regeneration nerves and to develop the mechanism and character of tracer transportation .two different color fluorescence probes were applied to detect the scource of regeneration of end-to- side anastomosis and to evaluate the influence of EPO on peripheral nerve regeneration . conventional behavioral testing, histological staining and Electrophysiology are also detected as the method in contrast at the same time.
Results: Neuro-DiI and Neuro-DiO for have the axonal specificity in peripheral nerve that provide the effective developing method in peripheral injury and regeneration in non-destructive manner.nerve tracer can reflect the immediate axoplasma flow to provide the direct nerve imaging index.
Conclusions:The tracer can persist 3 months in vivo Without interfering the nerve regerneration and fluorescence alternation ,this character can provide the nerve regeneration index directly. If Electrophysiology are detected at the same time ,we can get the accurate and complete information for nerve injury and regeneration .
引文
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