小脑轴突的激光Nanosurgery<em>在体内</em
Summary
双光子成像，其耦合到激光nanodissection，是有用的工具来研究退化和再生过程中对中枢神经系统具有亚细胞分辨率。该协议显示了如何标记，图像，并剖析单个登山纤维在体内小脑皮质。
Abstract
Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.
Introduction
从机械性损伤，有毒侮辱或者神经退行性疾病导致轴突切断术之后通常是从细胞体分离10-13轴突的远端部分变性。除了 少数例外2,7,14,15，在成年动物的中枢神经系统轴突切断通常无法激活再生程序16。
鲜为人知的是，在细胞和亚细胞水平退化性事件的实时动态。的新策略，用于限制神经元损伤，促进神经元的再生的发展需要，作为第一步，阐明由奇受伤的神经细胞退化和再生的机制。本研究是最直接处理通过监测体内的单个神经元的动态。而单光子荧光成像技术是将可见光的强烈散射的限制，双光子激发到达里深皮质层已经小鼠的亚细胞分辨率3,4,17。利用转基因小鼠中，荧光蛋白选择性地表达于神经元18-20的亚群，TPF显微镜已经发展在体内 21,22中应用到突触可塑性和轴突伸长的探索。奇受损神经元的T到他的能力再生后的损伤可通过耦合在体内监测通过双光子成像损伤的专门针对感兴趣的轴突的模型研究。飞秒激光脉冲的多光子吸收被用于破坏单 个树突甚至单棘5,23。此外，这种损伤模式允许单个切割轴突分支，而不会中断接触枝晶6。在解剖，允许特定的神经元群的特征再生轴突一旦受损，小脑登山纤维（CFS）的情况下是一个有用的模型SINCE他们保留显着的塑料性能，即使在成年动物24,25损伤后。最近，CF的长期成像表明，这些轴突能够再生长在遵循激光干切断6的日子。
本协议描述了如何通过顺行示踪标记olivocerebellar神经元及其轴突的伸长率。一旦所感兴趣的神经元被荧光标记，它们可以被重复地用于下一个窗口颅周或数月的任意时间点进行监测。由激光干切断体内解剖单个轴突分枝的要领进行说明。
这里介绍的技术提供了新的见解体内轴突重塑的机制，可能有助于治疗策略的开发，以限制神经元变性和促进轴突再生。
Protocol
Representative Results
Discussion
这个协议说明如何来标记下橄榄的神经元用荧光染料。随后，为了在小脑皮层进行颅窗口的方法进行说明。这种技术提供的光纤接入到olivocerebellar神经元，攀登纤维的端部。不幸的是，这两个标签和开颅手术的结果是相当低的，即使在熟练的操作人员的手（通常为1出3只小鼠的标记，和1出3颅窗1-2周后依然清晰）。
在活小鼠进行激光干切断的程序，然后提交。这种方法允许在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission’s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the “Stem Cells Call for Proposals.” This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by “Ente Cassa di Risparmio di Firenze”.
Materials
| Lab standard stereotaxic, rat and mouse | Stoelting | 51670 | |
| Borosilicate glass with filament | Sutter Instrument Inc | BF100-50-10 | |
| Germinator 500 (Glass bead sterilizer) | Roboz | ||
| Microinjection dispense system | Picospritzer | ||
| Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R) | Warner Instruments | 64-0720 | |
| Ti:Sapphire laser, 120 fs width pulses, 90 MHz repetition rate | Coherent | Chameleon | |
| Spongostan, haemostatic sponge | Ferrosan | MS0005 | |
| Galvanometric mirrors | GSI Lumonics | VM500+ | |
| Objective | Olympus | XLUMPLFLN 20XW | |
| Piezoelectric stage | Physik Instrumente | P-721 | |
| Photomultiplier modules | Hamamatsu Photonics | H7710-13 | |
| LabVIEW System Design Software | National Instruments | ||
| Voren, 1 mg/ml (dexamethasone-21- isonicotinate) | Boheringer Ingelheim | ||
| Rymadil (carprofen) | Pfizer | ||
| Lidocaine clorohydrate 2% | ATI | ||
| Alexa488 dextran | Life Technologies | D22910 |
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