Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows
Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows
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DOI：
10.1113/jphysiol.2012.230532
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发表时间：
2012-08-01
影响因子：
5.5
通讯作者：
Debarbieux, Franck
中科院分区：
文献类型：
--
作者：
Fenrich, Keith K.;Weber, Pascal;Debarbieux, Franck
成年哺乳动物脊髓的重复体内双光子成像，亚细胞分辨率，将是至关重要的了解正常和病理条件下的细胞机制。目前的方法是有限的，因为它们需要每次成像手术。在这里，我们报告了一个简单的玻璃窗方法，避免重复手术和随后的炎症。我们应用这种策略，通过对成年转基因小鼠进行多色成像，跟踪轴突完整性和炎症反应。我们发现玻璃窗对轴突数量或结构没有显著影响，引起短暂的炎症反应，并显着增加体内脊髓成像的吞吐量。此外，我们使用这种技术来跟踪收缩/变性和再生的切断轴突后，“针刺”的脊髓损伤具有较高的时间保真度。我们发现，再生轴突可以在4天内穿过损伤部位，并且它们的末端在损伤后数周内发生显著的形态学变化。总的来说，该技术可能适用于评估正常和患病脊髓中的细胞功能和治疗策略。
Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a 'pin-prick' spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.