Plastic Surgery Research Council
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Presenter: Joanna H Ng, MD
Co-Authors: Jung Y; Keating CP; Senthil-Kumar P; Meppelink AM; Randolph MA; Evans CL; Winograd JM
Massachusetts General Hospital

Purpose: It is well known that peripheral nerve regeneration is slow after insult, causing significant delay in function. Within 12-72 hours, growth occurs proximally and Wallerian degeneration ensues, but the ability to directly visualize individual axonal regeneration in three-dimensional space has been limited by light scattering and minimal depth penetration of current imaging techniques (100-200m maximum). We sought to resolve this dilemma by utilizing a tissue clearing method, previously applied to the central nervous system, which preserves axonal integrity and reduces opacity via chemical replacement of water. This creates the ability to identify individual axons and provide an alternative to traditional immunohistochemical and histomorphometric methods of evaluation. In combination with coherent anti-stokes Raman scattering (CARS) microscopy, a multifaceted image of both myelin sheaths and axons can be generated.

Methods: Twenty transgenic neural tissue gfp-1 and wild type rats underwent sciatic nerve transection and repair with standard microsurgery. Nerve restoration was evaluated 2, 4, 7 days, and 1 month after repair by walking track analysis, histology, and direct visualization after tissue clearing. Nerves were imaged using Fluoview 1000 and Image J on a time-course scale 5mm proximal and distal to the repair site with CARS and confocal fluorescence microscopy. The extensor digitorum longus muscle was also imaged for axonal connectivity at the neuromuscular junction in transgenic rats.

Results: Tissue clearing improved nerve depth penetration to >1000m, revealing a high resolution whole-nerve image of axons, including the regeneration front and Wallerian degenerative environment. Combining CARS myelin imaging and confocal axonal imaging with tissue clearing in whole nerve preparations is currently in progress.

Conclusions: This novel application of powerful imaging technologies (CARS and confocal microscopy) with tissue clearing will establish a three-dimensional imaging modality which will increase our understanding of the events of Wallerian degeneration and nerve regeneration.

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