The Northeastern Society of Plastic Surgeons

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3-Dimensional reconstruction of peripheral nerve regeneration using serial section electron microscopy: a new method of analyzing axonal regeneration.
Jonathan Leckenby, MD1, Jeff Lichtman, MD, PhD2, Adriaan Grobbelaar, MD, FRCS(Plast)3.
1URMC, Rochester, NY, USA, 2Harvard University, Boston, NY, USA, 3The Royal Free Hosptial, London, United Kingdom.

Introduction: Peripheral nerve assessment has traditionally been studied through histological and immunological staining techniques in a limited cross-sectional modality. The introduction of transgenic species, such as YFP-H mice, has greatly increased our ability to observe axonal regeneration and subjectively comment on behaviour. However, detailed analysis is still difficult to assess with either of these methods and understanding cellular interactions is almost impossible. A new application of serial section electron microscopy (SSEM) is presented to overcome these limitations. Methods: Direct nerve repairs (DNR) were performed on the posterior auricular nerve of transgenic YFP-H mice. Six weeks post-operatively the nerves were imaged using confocal fluorescent microscopy then excised and embedded in resin. Resin blocks were sequentially sectioned at 100nm and sections were serially imaged with an electron microscope (Magellan 400L, FEI). Images were aligned and auto-segmented to allow for 3D reconstruction. Results: Basic morphometry and axonal counts were fully automated. Using full 3D reconstructions, the relationships between the axons, the Nodes of Ranvier, and Schwann cells could be fully appreciated. The quality of regeneration could be examined throughout the entire dataset providing a comprehensive analysis. The interactions of individual axons with their surrounding environment could be visualised and explored in a virtual three dimensional space. Conclusions: SSEM allows the pathway of the regenerating axon to be visualized in detailed through a 3D virtual space. Fully automated histo-morphometry can now give accurate axonal counts, provide information regarding the quality of nerve regeneration throughout the whole dataset volume, and reveal the cell-to-cell interaction at a super-resolution scale. It is possible to fully visualize and ‘fly-through’ the nerve to help understand the behavior of a regenerating axon within its environment. Having established this technique, it provides future opportunities to evaluate the affect treatment modalities have on the neuro-regenerative potential and help us understand the impact different surgical techniques have when treating nerve injuries.


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