Imaging peripheral nerve regeneration using serial section electron microscopy: thinking like an axon
Jonathan I Leckenby, MBBS, BSc, MRCS; University College of London, London, United Kingdom; Adriaan O Grobbelaar, MBChB, MMed(Plast.), FCS(SA)(Plast.), FRCS(Plast.); The Royal Free Hospital, 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. However, detailed analysis is still difficult to assess with either of these methods. 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 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 interactions of individual axons with their surrounding environment could be visualised and explored in a virtual environment.
Conclusions: SSEM allows the detailed pathway of the regenerating axon to be visualised in a 3D virtual space. Fully automated histo-morphometry can now give accurate axonal counts and provide information regarding the quality of nerve regeneration. It is possible to fully visualise and ‘fly-through’ the regenerating nerve to help understand the behaviour of an axon with its environment.
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