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Three-Dimensional Wet-Mount Imaging of Peripheral Nerves by Two-Photon Microscopy: A Transgenic Murine Model with High-Throughput Automated Histomorphometric Assessment
Nate Jowett, MD; Ronit Malka, SB
Harvard Medical School, Boston, MA
Background: Traditional resin-embedded bright-field microscopy approaches to neural histomorphometric assessment are time consuming, necessitate a high-level of technical skill, and are fraught with bias due to sampling errors and limited resolution. Herein is described a high-resolution wet-mount model of peripheral nerve regeneration.
Method & Materials: Transgenic mice expressing yellow-fluorescent protein at high-levels uniquely in motor and sensory neurons (B6.Cg-Tg(Thy1-YFP)16Jrs/J, Joshua R. Sanes, The Jackson Laboratory) underwent unilateral transection and suture repair of the buccal branch of the facial nerve. Control- and injured-side nerves were harvested at 1, 3, 5, 7, 14, and 21 days, formalin-fixed, and three-dimensionally imaged using two-photon microscopy with broadband excitation between 1 and 1.04 µm and a long-pass dichroic mirror at 525 nm. An algorithm was developed using Imaris software (v 8.0, Bitplane Inc.) for automated calculation of histomorphometric parameters including axon counts, axon diameters, myelin thickness, and axon volumetric density.
Results: Three-dimensional reconstructions with sub-micron resolution capable of resolving small and large fiber axons were obtained in high-throughput fashion. Histomorphometric outcomes were calculated in automated fashion using the entire neural volume over the region of interest to eliminate sampling bias.
Conclusions: A high-throughput, label-free transgenic murine model of neural regeneration that allows for automated histomorphometric assessment at high resolution in three-dimensions with minimal bias has been described.
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