Real-time in vivo assessment of the nerve microenvironment with coherent anti-Stokes Raman scattering microscopy.

Abstract

BACKGROUND: Current analysis of nerve injury and repair relies largely on electrophysiologic and ex vivo histologic techniques. In vivo architectural assessment of a nerve without removal or destruction of the tissue would greatly assist in the grading of nerve injury and in the monitoring of nerve regeneration over time. Coherent anti-Stokes Raman scattering microscopy is an optical process with particular sensitivity for high-lipid-containing molecules such as myelin. This in vivo nonthermal technique offers high-resolution images that the authors aim to evaluate in both normal and injured nerves. METHODS: A demyelinating crush injury was reproduced in the sciatic nerves of Sprague-Dawley rats (n = 12). Animals were randomized into groups, and coherent anti-Stokes Raman scattering microscopy was undertaken at day 1 and weekly up to 4 weeks after injury. Functional analysis was undertaken weekly and histomorphometry was completed after imaging. RESULTS: All animals demonstrated loss of sciatic nerve function following injury. Recovery was documented, with functional data approaching normal at 3 and 4 weeks. Demyelination was confirmed in nerves up to 2 weeks after injury. Remyelination was observed in the 3-week group and beyond. Imaging of normal nerve revealed structured myelin bundles. These results were consistent with histologic findings that showed a statistically significant improvement in myelination over time. CONCLUSIONS: The authors conclude that coherent anti-Stokes Raman scattering microscopy has the ability to image the peripheral nerve following demyelinating crush injury. This technology, which permits in vivo, real-time microscopy of nerves at a resolution of 5 mum, could provide invaluable diagnostic and prognostic information regarding intraneural preservation and recovery following injury.