Optical Microscopy of the Peripheral Nerve following Injury; An In Vivo Structural and Quantitative Analysis of Myelination.
Francis P. Henry, M.D., Boris H. Park, PhD, Daniel Cote, PhD, David Wes, B.A., Mark A. Randolph, M.A.S., Irene E. Kochevar, PhD, Charles P. Lin, PhD, Johannes F. deBoer, PhD, Jonathan M. Winograd, M.D..
Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Electrophysiological and invasive ex vivo histological techniques remain the current method for assessing nerve injury. In vivo assessment without destruction of the tissue would advance both grading and monitoring following neural injury. CARS Microscopy is an optical microscope with sensitivity for high lipid containing molecules such as myelin. OCT functions in a method analogous to ultrasound where instead of soundwaves light is used to provide cross sectional images with the same scale and geometry as histology. These minimally invasive, non-thermal techniques offer high resolution images of neural microarchitecture, which we evaluated in both normal and injured nerves.
A demyelinating crush injury was reproduced in the sciatic nerves of Sprague Dawley rats (n=54). Animals were randomized into groups and both CARS microscopy and OCT imaging were undertaken at weeks 1, 2, 3 and 4 following injury. The uninjured nerve was used as a control. Functional analysis was undertaken weekly by calculation of a sciatic function index. Histomorphometry was undertaken following imaging to verify our findings.
All animals demonstrated loss of nerve function following injury. Recovery was documented with sciatic functional index data approaching normal at three weeks. Structural analysis using CARS Microscopy revealed demyelination in nerves up to two weeks post injury with remyelination in the three week group and beyond (Figure 1), consistent with histological findings. OCT analysis allowed for a direct non destructive quantitative analysis of the nerve resulting in a predictive scatter plot (Figure 2a & 2b) from which myelin thickness and G-Ratio of nerve can be estimated in vivo.
CARS Microscopy and OCT can be used to image the peripheral nerve following demyelinating injury in both structural and quantitative manners. These novel technologies which permit in vivo, real time microscopy of nerves provide invaluable diagnostic and prognostic information regarding intraneural preservation and recovery.