MD-PhD Student Department of Translational Neuroscience, Barrow Neurological Institute, Phoenix, AZ; Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE
Disclosure(s):
Margaret Mary McCann, MS: No financial relationships to disclose
Introduction: Peripheral nerve injury can cause devastating, lifelong disability and occurs in up to 5% of level 1 trauma patients [1]. Severe injuries require surgery, but up to 40% fail [2]. Early detection and correction of failed surgeries is crucial to long-term recovery. Current biomarkers (e.g., electrodiagnostics) are limited prior to end-organ reinnervation, hampering our ability to detect failed repairs early after surgery. Diffusion Tensor Imaging (DTI) is an MRI method that provides specific biomarkers that correlate with injury severity and de/regeneration [3], however, systematic validation in the presence of spatially heterogenous pathologies is lacking. The objective of this study is to develop novel 3D histology methods in peripheral nerves for this purpose.
Methods: Healthy rat sciatic nerves (N=20) were harvested from the hindlimb to systematically optimize whole-mount immunolabeling protocols (e.g., fixation times, methods to enhance penetration/uniformity). For the final protocol, nerve structure was protected with intramolecular epoxide linkages [4] prior to being cleared and fluorescently labeled for myelin/neurofilament using SDS mediated immunolabeling [5], which prevents binding during the penetration process and yields more uniform labeling. Labeled nerves were then whole mounted using a custom-built device and imaged at 15x on a confocal microscope (Nikon A1R). Finally, images were obtained at multiple locations/depths and stitched together to form a 3D volume.
Results: Preliminary results showed robust immunolabeling and improved uniform penetration of fluorescent markers throughout healthy nerves compared to published protocols [6,7]. These data will be correlated with ex vivo DTI for validation purposes following sham, cut/repair, and crush procedures.
Conclusion : We predict DTI biomarkers will identify sciatic nerve injury/recovery more specifically than behavioral measures, and that DTI acquired four weeks after injury (after Wallerian degeneration is complete) will predict 12-week recovery outcomes, results which the 3D immunolabeling will confirm.
