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Real-Time Visual Assessments of Nerve Damage: Second Harmonic Generation Microscopy as a Novel Imaging Modality
Matthew J Gluck, BS1; Christina M Beck, PhD2; Todd A Rubin, MD1; Paul J Cagle, MD1; Michael R Hausman, MD1; (1)Mount Sinai Hospital, New York, NY, (2)Icahn School of Medicine- Mount Sinai, New York, NY

Introduction: Orthopaedic injuries of the extremities are often complicated by confounding peripheral nerve trauma. Damaged nerves that appear macroscopically intact or exhibit an incomplete EMG disturbance pose the biggest clinical dilemma. This leaves care providers unable to predict the degree to which an injured nerve will recover, potentially delaying necessary surgical intervention. Second Harmonic Generation Microscopy (SHG) may provide a novel, reliable, real-time assessment of epineurial collagen damage, allowing surgeons to accurately predict the degree of axonal damage present in peripheral nerves. In this study, we demonstrate the utility of SHG microscopy to detect nerve damage in vivo.

Materials and Methods: Right and left median nerves of 16 Sprague-Dawley rats (n=16) were carefully exposed using micro-surgical techniques. Using a custom made stretch applicator right median nerves were stretched to 20% corresponding to a high strain injury (HS) and held for 5 minutes, while left median nerves (SC) served as a sham control, only being placed in the applicator for 5 minutes with no stretch applied. Subjects were then placed directly on a microscope stage and imaged using a multi-photon laser scanning microscope with a 25x/1.05 numerical aperture water immersion multi-photon lens. All rats were imaged at day 0 (immediately after injury), at which point half were sacrificed for conventional histology, and half were imaged again at 1 week following the injury, before being sacrificed for conventional histology.

Results: Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. HS nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers, with crossing fibers and tears, as well as a decrease in linear organization. At 1 week post injury, SC nerves demonstrated the same organization at later time points, while HS nerves exhibited increasing structural changes including increased fiber gapping, suggesting the presence of Wallerian degeneration as correlated to an increase in digestion chambers visible in histological sections.

Conclusion: SHG microscopy may offer the ability to detect not only an acute neural stretch injury, but also assess the longitudinal structural changes associated with Wallerian degeneration. SHG microscopy has demonstrated its utility as a tool for surgeons to predict the probability of spontaneous recovery, and thus allow for earlier intervention.


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