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Intraoperative Second Harmonic Generation Imaging Detects Structural Damage Associated with Poor Long-Term Outcomes After Stretch Injury in the Rat Median Nerve.
Christoph A Schroen, BS
1; Paul J Cagle, MD
2; Michael R. Hausman, MD
31Icahn School of Medicine at Mount Sinai, New York, NY; 2Mount Sinai Hospital, New York, NY; 3Orthopaedic Surgery, Mount Sinai Medical Center, New York, NY
Introduction: Recovery after peripheral nerve injury remains inherently unpredictable. This study investigates whether intraoperative Second Harmonic Generation (SHG) Imaging can detect and distinguish damage to major anatomical substructures of the rat median nerve and visualize long-term structural changes during nerve recovery.
Methods: Upon IACUC approval, 33 Sprague-Dawley rats were assigned to four groups: two mild-strain injury (MS) (n=8/group) and high-strain injury (HS) groups (n=8&9) with follow-up timepoints at 6- and 12-weeks. Left median nerves were injured, right served as sham-controls (SC). Nerves were exposed and stretched using a hook attached to a load-cell until a first (MS) or second (HS) rapid force reduction occurred.
After injury and at follow-up, rats were placed under an Olympus FVMPE-RS multiphoton microscope, exposing 1.5cm of nerve to a laser-wavelength of 910nm, whereupon SHG-signals were captured at 455nm. Multiple 54µm deep z-stack images were acquired along the nerve from proximal to distal. Conductivity was assessed using a Checkpoint nerve stimulator inducing digit movement post-injury and at 12-weeks.
Results: On day-0, SHG-Imaging of both strain levels revealed three distinct injury-zones: A zone with continuous epineurium, an area exhibiting epineurium rupture, and a zone showing exposed endoneurium. Both injury levels displayed epineurium rupture. The endoneurial SHG-Signal was substantially weaker than that of the epineurium. HS-nerves exhibited larger areas of exposed endoneurium, accompanied by endoneurial fiber disruptions. Epineurial collagen was heavily disorganized in both injuries, with short fiber fragments near the tear-zone and wavy, misaligned fibers further distal. SC-nerves exhibited normal nerve architecture. At 6- and 12-weeks post-injury, both strain levels persistently exhibited similar injury-zones on SHG-Imaging, indicative of no structural recovery and no closure of the epineurial gap at 12-weeks. HS-nerves exhibited a neuroma-like architecture at 6- and 12-weeks. HS-nerves required more electrical stimulation than MS-nerves on day-0 and at 12-weeks (p<0.01). Conductivity of MS-nerves improved after 12 weeks (p=0.04), but remained below pre-injury values (p<0.01). HS-nerves showed no functional recovery at 12-weeks (p=0.7727).
Conclusion: SHG-Microscopy offers precise differentiation between damage to the epineurium and endoneurium, detecting disruption of both structures as well as more subtle damage like fiber-disorganization and endoneurial tube disruption while macroscopic nerve continuity is maintained. Both injury severities lead to long-term impairment of nerve function and can be detected by intraoperative SHG-Imaging on the day of injury.
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