In Vivo Second Harmonic Generation Microscopy as a Novel Assessment of Nerve Damage

Abstract

Grant support received from: 2017 AFSH AFSH Basic Science Grant Royalty: Smith and Nephew (Hausman) Receipt of Intellectual Property Rights: Checkpoint Surgical (Hausman) Consulting Fee: Checkpoint Surgical (Hausman) Speaker's Bureau: Skeletal Dynamics, TriMed (Hausman) Ownership Interest: Checkpoint Surgical, NDI Medical (Hausman) We hypothesize second harmonic-generation (SHG) microscopy can be utilized as an in vivo, real-time imaging modality of peripheral nerve structural damage due to a stretch injury. Furthermore, we seek to validate these findings by using intraoperative nerve stimulation as a functional assessment of nerve damage. Following IACUC approval, Sprague-Dawley rats (N=5) were anesthetized and micro dissection was performed to separate median nerves from surrounding tissue. A biphasic handheld surgical nerve stimulator was used to probe median nerves before and after intervention to assess nerve function by monitoring for a twitch of the paw. 1cm of the right median nerve was placed in a custom-made stretch applicator and a 20% strain was applied to the nerve for 5 minutes, simulating a high-strain injury (HS). Left median nerves served as a sham control (SC), being treated exactly the same with the exception of applying a strain. Subjects were then brought to the microscopy core facility where they were imaged in vivo using a multiphoton microscope. In vivo SHG images of both HS and SC nerves demonstrated the ability to visualize epineurial collagen fibers. All (5 of 5) HS nerves at day 0 demonstrated undulating wavy fibers with general disorganization and large amounts of fiber crossing (Figure 46-1B). Conversely, 5 of 5 SC nerves at day 0 demonstrated intact, parallel, linearly organized collagen structure with no evidence of crossing fibers (Figure 46-1A). At 84 days postinjury, 4/5 HS nerves demonstrated complete recovery validated by linearly organized, parallel collagen fibers (Figure 46-1D). Nerve stimulation data corroborated with patterns of collagen structure observed via SHG microscopy. All nerves (HS and SC) required the same baseline stimulation value of 0.025 μC to produce a twitch of the paw. Following injury, HS nerves required a mean increase of 0.135 μC to induce a twitch (P = .0053) (Figure 46-2A) while SC nerves required no increase in stimulation. Comparing day 0 postintervention values with day 84 values, 4 of 5 HS nerves demonstrated complete recovery of function with a mean recovery of –0.12 μC (P = .0283) (Figure 46-2B). No SC nerves required an increase in stimulation at post-sham day 0 and day 84 conditions. •In vivo SHG microscopy provides high-resolution real-time visual assessment of nerve damage.•Intraoperative nerve stimulation is a novel and accurate functional assessment of nerve damage in animal models.•Epineurial collagen organization may be a reliable marker of nerve health and function.Figure 46-2AB(A) Day 0 intraoperative nerve stimulation data indicates no change in pre- and postsham values in SC nerves. A statistically significant increase in stimulation was observed between pre- and poststretch values in HS nerves. (B) A comparison of intraoperative nerve stimulation values at postintervention day 0 and at day 84 indicates a statistically significant recovery of stimulation values in HS nerves. No change was observed in SC nerves.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) This research was supported by a Basic Science Grant from the American Foundation for Surgery of the Hand.