Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury

Published:November 07, 2022DOI:


      Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage in vivo in an animal model.


      Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope.


      Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw.


      Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function.

      Clinical relevance

      In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

      Key words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Hand Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Cartwright M.S.
        • Chloros G.D.
        • Walker F.O.
        • Wiesler E.R.
        • Campbell W.W.
        Diagnostic ultrasound for nerve transection.
        Muscle Nerve. 2007; 35: 796-799
        • Menorca R.M.
        • Fussell T.S.
        • Elfar J.C.
        Nerve physiology: mechanisms of injury and recovery.
        Hand Clin. 2013; 29: 317-330
        • Carter G.T.
        • Robinson L.R.
        • Chang V.H.
        • Kraft G.H.
        Electrodiagnostic evaluation of traumatic nerve injuries.
        Hand Clin. 2000; 16 (1–vii)
        • Campbell W.W.
        Evaluation and management of peripheral nerve injury.
        Clin Neurophysiol. 2008; 119: 1951-1965
        • Grinsell D.
        • Keating C.P.
        Peripheral nerve reconstruction after injury: a review of clinical and experimental therapies.
        BioMed Res Int. 2014; 2014: 698256
        • Haftek J.
        Stretch injury of peripheral nerve. Acute effects of stretching on rabbit nerve.
        J Bone Joint Surg Br. 1970; 52: 354-365
        • Lorei M.P.
        • Hershman E.B.
        Peripheral nerve injuries in athletes. Treatment and prevention.
        Sports Med. 1993; 16: 130-147
        • Rosberg H.E.
        • Carlsson K.S.
        • Hojgard S.
        • Lindgren B.
        • Lundborg G.
        • Dahlin L.B.
        Injury to the human median and ulnar nerves in the forearm–analysis of costs for treatment and rehabilitation of 69 patients in southern Sweden.
        J Hand Surg Br. 2005; 30: 35-39
        • Robinson L.R.
        Traumatic injury to peripheral nerves.
        Suppl Clin Neurophysiol. 2004; 57: 173-186
        • Sunderland S.
        A classification of peripheral nerve injuries producing loss of function.
        Brain. 1951; 74: 491-516
        • Lee S.K.
        • Wolfe S.W.
        Peripheral nerve injury and repair.
        J Am Acad Orthop Surg. 2000; 8: 243-252
        • Robinson L.R.
        Predicting recovery from peripheral nerve trauma.
        Phys Med Rehabil Clin N Am. 2018; 29: 721-733
        • Robinson L.R.
        Traumatic injury to peripheral nerves.
        Muscle Nerve. 2000; 23: 863-873
        • Seddon H.J.
        Three types of nerve injury.
        Brain. 1943; 66: 237-288
        • National Institute of Neurological Disorders and Stroke
        Neurological Diagnostic Tests and Procedures. NIH publication 19-NS-5380.
        (Published April 10, 2020)
        Date accessed: November 8, 2021
        • Stoll G.
        • Wilder-Smith E.
        • Bendszus M.
        Imaging of the peripheral nervous system.
        Handb Clin Neurol. 2013; 115: 137-153
        • Ohana M.
        • Moser T.
        • Moussaouï A.
        • et al.
        Current and future imaging of peripheral nervous system.
        Diagn Interv Imaging. 2014; 95: 17-26
        • Toros T.
        • Karabay N.
        • Ozaksar K.
        • Sugun T.S.
        • Kayalar M.
        • Bal E.
        Evaluation of peripheral nerves of the upper limb with ultrasonography: a comparison of ultrasonographic examination and the intra-operative findings.
        J Bone Joint Surg Br. 2009; 91: 762-765
        • Ashwell G.J.
        • Jefferies G.
        • Hamilton D.G.
        • et al.
        Strong second-harmonic generation from centrosymmetric dyes.
        Nature. 1995; 375: 385-388
        • Li A.D.
        • Liu W.C.
        Optical properties of ferroelectric nanocrystal/polymer composites.
        in: Tjong S.C. Mai Y.W. Physical Properties and Applications of Polymer Nanocomposites. Woodhead Publishing, 2010: 108-158
        • Campagnola P.J.
        • Loew L.M.
        Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms.
        Nat Biotechnol. 2003; 21: 1356-1360
        • Sinclair E.B.
        • Andarawis-Puri N.
        • Ros S.J.
        • Laudier D.M.
        • Jepsen K.J.
        • Hausman M.R.
        Relating applied strain to the type and severity of structural damage in the rat median nerve using second harmonic generation microscopy.
        Muscle Nerve. 2012; 46: 899-907
        • Zipfel W.R.
        • Williams R.M.
        • Christie R.
        • Nikitin A.Y.
        • Hyman B.T.
        • Webb W.W.
        Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.
        Proc Natl Acad Sci U S A. 2003; 100: 7075-7080
        • Brown C.P.
        • Houle M.A.
        • Popov K.
        • et al.
        Imaging and modeling collagen architecture from the nano to micro scale.
        Biomed Opt Express. 2013; 5: 233-243
        • Gluck M.J.
        • Vijayaraghavan S.
        • Sinclair E.B.
        • Ashraf A.
        • Hausman M.R.
        • Cagle P.J.
        Detecting structural and inflammatory response after in vivo stretch injury in the rat median nerve via second harmonic generation.
        J Neurosci Methods. 2018; 303: 68-80
        • Vijayaraghavan S.
        • Huq R.
        • Hausman M.R.
        Methods of peripheral nerve tissue preparation for second harmonic generation imaging of collagen fibers.
        Methods. 2014; 66: 246-255
        • Jager S.B.
        • Ronchi G.
        • Vaegter C.B.
        • Geuna S.
        The mouse median nerve experimental model in regenerative research.
        Biomed Res Int. 2014; 2014: 701682
        • Ronchi G.
        • Gambarotta G.
        • Di Scipio F.
        • et al.
        ErbB2 receptor over-expression improves post-traumatic peripheral nerve regeneration in adult mice.
        PloS One. 2013; 8: e56282
        • Wang H.
        • Sorenson E.J.
        • Spinner R.J.
        • Windebank A.J.
        Electrophysiologic findings and grip strength after nerve injuries in the rat forelimb.
        Muscle Nerve. 2008; 38: 1254-1265