Second Harmonic Generation Imaging as a Virtual Biopsy for Upper Extremity Nerve Injuries: A Cadaver Study

A timely assessment of nerve damage is needed for early clinical decision-making. Second Harmonic Generation (SHG) microscopy visualizes collagen. This study investigated: Can SHG imaging distinguish collagenous substructures in human nerves? Can SHG imaging detect structural damage in human nerves following destructive stretch injury?

Six human upper extremities were equally divided into 2 groups: A no-injury and a load-to-failure (LTF) group. The median, radial, and ulnar nerves were surgically exposed. Arms were placed on an Instron material testing system. Eight centimeters of each nerve was secured under 2 pins. A hook was raised from beneath the nerve until complete nerve transection occurred. After the experiment, LTF nerves were excised along their full length. No-injury and LTF nerves were placed in isotonic saline under an FVMPE-RS Multiphoton Microscope using a laser wavelength of 900 nm to induce an SHG signal. Z-stack images were acquired using a wavelength of 45 nm. Nerves were then harvested for histology.

Histology sections of NI nerves exhibited normal peripheral nerve architecture. All collagenous substructures visible on histology were clearly identifiable and distinguishable on SHG images of uninjured nerves. In LTF nerves, epineurium rupture and exposed fascicles were clearly identifiable on SHG imaging. Epineurial collagen of LTF nerves appeared heavily disorganized, with short fiber fragments following no clear trajectory. Findings were consistent among nerve types.

This is the first study to visualize human nervous tissue using SHG microscopy. Second Harmonic Generation imaging offers detailed visualization of all collagenous substructures of peripheral nerves and detects structural damage, like epineurial collagen-disorganization, and exposure of individual fascicles in unprecedented detail.