Second Harmonic Generation Imaging Reveals Extent of Damage in Acute Nerve Stretch Injuries in Rats

In-continuity nerve injuries often result in poor long-term outcomes, as recovery remains unpredictable in the acute setting. Second-harmonic generation (SHG) imaging offers a label-free method to visualize collagen fibers intraoperatively. We investigated whether intraoperative SHG imaging can be used to distinguish two degrees of acute stretch injury, epineuroclasis and endoneuroclasis, in a rat model.

Forty-five adult Sprague-Dawley rats were randomized into two groups: epineuroclasis (n = 22) and endoneuroclasis (n = 23). A stretch injury was applied to the left median nerve, the right served as a sham-control. Before and after injury, nerve function was evaluated via electrical stimulation thresholds. Intraoperative SHG imaging was performed to visualize the collagenous framework of the nerve.

SHG imaging revealed three distinct zones in injured nerves from proximal to distal: a zone with an exposed endoneurial core, a transition zone showing epineurial disruption, and an epineurial zone displaying disorganized, wavy collagen, despite an intact epineurium. Endoneuroclasis demonstrated worse endoneurial damage than epineuroclasis. Following epineuroclasis, stimulation thresholds increased from median 25 nanocoulomb (nC) to 100 nC (range 50-300 nC) after injury. The threshold following endoneuroclasis increased from 25 nC-300 nC (range 100-400 nC), demonstrating a greater reduction of conductivity (difference 200 nC, P < 0.001).

Intraoperative SHG imaging effectively reveals epineurium rupture and endoneurial collagen disorganization. It allows visual evaluation of the extent of structural damage and thus injury severity. These findings highlight the potential of SHG imaging as an intraoperative diagnostic tool that could guide surgical decision-making in managing acute nerve trauma.