Rat Hindlimb Below-Knee Amputation Model and Immediate Targeted Muscle Reinnervation

Amputation-related pain reduces patient quality of life, contributes to opioid use, and can reduce functional rehabilitation and prosthesis use. Preclinical models that reliably produce post-amputation pain while preserving mobility are essential for studying the biological mechanisms underlying these outcomes. Here, we present a detailed, reproducible, rodent surgical protocol using Sprague-Dawley rats to model hindlimb below-knee amputation. The procedure involves isolating and transecting the sciatic nerve branches, including the tibial, common peroneal, and sural nerves, producing sustained post-amputation nerve pathology and pain-related behaviors. Rats recover well from this procedure and can ambulate immediately post-op with no difficulty eating or drinking. Animals can subsequently be evaluated with reflexive and spontaneous pain measures. The pain behaviors resulting from this procedure are robust over time, and standard reflexive pain behaviors can be obtained from the stump. Importantly, preserved mobility enables longitudinal behavioral testing without confounding motor impairment. While this protocol is centered on amputation, it may be readily adapted to incorporate targeted muscle reinnervation (TMR) as a post-amputation intervention by coapting transected nerves to nearby motor branches of the nerves to the biceps femoris and semimembranosus, enabling investigation of reinnervation interventions and their effects on post-amputation pain. We have successfully used this model previously to evaluate sex-specific differences in pain behaviors following amputation and to study the effects of TMR. This protocol provides a robust and flexible platform for investigating amputation-related pain, neuroma formation, and functional outcomes, with applicability to translational studies aimed at improving post-amputation pain.