5. A Novel Animal Model of Hindlimb Amputation to Evaluate Chronic Pain

PURPOSE: Over 150,000 individuals undergo amputation in the United States each year, many of whom experience significant physical impairments and impaired quality of life. Following amputation, effective pain management and functional recovery remains challenging, highlighting the need for practical animal models investigating the pathophysiology of postamputation pain. Current animal models and assessment strategies often inadequately replicate the complexities of human amputations, limiting the development of effective therapeutic interventions. In this study, we established a novel animal model of hindlimb amputation to induce neuroma associated stump pain, simulating above-knee-amputation. METHODS: Eighteen rats were randomly assigned to one of three groups (n=6/group): (1) amputation, (2) tibial neuroma creation without amputation, and (3) sham. Following identification, isolation, and transection of the tibial, common peroneal, sural, and saphenous nerves, hindlimb amputation was created at the knee joint. Myodesis was performed, and transected nerves were attached to the skin for accurate pain measurement. Weekly pain assessments, including mechanical hypersensitivity, neuroma site pain, and cold and thermal allodynia were conducted for 8 weeks. Immunohistochemistry was used to evaluate the architecture of the distal nerve and neuroma. RESULTS: Rats that underwent amputation exhibited significantly greater mechanical pain at the stump compared to pain levels in the hind paw of both the control and tibial neuroma groups. No significant differences in mechanical hypersensitivity were observed between the control and tibial neuroma groups (p>0.05). Neuroma formation was observed in both the amputation and tibial neuroma groups, resulting in marked pain at the neuroma site compared to controls. Rats in the amputation and tibial neuroma groups developed significant cold hypersensitivity by postoperative week 1 compared to the control group. Amputation led to late-onset thermal hypersensitivity on day 28, which was absent in the tibial neuroma and control groups. Immunohistochemical analysis showed disorganized axonal sprouting at week 1, with neuroma formation observed by week 3. CONCLUSION: Our model provides a robust tool for accurate investigation of the impact of the innovative strategies on postamputation pain, including Regenerative Peripheral Nerve Interface (RPNI) and Targeted Muscle Reinnervation (TMR).