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March 7th, 2025
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Medical College of Wisconsin
animal behavior and cognition
biorxiv

Rat Hindlimb Amputation Model Shows Analgesia and Sexually Dimorphic Cold Hypersensitivity with Immediate Targeted Muscle Reinnervation

Zepeda, J. L.Open in Google Scholar•Mraz, G.Open in Google Scholar•Roth, E.Open in Google Scholar•Weihrauch, D.Open in Google Scholar•Hoben, G.Open in Google Scholar

Introduction: Targeted muscle reinnervation (TMR) has been used clinically to reduce pain in amputees but the mechanisms producing analgesia are unknown. Here we employ rat hindlimb amputation as an appropriate model to evaluate algogenic processes affected by TMR, performed at the time of amputation in subjects of both sex. Methods: Adult male and female Sprague-Dawley rats were assigned to amputation-only or amputation + immediate TMR (iTMR). Reflexive pain behaviors (von Frey, pin, and cold hypersensitivity testing) and spontaneous pain behaviors (guarding, flinching, anhedonia from loss of sucrose preference) were assessed. Retrograde labeling distinguished sensory and motor neurons and identified nerve coaptation sites. Results: iTMR reduced hyperalgesia and anhedonia that followed amputation. By 4 weeks, pin testing showed reduced pain responses in iTMR rats (25%) versus amputation-only (51%, p=0.01). Sucrose preference testing revealed reduced anhedonia in iTMR rats (76% vs. 43%, p=0.01). Response to cold diminished in male iTMR rats (49% vs. 100% in amputation-only males, p<0.001), but not in females. Histology showed neuroma formation in controls but not after iTMR. Both sensory and motor fibers entered the motor branch after iTMR. Conclusions: These findings show iTMR prevents neuroma formation and provides analgesia after amputation and, while its effect on cold hypersensitivity is sexually dimorphic. This supports further investigation into TMR analgesic mechanisms, the utility of the hindlimb amputation model, and the need for sex-specific evaluations.

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