Rat Hindlimb amputation model shows analgesia and sexually dimorphic cold hypersensitivity with immediate targeted...

Targeted muscle reinnervation (TMR) has been used clinically to reduce pain in amputees, but its underlying analgesic mechanisms remain unclear. We developed a rat below-knee amputation model to evaluate pain outcomes following immediate TMR (iTMR) in both male and female Sprague-Dawley rats. Animals were randomized to amputation-only or amputation with iTMR, and assessed for reflexive pain behaviors (von Frey, pin, and cold hypersensitivity) and spontaneous pain behaviors (guarding, flinching, and sucrose preference as a measure of anhedonia). Retrograde labeling was used to trace sensory and motor neurons and confirm nerve coaptation patterns. iTMR significantly reduced hyperalgesia and anhedonia compared to amputation-only. By four weeks, pin testing showed decreased responses in iTMR rats (25%) compared to controls (51%, p=0.01), and sucrose preference was higher in iTMR rats (76% vs. 43%, p=0.01). Cold sensitivity responses were reduced in iTMR-treated males (49% vs. 100% in amputation-only males, p<0.001), but not in females, indicating sex-specific differences. Histologic analysis demonstrated neuroma formation in amputation-only rats but not in iTMR rats. Retrograde labeling confirmed both sensory and motor axons entered the motor branch post-iTMR. These findings demonstrate that iTMR provides analgesia and prevents neuroma formation following amputation, with cold hypersensitivity responses differing by sex. This supports the utility of the hindlimb iTMR model and highlights the need for continued mechanistic investigation and sex-specific analyses in future studies. PERSPECTIVE: Using a rat hindlimb amputation model to evaluate iTMR, we demonstrate its potential to mitigate neuropathic pain and symptomatic neuroma formation and reveal sex-specific responses to cold hypersensitivity. These findings indicate the potential clinical utility of iTMR in improving pain outcomes, providing more tailored approaches to pain management in amputees.