Purpose: Following peripheral nerve injury, denervated tissues undergo several progressive degenerative processes that impede regeneration and minimize the degree of functional return achievable on reinnervation. Our group has previously demonstrated the individual efficacy of IGF-1 nanoparticles (IGF-1 NPs) and agrin nanoparticles (agrin NPs) in improving functional recovery following nerve injury. We hypothesize IGF-1 and agrin will have synergistic benefits in promoting functional recovery as IGF-1 accelerates axonal regeneration and preserves Schwann cells and myocytes while agrin preserves neuromuscular junction morphology and promotes receptivity to reinnervation. This study aimed to evaluate the efficacy of co-treatment with IGF-1 NPs and agrin NPs in a rodent chronic denervation model.
Methods: IGF-1 and agrin were encapsulated in biodegradable NPs and then embedded within a hyaluronic acid/PCL nanofiber hydrogel composite (NHC). The effects of locally delivered treatment were assessed using a chronic denervation nerve injury rodent model in which the median nerve was denervated for 12 weeks until ulnar-to-median nerve transfer at Week 0. An additional control group received sham surgery instead of median nerve denervation. Following nerve transfer, all animals underwent 16 weeks of regeneration and grip strength assessment. Animals were injected with 1) IGF-1/agrin NP co-treatment, 2) IGF-1 NP, 3) agrin NP, 4) free IGF-1/agrin, 5) empty NP, or 6) saline (No Denervation) every six weeks from time of median nerve injury until Week 12. To identify the optimal timing of co-treatment, two groups alternated treatment during denervation and regeneration between either IGF-1/agrin NP co-treatment or IGF-1 NP. Upon sacrifice at Week 16, flexor muscles, median nerve, cervical DRGs, and palmar cutaneous skin were harvested for histologic analyses.
Results: All nanoparticle-encapsulated drug treated animals exhibited increased functional recovery compared to free IGF-1/agrin co-treatment (p<0.01, 2-way ANOVA) and empty-NP treated animals (p<0.001 2-way ANOVA), with the greatest benefit seen in the full IGF-1/agrin NP co-treatment group. At Week 16, full IGF-1/agrin NP co-treatment animals demonstrated an increase in functional recovery of 36.5% compared to empty-NP. Full IGF-1/agrin NP co-treatment and Denervation IGF-1/Reinnervation Co-treatment groups demonstrated greater functional recovery than IGF-1 NP only and agrin NP only groups. All IGF-1/agrin NP treated animals demonstrated significantly greater NMJ reinnervation than empty NP animals, with the greatest benefit seen in the Full IGF-1/agrin NP co-treatment group (p<0.001). Similarly, IGF-1/agrin NP treatment significantly improved axonal regeneration and reduced muscle atrophy as compared to empty NP treatment (p<0.001 and p<0.01 respectively).
Conclusion: IGF-1/agrin co-treatment in vivo significantly improves functional recovery of forelimb grip strength. This combined treatment promotes neuromuscular junction reinnervation, increases axonal regeneration, and reduces chronic denervation-induced myofibril atrophy. These effects were greater than treatment with a single therapeutic, suggesting a synergistic effect. Additionally, co-treatment during reinnervation appeared to improve functional recovery greater than co-treatment during denervation, which provides insight into role of neuromuscular junction stabilization in functional recovery. Histological analyses of Schwann cell proliferation and sensory axon regeneration are currently ongoing.