15. Sustained Agrin Nanoparticle Delivery Improves Neuromuscular Junction Reinnervation and Functional Recovery after Chronic Denervation

PURPOSE: One of the most critical factors contributing to poor outcomes after peripheral nerve injury is the prolonged period of latency prior to reinnervation. Acetylcholine receptors (AChRs) within denervated muscle rapidly destabilize and consequently degrade neuromuscular junctions (NMJs); thereby limiting meaningful functional motor recovery. Agrin, a proteoglycan essential to NMJ formation and AChR aggregation, has been found to preserve NMJ morphology in the setting of denervation and may have an essential role in preserving NMJ receptivity to reinnervation. Our group has previously demonstrated over 6 weeks of sustained release of bioactive agrin encapsulated within biodegradable nanoparticles (NPs). This study aimed to evaluate the efficacy of agrin-NP therapy on functional motor recovery in a rodent forelimb chronic denervation model. METHODS: Agrin was encapsulated in biodegradable polyelectrolyte:polymer NPs and then embedded within a hyaluronic acid/PCL nanofiber hydrogel composite (NHC). The effects of locally delivered agrin-NP/NHC on NMJ reinnervation were assessed using a Lewis-Norway rodent chronic denervation nerve injury model in which the median nerve was denervated for 12 weeks until ulnar-to-median nerve transfer. Following nerve transfer, all animals underwent 16 weeks of regeneration and grip strength assessment. Denervated animals were injected with agrin-NP/NHC, free agrin, or empty-NP/NHC every six weeks from time of median nerve injury. A total of five injections were given: 1) at the time of median nerve injury, 2) after 6 weeks of denervation, 3) at the time of nerve transfer after 12 weeks of denervation, 4) week 6 post nerve transfer, 5) week 12 post nerve transfer. The “no denervation” control group only received the ulnar-to-median nerve transfer and saline injections. Upon sacrifice at Week 16, tissues were assessed for NMJ reinnervation, muscle atrophy, and axonal regeneration. RESULTS: Agrin-NP/NHC treated animals exhibited significantly increased functional recovery throughout the regeneration period compared to free agrin (p<0.01 2-way ANOVA) and empty-NP/NHC treated animals (p<0.01 2-way ANOVA) At Week 15, Agrin-NP/NHC treated animals demonstrated an increase of 21.2% compared to free agrin (p=0.056) and of 26.8% compared to empty-NP/NHC animals (p<0.05). Agrin-NP/NHC treated animals demonstrated significantly greater NMJ reinnervation than free agrin (78.9% vs 66.8%, p<0.05) and empty-NP/NHC groups (78.9% vs 39.2%, p<0.001). No significant differences were seen in myofibril cross-sectional area between agrin-NP/NHC, free agrin, and empty-NP/NHC groups. No foreign body response was detected in empty-NP/NHC or agrin-NP/NHC treated animals. Upon ELISA, agrin levels were undetectable in serum and were significantly higher in agrin-NP/NHC animals than free agrin-treated animals. CONCLUSION: Agrin-NP/NHC treatment in vivo promotes neuromuscular junction reinnervation and improves functional recovery of forelimb grip strength. Our group has previously established the benefits of insulin-like growth factor 1 (IGF-1) nanoparticles on ameliorating the effects of chronic denervation. Future studies will combine Agrin-NPs and IGF-1-NPs and evaluate their hypothesized synergistic benefits.