From Motor Neurons to Muscle: Regenerative Strategies to Babysit both Proximal and Distal to a Primary Nerve Repair
Zarina S Ali, MD, MS1, Franco A. Laimo, BS1, Justin C Burrell, MS1, Kritika Katiyar, PhD1, C. Joseph Maggiore, BS1, Kevin Browne, BA1, Joseph M. Rosen, MD2 and Daniel Kacy Cullen, PhD1, (1)University of Pennsylvania, Philadelphia, PA, (2)Surgery / Plastic Surgery, Dartmouth Hitchcock Medical Center, Lebanon, NH
Introduction: Following segmental nerve repair, functional recovery depends on the ability of proximal neurons to regenerate axons across the defect and span the entire denervated nerve to reach the distal muscle target. After injury, Schwann cells (SCs) provide a temporary pro-regenerative environment that supports the injured neurons and promotes axonal guidance. However, prolonged periods without axonal contact diminishes the potential for successful recovery. Current approaches to mitigate prolonged denervation, such as nerve transfers, are only possible in specific scenarios and may result in surgical complications. To address this need, our group has pioneered the development of tissue engineered nerve grafts (TENGs) as living scaffolds comprised of "stretch-grown" axons. Building on this concept, we report several advanced TENG "babysitting" strategies to sustain the regenerative capacity of proximal neurons and facilitate functional recovery by preventing prolonged denervation of distal nerve and muscle.
Materials and Methods: Proximal neuron babysitting was evaluated following a 1 cm repair using an autograft, nerve guidance tube (NGT) or TENG in rats. Host spinal motor neuron (MN) and dorsal root ganglia (DRG) health were compared by measuring FluoroRuby (FR) uptake and staining for NeuN. Distal nerve babysitting was assessed by studying SC presence in a rodent chronic axotomy model. Lastly, functional recovery was tested in rats (1 cm) and pigs (5 cm). Host axon regeneration, myelination, and muscle reinnervation were quantified. Nerve and muscle electrophysiological recovery were measured.
Results: In the proximal neuron babysitting study, increased FR+ and NeuN+ MNs were detected following repairs with TENGs or autografts compared to NGTs. No statistical differences were observed in DRG. In the distal babysitting study, TENG axons were found in the otherwise denervated nerve closely interacting with the host SCs at 16 weeks post transplantation compared to minimal SC expression in controls. Next, we assessed the efficacy on nerve regeneration and functional recovery in rats and pigs. Similar axon counts, myelination, muscle fiber diameter distribution, and muscle reinnervation were found in rats and pigs following TENG repair compared to autografts.
Conclusions: TENGs may represent a novel nerve repair strategy designed to maximize the potential for functional recovery. Our findings suggest TENGs preserve proximal neuronal regenerative capacity, maintain pro-regenerative capacity of distal SCs, and prevent muscle fiber atrophy. Moreover, TENGs appear to be comparable to autografts in long gap nerve injury model in pigs. Future TENG development will further investigate efficacy in large animals and protocols necessary for clinical translation.
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