Acellularized Nerve Allografts Guide Axon Regeneration to a Controlled Termination in a Rat Model
Miles J. Bichanich, BS1, Thomas Hong, MS1, Daniel A. Hunter, RA1, Lauren Schellhardt, BA1, Ying Yan, MD, PhD1, Susan E. Mackinnon, MD1, Thomas Davis, PhD2,3, Scott Tintle, MD2,3, Matthew D. Wood, PhD1, Amy M. Moore, MD1.
1Washington University in St. Louis School of Medicine, St. Louis, MO, USA, 2USU Walter Reed Department of Surgery, Uniformed Services University, Bethesda, MD, USA, 3Naval Medical Research Center, Regenerative Medicine Department, Silver Spring, MD, USA.
PURPOSE: The myriad of existing management approaches to neuroma highlight the difficulty in treating this clinical entity, and no satisfactory approach has been developed. Evaluation of “off-the-shelf” acellular nerve allografts (ANAs) as a means to bridge nerve gaps has shown an unintentional, controlled termination of axonal regrowth within long (>3cm) ANAs. We hypothesized that long ANAs can be beneficially utilized to “cap” injured nerve and guide regenerating axons to a gradual termination effectively neutralizing neuroma formation.
METHODS: Thy1-GFP and Lewis rats were randomized to eight groups which received: 1) nerve transection alone, 2) traction neurectomy, 3) transection and 0.5 cm closed end silicone conduit, 4) transection and 0.5 cm ANA, 5) transection and 2.5 cm ANA, 6) transection and 5.0 cm ANA, 7) transection and proximal nerve crush, or 8) transection, proximal nerve crush and 5.0 cm ANA. In all groups, the distal nerve stump was ligated and the distal nerve turned from the proximal end to remove any trophic influence. The Thy1-GFP rat nerves were serially imaged at 4, 8, and 20 weeks to provide a visual history of regeneration. Lewis rats were sacrificed at 5 and 20 weeks for quantitative nerve histology and IHC. ANOVA with post hoc analysis were performed to evaluate significance (p<0.05).
RESULTS: GFP animals that received transection alone, traction neurectomy, or transection and crush showed signs of neuroma with chaotic nerve regeneration (multidirectional axonal regrowth confirmed by histology) extending from the proximal stump as early as 4 weeks (Figure 1. Neuroma formation 4 weeks following traction neurectomy.).
At 5 weeks, axons grew through the entirety of the 0.5 cm ANAs, with neuroma formation extending beyond the grafts. In the 2.5 and 5.0 cm ANAs, robust axonal regeneration was demonstrated in the proximal portions of the grafts with a gradual tapering of regeneration as it moved distally, and axons failed to grow beyond the grafts. At 20 weeks, gross visualization of Thy1-GFP labeled axons demonstrates that regeneration dwindles and terminates within 5.0 cm ANAs without neuroma formation (Figure 2. Organized regeneration at 20 weeks within 5.0 cm ANA.).
Further histological analysis is ongoing, as are additional 20 week experiments to evaluate controlled termination with histology and IHC.
CONCLUSION: Following nerve transection, long ANA “caps” can be used to control disorganized axonal regrowth, and therefore prevent the formation of a neuroma. As such, the “capping” of a transected nerve with a long ANA is a potential surgical tool in the future of neuroma management. Based upon these results, further studies are underway in a swine model to evaluate the use of ANAs in neuroma prevention in a neuroma model more similar to the human.
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