Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Senescent Schwann Cells Inhibit Nerve Regeneration in a Short Conduit Model
Gwendolyn M. Hoben, MD, PhD, Piyaraj Newton, BS, Dan A. Hunter, RA, Sheila Stewart, PhD, Philip J. Johnson, PhD, Matthew D. Wood, PhD, Susan E. Mackinnon, MD.
Washington University, St.Louis, MO, USA.

PURPOSE: Nerve injuries requiring long distances for axonal regeneration are associated with poor functional outcomes. Our laboratory has shown that increased nerve graft length (both isografts and acellular nerve allografts) results in decreased regeneration and accumulation of senescent SCs (SenSCs) both in the graft and in the nerve distal to the graft. Senescent cells activate the senescent associated secretory phenotype (SASP), which is rich in inflammatory proteins, ECM remodeling proteins, and other factors. We hypothesize that SenSCs impede axonal regeneration after injury. To test this hypothesis, we examined whether SenSCs directly limit nerve regeneration by transplanting cultured SCs induced to a senescent state into a nerve gap injury model. We measured changes to gene expression associated with nerve regeneration in these SenSCs to explore a mechanism for the limited regeneration.

METHODS: A rat sciatic nerve transection model was repaired utilizing 5mm conduits. The experimental groups were: 1) empty conduits; conduits filled with 1x106 2) normal SCs in fibrin, 3) H2O2 induced SenSCs in fibrin, and 4) aphidicolin induced SenSCs in fibrin. After 4 weeks, the conduits were excised and examined for nerve regeneration with histomorphometry. To confirm Schwann cell survival and examine migration following transplantation, Schwann cells transduced to express red fluorescent protein were used in groups 2, 3, and 4 and examined macroscopically after 1 wk, 2 wks, and 4 wks. Schwann cell gene expression was analyzed using qRT- PCR for markers of cell senescence (P16, IL-6, and IL-8) and nerve regeneration proteins.

RESULTS: Based on histomorphometric analysis, nerve regeneration was greatest in conduits filled with normal SCs. Rats receiving empty conduits demonstrated sporadic and poor nerve regeneration with few axons reaching the distal portion of the conduit. Although there was nerve regeneration more consistently in the conduits filled with the senescent cells, the difference was not significant compared to the empty conduits. Fluorescence microscopy confirmed the retention of both normal SCs and SenSCs for at least 2 weeks. SenSC gene expression differed from normal SCs with regard to an upregulation of cell senescence markers and differing nerve regeneration protein profiles.

CONCLUSION: It has been previously shown that seeding a conduit with normal SCs greatly enhanced nerve regeneration. Here we show that SCs induced to a senescent state reduces nerve regeneration in a conduit model. As SenSCs survived transplantation and affected gene expression of nerve regeneration proteins, SenSCs were having a direct effect on regenerating axons. This result significantly strengthens the case for senescence as a predominant cause of poor regeneration in long acellular nerve allografts. Thus, strategies to reduce SenSCs or their effects could be an important goal in future nerve regeneration studies.

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