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Schwann Cell Senescence: A Key Role in Reducing Axon Regeneration
Gwendolyn Hoben, MD, PhD1; Xueping Ee, MD1; Amy M. Moore, MD1; Yuewei Wu-Feinberg, MD1; Piyaraj Newton, BS1; Dan A. Hunter, RA1; Philip J. Johnson, PhD1;Sheila Stewart, PhD2; Susan E. Mackinnon, MD1;Matthew D. Wood, PhD1
1Department of Surgery, Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, Saint Louis, MO; 2Department of Cell Biology and Physiology, Washington University, Saint Louis, MO
Introduction: Nerve injuries repaired with long nerve grafts (autograft or processed/acellular nerve allograft) are associated with poor axonal regeneration and the accumulation of senescent Schwann cells (SenSCs). Senescent cells are commonly characterized by altered gene expression and protein secretion. We hypothesize that SenSCs have gene expression changes that are deleterious to regeneration. SenSCs impede axonal growth and are thus, a direct contributor to limited regeneration in nerve grafts. Using SenSC culture, we assessed gene expression changes in a neuroregulatory specific array and measured neuronal neurite extension in co-culture with SenSCs. In an in vivo model, we measured axonal regeneration through a conduit filled with normal or senescent SCs.
Materials and Methods: SCs from rat sciatic nerves were treated with aphidicolin (damages DNA) to induce senescence. A custom PCR array of 91 neuroregulatory genes (axonal inhibitors and growth factors) was used to screen for gene expression changes compared to normal SCs. To assess the effect on neurite outgrowth, rat dorsal root ganglia (DRG) neurons were co-cultured with varying densities of SCs and SenSCs and maximal neurite length was measured over time. In vivo, a 5mm conduit was implanted into the transected sciatic nerve of rats containing normal or SenSCs. After 4 weeks, axonal regeneration was measured by retrograde labeling of motoneurons regenerating axons distal to the conduit and histomorphometric analysis of the mid-conduit and distal nerve.
Results: Gene expression was significantly upregulated in 19 genes and downregulated in 17. The greatest increases in expression changes occurred with Notch ligands, semaphorins, and cell surface proteins associated with inhibition of nerve regeneration. Co-culture in the presence of SenSCs showed a consistent significant 21-28% decrease in neurite extension from DRG neurons. In vivo studies showed reduced axon regeneration in the mid-conduit in the presence of SenSCs compared to normal SCs. However, the difference diminished at the distal nerve. Retrograde labeling showed no difference in the number of motoneurons regenerating axons.
Conclusions: Neurite outgrowth in SenSC co-culture and midgraft histomorphometry showed reduced axon growth when the axons are directly in contact with SenSCs. As the number of motoneurons regenerating axons was not affected, these results suggest SenSCs reduce axonal sprouting. Upregulation of Notch ligands and cell surface proteins support mechanistic pathways for this reduction in sprouting. This model suggests targeting the changes resultant from SenSCs could improve axonal regeneration in long nerve grafts.
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