Discrepancies in Senescence and Protein Expression from Cells in Nerve Autografts Compared to Injured Nerve
Gwendolyn M. Hoben, MD PhD1, Xueping Ee, MD1, Lauren Schellhardt, BA1, Ying Yan, MD PhD1, Daniel Hunter, RA1, Amy M. Moore, MD2, Alison Snyder-Warwick, MD1, Sheila Stewart, PhD1, Susan E. Mackinnon, MD1, Matthew D. Wood, PhD1.
1Washinton University, St.Louis, MO, USA, 2Washinton University, Saint Louis, MO, USA.
PURPOSE: Outcomes of nerve autografting are often merely adequate to poor, especially as the nerve gap increases in length. We hypothesize senescent cells accumulate in longer grafts and resultant changes in protein expression contribute to reduced regeneration.
METHODS: A rat sciatic nerve transection model was used to compare short (2cm) and long (6cm) isografts (equivalent to autograft in syngeneic animals), while injured (no repair) and cut-repair nerves were used as controls.Grafts were analyzed after 4 and 8 weeks in vivo with histology, gene expression, histomorphometry, and retrograde labeling.
RESULTS: Axon counts clearly demonstrated disproportionately reduced regeneration in the longer graft compared to the short graft when measuring at equivalent distances to the spinal cord: the number of regenerated axons in the long graft was 57% of those regenerated in the short graft. Additionally, retrograde labeling showed significantly fewer motoneurons were found to be regenerating axons to long grafts compared to the short graft repairs. Cell composition amongst the different grafts and injured nerve were surprisingly consistent: groups had similar total numbers of cells and similar proportions of fibroblasts, Schwann cells, and macrophages. Immunohistochemical analysis showed an increased percentage of cells with senescent markers in the long grafts and in the “normal” nerve proximal to the graft. While gene expression for senescent markers was increased in both long and short nerve grafts, these markers remained elevated over time in the long grafts. Interestingly, GDNF and IL-6 expression was elevated in the long grafts compared to the short. Finally, immunohistochemistry revealed increased NOTCH signalling in the long grafts.
CONCLUSION: Comparison of short and long grafts, at points equidistant to the spinal cord, showed reduced axon regeneration and reduced number of motoneurons regenerating axons. Given this reduced regeneration in the presence of a long graft and the finding that cellular composition was unchanged, immunohistochemistry and gene expression were used to identify gene and protein expression differences in the grafts.The longer grafts were found to have a greater proportion of senescent cells and the nerve proximal to the graft even showed senescent changes. Gene expression associated with senescent was increased in the grafts but retained over time in the longer grafts. Prior work has linked increased NOTCH signalling to reduced neurite extension from dorsal root ganglia neurons and the current work links increasing graft length with increased NOTCH signalling. These protein and gene expression changes give insight as to the poor clinical outcomes associated with autografts and provide targets for improvement.
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