Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Cellular Senescence Onset Coincides With Axonal Regenerative Arrest In Long Acellular Nerve Grafts
Louis H. Poppler, MD, Xueping Ee, MD, Lauren Prange, BS, Gwendolyn Hoben, MD, Daniel A. Hunter, RA, Ying Yan, M.D., Ph.D., Sheila Stewart, Ph.D., Susan E. Mackinnon, MD, Matthew D. Wood, Ph.D..
Washington University in St. Louis School of Medicine, St. Louis, MO, USA.

Purpose:
Acellular nerve allografts (ANA) poorly facilitate axonal regeneration when grafts surpass 3cm. We recently correlated cellular senescence with lack of axonal regeneration across long ANAs ten weeks after nerve repair. Although this work suggests senescence is involved in regenerative failure, it is unknown if cellular senescence precedes regenerative failure and could therefore be causal. This study determined the timing of axonal arrest in long ANAs. We then differentially assessed cellular populations and senescence accumulation between long and short ANAs to identify candidates causing axonal arrest in long ANAs. We hypothesize senescence precedes regenerative failure and is therefore a candidate cause for axonal arrest.
Methods:
Thy-1-GFP rats (axons fluoresce) were imaged bi-weekly after sciatic repair with 3 (short) and 6cm (long) ANAs for 10 weeks to measure axonal extension. After the time of axonal arrest was identified, tissue was harvested before (2 weeks) and during (4 weeks) arrest (Table 1). Cellular senescence was assessed using senescence associated β-galactosidase (SA-βgal) activity and expression of cell-cycle inhibitors. Cellular populations were characterized with immunohistochemistry and histology. Molecular expression due to senescence (Notch ligands) was quantified in ANAs, and receptor activation confirmed in DRG. One-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis (α=0.05).
Results:
Axonal regeneration arrested at 4 weeks in 6cm grafts while axons crossed 3cm ANAs successfully revealing a time-frame for analysis. Schwann Cells were the predominant population in either ANA at 4 weeks (75±13%), similar to normal nerve. Before axonal arrest (2 weeks), fibroblast accumulation based on CD90 staining was comparable in short ANAs, long ANAs, and normal nerve. During axonal arrest (4 weeks), CD90 expression was 2x normal nerve (p<0.05) in both ANA groups. Therefore, cellular population differences were unlikely candidates for axonal arrest. Expression of senescence markers was significantly elevated in long ANAs at 4 weeks but not 2 weeks indicating senescence may cause arrest as axons arrested at 4 weeks (Figure 1). Furthermore, long ANAs demonstrated significantly higher levels of senescence markers compared to short ANAs suggesting senescence could be a candidate cellular phenotype causing axonal arrest in long ANAs. Additionally, notch ligands, proteins known to inhibit axonal growth, were significantly elevated at 4, but not 2 weeks, in long ANAs compared to short ANAs.
Conclusions:
Cellular senescence occurs concurrently with axonal arrest in long ANAs, and may inhibit axonal regeneration through the Notch signalling pathway. Further studies are underway to demonstrate causality based on this strong correlation.


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