Discrepancies In Cellular Composition And Gene Expression In Long Acellular Allografts
Deng Pan, B.S., Dan Hunter, Ellen Larson, B.S., Anja Fuch, PhD, Sally Jo, B.S., Xueping Ee, M.D/, Katherine Santosa, M.D, Alison Snyder-Warwick, M.D., Susan Mackinnon, M.D., Matthew Wood, PhD.
Washington University in St Louis, Saint Louis, MO, USA.
Purpose: Acellular nerve grafts (ANAs) represent clinical alternatives to nerve autografting. However, as ANAs increase in size and length, these alternatives exhibit reduced capability to facilitate axon regeneration across the graft. Understanding why this deficiency develops is critical to designing autograft alternatives.
Methods: Rat sciatic nerves were transected and repair with either short (2 cm) or long (4 cm) ANAs. Grafts were analyzed after 2, 4 and 8 weeks in vivo with histology, lectin perfusion, gene expression, histomorphometry, and immunohistochemistry. Results are presented as means with a student's two-tailed t-test to assess statistical significance (p<0.05).
Results: Histomorphometry assessed the extent of axon regeneration across 2 cm and 4 cm ANAs. The number of myelinated axons that crossed the graft mediated by 2 cm ANAs were significantly greater than 4 cm ANAs (6449 vs 44 myelinated axons) by 8 weeks. Considering early gene expression, analysis of a select panel of inflammatory and angiogenetic genes showed that at 2 weeks, long ANAs experienced reduced VEGFa, IL-4, and IL-10 gene expression, while having an elevated iNOS gene expression. Considering angiogenesis as VEGFa was decreased in long compared to short ANAs, at 2 weeks, total blood vessel length (indicated by RECA-1+ immunohistochemistry) of long ANAs were one third the value of short ANAs at 2 weeks, and only reached half the RECA-1+ values by 4 weeks. Considering functional blood vessels, lectin intravenous perfusion also showed similar reduction of blood vessels in long ANAs compared to short ANAs at 4 weeks. To further determine the impact of the altered inflammatory profile between long and short ANAs, we considered leukocyte populations within ANAs. While macrophage composition was similar between the long and short ANAs at 2 weeks (38% vs 45%), there was a reduction in the proportion of macrophages at 4 weeks between long and short ANAs (10.6% vs 21.9%). Additionally, while T cells represented a small proportion of cells within short and long grafts at 2 weeks (4.2% vs 0.9%), their relative proportion became significant by 4 weeks with stark differences between short and long ANAs (16.3% vs 4.3%).
Conclusions: Long ANAs mediated significantly reduced axonal regeneration in rat model of sciatic repair. Gene expression studies demonstrated a different inflammatory environment within the long ANAs compared to short ANAs, with elevated inflammatory gene (iNOS) and reduced anti-inflammatory genes (IL-4, IL-10). VEGF, important for angiogenesis, was also reduced. Histologically, we observed reduced angiogenesis in long ANAs, as demonstrated by reduction of total blood vessel length, at 2 weeks and 4 weeks, which was confirmed by intravenous infusion of fluorescently labeled lectin at 4 weeks. Finally, T cell accumulation is reduced in long ANAs. Previous studies have suggested that T cells may play a role in mediating regeneration through tissue engineered scaffolds. Therefore, our future studies will assess these associations to determine whether a causal relationship is present.
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