Poppler Louis H, Ee Xueping, Schellhardt Lauren, Hoben Gwendolyn M, Pan Deng, Hunter Daniel A, Yan Ying, Moore Amy M, Snyder-Warwick Alison K, Stewart Sheila A, Mackinnon Susan E, Wood Matthew D
1 Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine , St. Louis, Missouri.
2 Division of Cell Biology and Physiology, Washington University , St. Louis, Missouri.
Tissue Eng Part A. 2016 Jul;22(13-14):949-61. doi: 10.1089/ten.TEA.2016.0003. Epub 2016 Jul 7.
Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated β-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100β(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100β(-)/CD68(-) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest ("stressed" ANAs). These stressed ANAs contained mainly S100β(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits in axonal extension. Overall, this evidence demonstrates that long ANAs are repopulated with increased p16(+) Schwann cells and stromal cells compared to short ANAs, suggesting a role for these cells in poor axonal regeneration across nerve constructs.
脱细胞神经同种异体移植物（ANA）和其他神经构建体不能可靠地促进轴突在长缺损（>3厘米）处的再生。对这种缺陷的原因了解甚少。在本研究中，我们确定了在神经构建体中轴突生长停滞之前ANA内存在哪些细胞，以及这些细胞是否表达细胞应激和衰老标志物。使用Thy1-GFP大鼠和连续成像，我们确定了长（6厘米）ANA中轴突生长停滞的时间和位置。轴突在4周内停止在长ANA内生长，而轴突成功地穿过短（3厘米）ANA再生。使用免疫组织化学、组织学和衰老相关β-半乳糖苷酶染色来确定细胞群体和衰老标志物。到2周时，短ANA和长ANA都被雪旺细胞（SC）和基质细胞大量重新填充。雪旺细胞（S100β(+)）是重新填充两种ANA的主要细胞类型。总体而言，除了长ANA中基质细胞（纤连蛋白(+)/S100β(-)/CD68(-)细胞）增加外，两种ANA的细胞群体相似。对ANA进行细胞衰老标志物的表征显示，与短ANA相比，长ANA积累了更高水平的衰老标志物，并且表达衰老标志物p16的雪旺细胞百分比更高。为了确定长ANA环境对轴突再生的影响，在轴突生长停滞时从长ANA中制备通常支持轴突再生的短ANA（2厘米）（“应激”ANA）。这些应激ANA主要包含S100β(+)/p16(+)细胞，并且轴突再生明显减少。在额外的实验中，在轴突生长停滞时切除长ANA的远端部分（4厘米）并用长的同基因移植物（4厘米）替代，挽救了跨缺损的轴突再生。来自长ANA中轴突生长停滞后的神经的神经元培养显示轴突延伸没有缺陷。总体而言，这一证据表明，与短ANA相比，长ANA中p16(+)雪旺细胞和基质细胞增加，表明这些细胞在神经构建体中轴突再生不良中起作用。
