Graham James B, Muir David
Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2016 Dec 14;11(12):e0167682. doi: 10.1371/journal.pone.0167682. eCollection 2016.
The success of peripheral nerve regeneration is highly dependent on the regrowth of axons within the endoneurial basal lamina tubes that promote target-oriented pathfinding and appropriate reinnervation. Restoration of nerve continuity at this structural level after nerve transection injury by direct repair and nerve grafting remains a major surgical challenge. Recently, biological approaches that alter the balance of growth inhibitors and promoters in nerve have shown promise to improve appropriate axonal regeneration and recovery of peripheral nerve function. Chondroitin sulfate proteoglycans (CSPGs) are known inhibitors of axonal growth. This growth inhibition is mainly associated with a CSPG's glycosaminoglycan chains. Enzymatic degradation of these chains with chondroitinase eliminates this inhibitory activity and, when applied in vivo, can improve the outcome of nerve repair. To date, these encouraging findings were obtained with chondroitinase ABC (a pan-specific chondroitinase). The aim of this study was to examine the distribution of CSPG subtypes in rodent, rabbit, and human peripheral nerve and to test more selective biological enzymatic approaches to improve appropriate axonal growth within the endoneurium and minimize aberrant growth. Here we provide evidence that the endoneurium, but not the surrounding epineurium, is rich in CSPGs that have glycosaminoglycan chains readily degraded by chondroitinase C. Biochemical studies indicate that chondroitinase C has degradation specificity for 6-sulfated glycosaminoglycans found in peripheral nerve. We found that chondroitinase C degrades and inactivates inhibitory CSPGs within the endoneurium but not so much in the surrounding nerve compartments. Cryoculture bioassays (neurons grown on tissue sections) show that chondroitinase C selectively and significantly enhanced neuritic growth associated with the endoneurial basal laminae without changing growth-inhibiting properties of the surrounding epineurium. Interestingly, chondroitinase ABC treatment increased greatly the growth-promoting properties of the epineurial tissue whereas chondroitinase C had little effect. Our evidence indicates that chondroitinase C effectively degrades and inactivates inhibitory CSPGs present in the endoneurial Schwann cell basal lamina and does so more specifically than chondroitinase ABC. These findings are discussed in the context of improving nerve repair and regeneration and the growth-promoting properties of processed nerve allografts.
周围神经再生的成功高度依赖于神经内膜基膜管内轴突的生长，这些基膜管促进靶向性路径寻找和适当的再支配。在神经横断损伤后，通过直接修复和神经移植在这个结构水平上恢复神经连续性仍然是一项重大的外科挑战。最近，改变神经中生长抑制剂和促进剂平衡的生物学方法已显示出有望改善轴突的适当再生和周围神经功能的恢复。硫酸软骨素蛋白聚糖（CSPGs）是已知的轴突生长抑制剂。这种生长抑制主要与CSPG的糖胺聚糖链有关。用软骨素酶对这些链进行酶促降解可消除这种抑制活性，并且在体内应用时可改善神经修复的结果。迄今为止，这些令人鼓舞的发现是通过软骨素酶ABC（一种泛特异性软骨素酶）获得的。本研究的目的是检查CSPG亚型在啮齿动物、兔子和人类周围神经中的分布，并测试更具选择性的生物学酶促方法，以改善神经内膜内轴突的适当生长并最小化异常生长。在这里，我们提供证据表明，神经内膜而非周围的神经外膜富含CSPGs，其糖胺聚糖链很容易被软骨素酶C降解。生化研究表明，软骨素酶C对周围神经中发现的6-硫酸化糖胺聚糖具有降解特异性。我们发现软骨素酶C可降解并使神经内膜内的抑制性CSPGs失活，但对周围神经区域的作用不大。冷冻培养生物测定（在组织切片上生长的神经元）表明，软骨素酶C选择性且显著地增强了与神经内膜基膜相关的神经突生长，而没有改变周围神经外膜的生长抑制特性。有趣的是，软骨素酶ABC处理极大地增加了神经外膜组织的生长促进特性，而软骨素酶C几乎没有影响。我们的证据表明，软骨素酶C有效地降解并使存在于神经内膜施万细胞基膜中的抑制性CSPGs失活，并且比软骨素酶ABC更具特异性。这些发现将在改善神经修复和再生以及处理后的神经同种异体移植物的生长促进特性的背景下进行讨论。
