1 Department of Anesthesiology, Emory University School of Medicine , Atlanta, Georgia .
2 Department of Neurology, Emory University School of Medicine , Atlanta, Georgia .
J Neurotrauma. 2018 Mar 1;35(5):802-813. doi: 10.1089/neu.2016.4871.
Traumatic brain injury (TBI) is a prevalent disorder, but no effective therapies currently exist. An underlying pathophysiology of TBI includes the pathological elevation of autophagy. β-Catenin, a downstream mediator of the canonical Wnt pathway, is a repressor of autophagy. The Wnt/β-catenin pathway plays a crucial role in cell proliferation and neuronal plasticity/repair in the adult brain. We hypothesized that activation of this pathway could promote neuroprotection and neural regeneration following TBI. In the controlled cortical impact (CCI) model of TBI in C57BL/6 mice (total n = 160), we examined intranasal application of recombinant Wnt3a (2 μg/kg) in a short-term (1 dose/day for 2 days) and long-term (1 dose/day for 7 days) regimen. Immunohistochemistry was performed at 1 to 14 days post-TBI to assess cell death and neurovascular regeneration. Western blotting measured canonical Wnt3a activity, expression of growth factors, and cell death markers. Longitudinal behavior assays evaluated functional recovery. In short-term experiments, Wnt3a treatment with a 60-min delay post-TBI suppressed TBI-induced autophagic activity in neurons (44.3 ± 6.98 and 4.25 ± 2.53 LC3+/NeuN+ double positive cells in TBI+Saline and TBI+Wnt3a mice, respectively; p < 0.0001, n = 5/group), reduced autophagic markers light chain 3 (LC3)-II and Beclin-1, as well as injury markers caspase-3 and matrix metalloproteinase 9 (MMP-9). The Wnt3a treatment reduced cell death and contusion volume (0.72 ± 0.07 mm and 0.26 ± 0.04 mm in TBI+Saline and TBI+Wnt3a mice, respectively; p < 0.001, n = 5/group). The 7-day Wnt3a treatment increased levels of β-catenin and growth factors glial-derived growth factor (GDNF) and vascular endothelial growth factor (VEGF). This chronic Wnt3a therapy augmented neurogenesis (0.52 ± 0.09 and 1.25 ± 0.13 BrdU+/NeuN+ co-labeled cells in TBI+Saline mice and TBI+Wnt3a mice, respectively; p < 0.01, n = 6/group) and angiogenesis (0.26 ± 0.07 and 0.74 ± 0.13 BrdU+/GLUT1+ co-labeled cells in TBI+Saline and TBI+Wnt3a mice, respectively; p = 0.014, n = 6/group). The treatment improved performance in the rotarod test and adhesive removal test. Targeting the Wnt pathway implements a unique combination of protective and regenerative approaches after TBI.
创伤性脑损伤（TBI）是一种普遍存在的疾病，但目前尚无有效的治疗方法。TBI 的一个潜在病理生理学包括自噬的病理性升高。β-连环蛋白是经典 Wnt 途径的下游介质，是自噬的抑制剂。Wnt/β-连环蛋白途径在成年大脑中的细胞增殖和神经元可塑性/修复中起着至关重要的作用。我们假设激活该途径可以在 TBI 后促进神经保护和神经再生。在 C57BL/6 小鼠的控制性皮质撞击（CCI）TBI 模型中（总 n=160），我们研究了重组 Wnt3a（2μg/kg）的鼻内应用，包括短期（每天 1 次，持续 2 天）和长期（每天 1 次，持续 7 天）方案。在 TBI 后 1 至 14 天进行免疫组织化学染色，以评估细胞死亡和神经血管再生。Western blot 检测了经典 Wnt3a 活性、生长因子表达和细胞死亡标志物。纵向行为测定评估了功能恢复。在短期实验中，TBI 后 60 分钟给予 Wnt3a 治疗抑制了 TBI 诱导的神经元自噬活性（TBI+生理盐水和 TBI+Wnt3a 小鼠中的 LC3+/NeuN+双阳性细胞分别为 44.3±6.98 和 4.25±2.53；p<0.0001，n=5/组），降低了自噬标志物 LC3-II 和 Beclin-1 以及损伤标志物半胱天冬酶-3 和基质金属蛋白酶 9（MMP-9）。Wnt3a 治疗减少了细胞死亡和挫伤体积（TBI+生理盐水和 TBI+Wnt3a 小鼠分别为 0.72±0.07 和 0.26±0.04mm；p<0.001，n=5/组）。7 天的 Wnt3a 治疗增加了β-连环蛋白和神经胶质衍生生长因子（GDNF）和血管内皮生长因子（VEGF）等生长因子的水平。这种慢性 Wnt3a 治疗增强了神经发生（TBI+生理盐水和 TBI+Wnt3a 小鼠中的 BrdU+/NeuN+共标记细胞分别为 0.52±0.09 和 1.25±0.13；p<0.01，n=6/组）和血管生成（TBI+生理盐水和 TBI+Wnt3a 小鼠中的 BrdU+/GLUT1+共标记细胞分别为 0.26±0.07 和 0.74±0.13；p=0.014，n=6/组）。治疗改善了旋转棒试验和粘连去除试验的表现。靶向 Wnt 途径在 TBI 后实施了一种独特的保护和再生方法的组合。
