Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, Ohio 44195
J Neurosci. 2018 Jul 11;38(28):6247-6266. doi: 10.1523/JNEUROSCI.3017-17.2018. Epub 2018 Jun 11.
White matter (WM) damage following a stroke underlies a majority of the neurological disability that is subsequently observed. Although ischemic injury mechanisms are age-dependent, conserving axonal mitochondria provides consistent post-ischemic protection to young and aging WM. Nitric oxide synthase (NOS) activation is a major cause of oxidative and mitochondrial injury in gray matter during ischemia; therefore, we used a pure WM tract, isolated male mouse optic nerve, to investigate whether NOS inhibition provides post-ischemic functional recovery by preserving mitochondria. We show that pan-NOS inhibition applied before oxygen-glucose deprivation (OGD) promotes functional recovery of young and aging axons and preserves WM cellular architecture. This protection correlates with reduced nitric oxide (NO) generation, restored glutathione production, preserved axonal mitochondria and oligodendrocytes, and preserved ATP levels. Pan-NOS inhibition provided post-ischemic protection to only young axons, whereas selective inhibition of NOS3 conferred post-ischemic protection to both young and aging axons. Concurrently, genetic deletion of NOS3 conferred long-lasting protection to young axons against ischemia. OGD upregulated NOS3 levels in astrocytes, and we show for the first time that inhibition of NOS3 generation in glial cells prevents axonal mitochondrial fission and restores mitochondrial motility to confer protection to axons by preserving Miro-2 levels. Interestingly, NOS1 inhibition exerted post-ischemic protection selectively to aging axons, which feature age-dependent mechanisms of oxidative injury in WM. Our study provides the first evidence that inhibition of glial NOS activity confers long-lasting benefits to WM function and structure and suggests caution in defining the role of NO in cerebral ischemia at vascular and cellular levels. White matter (WM) injury during stroke is manifested as the subsequent neurological disability in surviving patients. Aging primarily impacts CNS WM and mechanisms of ischemic WM injury change with age. Nitric oxide is involved in various mitochondrial functions and we propose that inhibition of glia-specific nitric oxide synthase (NOS) isoforms promotes axon function recovery by preserving mitochondrial structure, function, integrity, and motility. Using electrophysiology and three-dimensional electron microscopy, we show that NOS3 inhibition provides a common target to improve young and aging axon function, whereas NOS1 inhibition selectively protects aging axons when applied after injury. This study provides the first evidence that inhibition of glial cell NOS activity confers long-lasting benefits to WM structure and function.
中风后的白质（WM）损伤是随后观察到的大多数神经功能障碍的基础。尽管缺血性损伤机制与年龄有关，但保留轴突线粒体为年轻和衰老的 WM 提供一致的缺血后保护。一氧化氮合酶（NOS）的激活是缺血期间灰质中氧化和线粒体损伤的主要原因；因此，我们使用纯 WM 束，即分离的雄性小鼠视神经，来研究 NOS 抑制是否通过保留线粒体来提供缺血后的功能恢复。我们发现，在氧葡萄糖剥夺（OGD）之前应用全 NOS 抑制可促进年轻和衰老轴突的功能恢复并维持 WM 细胞结构。这种保护与减少一氧化氮（NO）生成、恢复谷胱甘肽生成、保留轴突线粒体和少突胶质细胞以及维持 ATP 水平相关。全 NOS 抑制仅对年轻轴突提供缺血后保护，而选择性抑制 NOS3 则对年轻和衰老轴突均提供缺血后保护。同时，NOS3 的基因缺失赋予年轻轴突对缺血的持久保护。OGD 上调星形胶质细胞中的 NOS3 水平，我们首次表明，抑制胶质细胞中 NOS3 的产生可防止轴突线粒体裂变，并通过维持 Miro-2 水平恢复线粒体运动，从而为轴突提供保护。有趣的是，NOS1 抑制选择性地对衰老轴突发挥缺血后保护作用，衰老轴突具有 WM 中氧化损伤的依赖于年龄的机制。我们的研究首次提供证据表明，抑制胶质细胞 NOS 活性可长期有益于 WM 功能和结构，并表明在血管和细胞水平上定义 NO 在脑缺血中的作用时应谨慎。中风期间的 WM 损伤表现为存活患者随后出现的神经功能障碍。衰老主要影响中枢神经系统 WM，并且缺血性 WM 损伤的机制随年龄而变化。一氧化氮参与各种线粒体功能，我们提出抑制胶质特异性一氧化氮合酶（NOS）同工型可通过维持线粒体结构、功能、完整性和运动性来促进轴突功能恢复。我们使用电生理学和三维电子显微镜显示，NOS3 抑制为改善年轻和衰老轴突功能提供了共同靶点，而 NOS1 抑制在损伤后应用时选择性地保护衰老轴突。这项研究首次提供证据表明，抑制神经胶质细胞 NOS 活性可长期有益于 WM 结构和功能。
