Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
Stroke. 2012 Jan;43(1):199-204. doi: 10.1161/STROKEAHA.111.626911. Epub 2011 Oct 6.
Cerebral preconditioning provides insights into endogenous mechanisms that protect the brain from ischemic injury. Hypoxia and the anesthetic isoflurane are powerful preconditioning agents. Recent data show that sphingosine 1-phosphate receptor stimulation improves outcome in rodent models of stroke. Endogenous sphingosine 1-phosphate levels are controlled by the expression and activity of sphingosine kinases (SPK). We hypothesize that SPK upregulation mediates preconditioning induced by isoflurane and hypoxia and reduces ischemic injury.
Male wild-type C57BL/J, SPK1(-/-) and SPK2(-/-) mice were exposed to isoflurane or hypoxia preconditioning before transient middle cerebral artery occlusion. Infarct volume and neurological outcome were measured 24 hours later. SPK inhibitors (SKI-II and ABC294640) were used to test the involvement of SPK2. Expressions of SPK1, SPK2, and hypoxia-inducible factor 1α were determined. Primary cultures of mouse cortical neurons were exposed to isoflurane before glutamate- or hydrogen peroxide-induced cell death.
Isoflurane preconditioning and hypoxia preconditioning significantly reduced infarct volume and improved neurological outcome in wild-type and SPK1(-/-) mice but not in SPK2(-/-) mice. Pretreatment with SKI-II or ABC294640 abolished the isoflurane preconditioning-induced tolerance. Western blot showed a rapid and sustained increase in SPK2 level, whereas SPK1 level was similar between preconditioned mice and controls. Hypoxia-inducible factor 1α was upregulated in wild-type isoflurane-preconditioned mice but not in SPK2(-/-). Isoflurane preconditioning protected primary neurons against cell death, which was abolished in ABC294640-treated cells.
Applying genetic and pharmacological approaches, we demonstrate that neuronal SPK2 isoform plays an important role in cerebral preconditioning.
脑预处理为了解内源性机制提供了思路，这些机制可以保护大脑免受缺血性损伤。缺氧和麻醉剂异氟烷是强大的预处理剂。最近的数据表明，鞘氨醇 1-磷酸受体的刺激改善了中风啮齿动物模型的结果。内源性鞘氨醇 1-磷酸水平受鞘氨醇激酶（SPK）的表达和活性控制。我们假设 SPK 上调介导异氟烷和缺氧诱导的预处理，并减少缺血性损伤。
雄性野生型 C57BL/J、SPK1(-/-)和 SPK2(-/-)小鼠在短暂性大脑中动脉闭塞前接受异氟烷或缺氧预处理。24 小时后测量梗死体积和神经功能结果。使用 SPK 抑制剂（SKI-II 和 ABC294640）测试 SPK2 的参与情况。测定 SPK1、SPK2 和缺氧诱导因子 1α的表达。在谷氨酸或过氧化氢诱导的细胞死亡之前，将原代培养的小鼠皮质神经元暴露于异氟烷中。
异氟烷预处理和缺氧预处理显著减少了野生型和 SPK1(-/-)小鼠的梗死体积并改善了神经功能结果，但在 SPK2(-/-)小鼠中没有。SKI-II 或 ABC294640 的预处理消除了异氟烷预处理诱导的耐受。Western blot 显示 SPK2 水平迅速且持续增加，而预处理小鼠和对照之间的 SPK1 水平相似。野生型异氟烷预处理小鼠中缺氧诱导因子 1α上调，但 SPK2(-/-)小鼠中没有。异氟烷预处理可保护原代神经元免受细胞死亡，而 ABC294640 处理的细胞则消除了这种保护作用。
通过应用遗传和药理学方法，我们证明神经元 SPK2 同工型在脑预处理中起重要作用。
