Department of Neurology, The Chaim Sheba Medical Center, Ramat Gan, Israel.
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
J Neurosci Res. 2021 Mar;99(3):966-976. doi: 10.1002/jnr.24764. Epub 2020 Dec 9.
Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24 hr following stroke in comparison to healthy mice (F(3, 72) = 39.5, p < 0.0001, repeated measures ANOVA) after the induction of permanent middle cerebral artery occlusion (PMCAo). Plasmin activity was higher in the ischemic hemisphere (F(1,36) = 9.1, p = 0.005) and there was a significant interaction between time and ischemic hemisphere (F(3,36) = 4.4, p = 0.009). Plasmin activity was correlated with infarct volume (R = 0.5289, p = 0.0009 by Spearman). The specificity of the assay was verified utilizing tissue-type plasminogen activator (tPA)-deficient mice which, as expected, had significantly lower levels of plasmin 24 hr following ischemia compared to wild-type mice (ischemic (0.6 ± 0.23 and 1.94 ± 0.5, respectively), p = 0.049 and contralateral hemispheres (0.13 ± 0.14 and 0.75 ± 0.10, respectively), p = 0.018 by t test). There is a time-dependent increase in plasmin levels and an association of higher levels of plasmin with larger infarct volumes in an experimental stroke model. This suggests caution in the use of recombinant tPA (rtPA) and that plasmin inhibition in the brain may be a therapeutic target in acute ischemic stroke.
许多凝血因子蛋白酶在缺血性中风期间在大脑中增加。这些蛋白酶之一是纤溶酶。在这项研究中，我们建立了一种新的方法，使用灵敏的荧光底物和特定的蛋白酶抑制剂，直接定量测量雄性小鼠脑切片中的纤溶酶活性。与健康小鼠相比，在永久性大脑中动脉闭塞（PMCAo）诱导后 3、6 和 24 小时，缺血半球和对侧半球中的纤溶酶活性均增加（F(3,72) = 39.5，p < 0.0001，重复测量方差分析）。缺血半球中的纤溶酶活性更高（F(1,36) = 9.1，p = 0.005），时间和缺血半球之间存在显著的相互作用（F(3,36) = 4.4，p = 0.009）。纤溶酶活性与梗死体积相关（Spearman 相关系数 R = 0.5289，p = 0.0009）。利用组织型纤溶酶原激活物（tPA）缺陷型小鼠验证了该测定的特异性，正如预期的那样，与野生型小鼠相比，缺血后 24 小时的纤溶酶水平明显降低（缺血半球（分别为 0.6 ± 0.23 和 1.94 ± 0.5），p = 0.049 和对侧半球（分别为 0.13 ± 0.14 和 0.75 ± 0.10），p = 0.018，t 检验）。在实验性中风模型中，纤溶酶水平呈时间依赖性增加，并且较高水平的纤溶酶与较大的梗死体积相关。这表明在使用重组 tPA（rtPA）时需要谨慎，并且脑内纤溶酶抑制可能是急性缺血性中风的治疗靶点。
