Comprehensive Stroke Center, the Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Tel-Hashomer, Israel,
J Mol Neurosci. 2013 Nov;51(3):844-50. doi: 10.1007/s12031-013-0072-y. Epub 2013 Jul 31.
Thrombin, a central factor in thrombogenesis, affects cells in the brain through protease activated receptors. Low levels of thrombin activity are neuroprotective while higher levels are deleterious, and we have therefore developed a new method for its direct quantitative measurement in brain slices following stroke. Thrombin activity was measured by a fluorescent substrate on fresh coronal slices taken from the ipsilateral and contralateral hemispheres 24-72 h following permanent right middle cerebral artery occlusion. Prolyl endopeptidase and aminopeptidases were inhibited as a critical step to insure the specificity of the assay for thrombin detection. Infarct volume was assessed using TTC staining. Thrombin activity in the right ischemic hemisphere was significantly higher compared to the contralateral hemisphere (32 ± 6 and 27 ± 10 mU/ml, mean ± SE in the two most affected slices from the ischemic hemisphere vs. 21 ± 6 and 8 ± 2 mU/ml in corresponding contralateral slices; p < 0.05). Thrombin levels in the ischemic and contralateral hemispheres were significantly higher compared to healthy control mice and were above the range known to be protective to brain cells. A significant correlation was found between thrombin activity in the ischemic hemisphere and the infarct volume. Results of studies based on this method may translate into potential thrombin based therapies.
凝血酶是血栓形成的核心因素，通过蛋白酶激活受体影响大脑细胞。低水平的凝血酶活性具有神经保护作用，而高水平的凝血酶活性则具有有害作用，因此我们开发了一种新方法，用于在中风后直接定量测量脑切片中的凝血酶活性。在永久性右侧大脑中动脉闭塞后 24-72 小时，通过对取自同侧和对侧半球的新鲜冠状切片上的荧光底物来测量凝血酶活性。作为确保检测凝血酶特异性的关键步骤，抑制脯氨酰内肽酶和氨肽酶。通过 TTC 染色评估梗死体积。与对侧半球相比，右侧缺血半球中的凝血酶活性明显升高（缺血半球的两个最受影响的切片中为 32±6 和 27±10 mU/ml，而相应的对侧切片中为 21±6 和 8±2 mU/ml；p<0.05）。与健康对照小鼠相比，缺血和对侧半球中的凝血酶水平明显升高，且高于已知对脑细胞具有保护作用的范围。在缺血半球中的凝血酶活性与梗死体积之间发现了显著的相关性。基于该方法的研究结果可能转化为潜在的基于凝血酶的治疗方法。
