London, United Kingdom 2013 Fig 1. Thrombin activity measured in brain slices after right permanent MCAo. Slices were numbered from anterior (#3) to posterior (#11), slices’ thickness =1mm. For calibration, known concentrations of thrombin were used in the same assay. R=Right (ipsilateral) hemisphere, L=left (contralateral) hemisphere. Example of relevant slice stained by TTC are presented above each column when Intact brain regions (red), infarcted regions (white). Mean±SE. Cerebrovasc Dis 2013; 35 (suppl 3)1-854 81 15 Experimental studies B 17:20 - 17:30 A NEW METHOD FOR QUANTITATIVE THROMBIN DETECTION IN BRAIN SLIC-ES- FIRST RESULTS IN AN ISCHEMIC STROKE MODEL D. Bushi1, J. Chapman2, A. Katzav3, E. Shavit -Stein4, N. Molshatzki5, D. Tanne6 Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL1,Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL2, Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL3, De-partment of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL4, Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL5, Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Israel, Tel Hashomer, ISRAEL6 BACKGROUND Thrombin has in addition to its role in thrombogenesis, also important hor-mone- like activities that affect various cells in the brain through protease activated receptors. Activation of these receptors by low concentrations of thrombin may have neuroprotective ef-fects while at higher concentrations thrombin has deleterious effects. This study presents a new method for direct quantitative measurement of thrombin activity in brain slices. The method is being used to quantitatively measure thrombin activity in mice brains following MCA occlu-sion. METHODS We first developed a new method for measuring thrombin activity in brain slic-es based on a fluorometric assay. Permanent focal cerebral ischemia was induced in C57Bl/6J mice (n=9) by endovascular suture occlusion of the right MCA. After 24-72 hours thrombin activity was measured on 1 mm thickness fresh coronal slices taken from the ipsilateral and contralateral hemisphere. Infarct assessment was performed using TTC. The distribution of thrombin activity in the brain was determined using tissue punches taken from different slice locations. Results were compared to control healthy mice (n=5). RESULTS The specificity of the assay for thrombin activity was verified using NAPAP-a spe-cific thrombin inhibitor. Thrombin activity in the right ischemic hemisphere was significantly higher compared to the left contralateral hemisphere (9.7+/-1.5 vs. 6.0+/-1.1 mU respectively; Mean±SE; p<0.05). In addition thrombin activity in the left contralateral hemisphere was sig-nificantly higher compared to healthy control mice (1.7+/-0.4 mU; p<0.05) (Fig1). Thrombin activity was higher in the ischemic core compared to the surrounding area. CONCLUSIONS A novel and specific method for quantitative measurement of thrombin in brain slices is presented. Results of studies based on this method may translate into potential thrombin based therapies.
Karger_ESC London_2013
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