| AimBlood brain barrier ( BBB) is constituted with tight junction of endothelial cell, basal lamina and astrocyte feet . It is a interface to regulate materials that enter in the brain tissues. The destruction of BBB is an important mechanism of cerebral ischemia - reperfusion injury. Inflammation and immune response involving in the infiltration of leucocyte and the activation of macrophage, producing much proteolytic enzymes, oxygen derived free radicals and other factors, causing the destruction of capillary endothelium and basal membrane, and increasing the permeability of BBB. Erigeron breviscapus is extracted from Eriger-on Breviscapus ( Vant ). Hand - Mazz , it is 4, 5, 6 - 3 hydroxy methanone - 7 - glucose aldehyde acid anhydride, it already studied to improve microcir-culation , increase brain blood flow and resist the roles of free radicals. On clinical usage, it is applied extensively in the treatment of the brain blood vessel disease . There already has report that erigeron breviscapus can treat ischemic reperfusion injury, but its mechanism still known little. This test investigated the destruction of BBB in brain ischemia reperfusion injury and some mechanism of erigeron breviscapus.Methods1. The replication of animal models;Conscious mice , performing operation on the neck to separate the double common carotid artery and fixxed with artery clamp, 10minutes later,loosing the clamp and suturing the skin sterily. Erigeron breviscapus group were given injection 10mg/kg.2. The cerebral blood flow measurements:Opening the cranial cavity, using laser doppler blood - flow measuring e-quipment to detect brain cortex blood flow of the normal brain tissue and the is-chemia 10 min, IR 10min, 4h, 12h, 24h, 48h, 72h.3. The content of evans blue in brain tissues;Before 1 hour of death, the mouse was injected 2% evans blue through tail vein , 20 minutes before examination, using saline to filling the heart before the atrium run ofif clear liquid , using the electronics weighing scoles to weight the brain and put them into tube. 3 ml formamide was adding into them. Tube was covered and put in 45 C water bath for 48 h, lightly shaken and centrifugated for 15 min (3000 rs/min) , taken out the pure liquid and detected at 721spectrophotometer ( X =632 run).4. Gelatin zymography to detect the activity of gelatinase :Taking the hippocampus of brain tissue and making 10% tissue homogeni-zer, then centrifugation to obtain protein extraction. Taking out of 20ul and add in the gel(5rag/ml gelatin) ,20mA constant current electrophoresis for 5h,then letting the gel in the fluid of 2.5% Triton -X 100 shaking for 371 ,lh. ,then putting in the enzyme buffer to incubate for 37 C ,42h. When the incubation was o-ver ,put the gel in the staining fluid for 3h,then discoloration for 0. 5,l,2h, MMP - 2 and MMP - 9were the clearing zone on the blue background .5. The measurement of antioxidation enzymes ( GSH - Px, SOD, CAT) and NOS ' s activation and the content of MDA ;The mice were put to death and quickly took out of the hippocampus on a ice dish, immediately weight and make 10%tissue homogenizer , 4 C 10000 r/ min centrifugation for 5 minutes, and taking the pure liquid, using GSH - Px, SOD,CAT, NOS and MDA' s test box . Taking the pure liquid at 721 Spectro-photometer( = 550nm, = 412nm, X = 530nm, X = 405nm, andX = 532nm ) to detect the activation of enzymes and the content of MDA.Results1. The brain cortex blood flow of different time during brain ischemia reper-fusion injuryBrain cortex blood flow declined in ischemia 10min, then increased at reperfusion 10min,while 4h, decreased, 12 h and 24 h increased, reperfusion48h,72h has no difference as to normal.2. The influence of erigeron breviscapus on contents of Evans blue in cerebral ischemia - reperfusion animal brain tissueThe contents of Evans blue in cerebral ischemia - reperfusion group increased from 4h reperfusion,and reach peak at 48 h, then decreased gradually. Erigeron breviscapus group s was more descent t... |
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