Interventional effect of flunarizine on the expression of cyclooxygenase-2 and plasminogen activator inhibitor type-1 during experimental Cerebral ischemia/reperfusion in gerbils

Wensheng Zhou; Zhiping Hu; Yan Hong

摘要：BACKGROUND:Some researches suggest that induced cyclooxygenase-2 (COX-2) can cause brain injury through a series of ways at the phase of cerebral ischemia/hypoxia.Plasminogen activator inhibitor type-1(PAI-1)is a kind of inhibitor of serine stretch protein enzyme and is able to protect cell surface and microvascular basement membrane from degradation of protease and also protect contact surface among cells so as to maintain integrality of tissue structure.However,correlation of protective effect of flunarizine on brain with COX-2 and PAI-1 should be studied further.OBJECTIVE:To observe the effect of flunadzine on expressions of COX-2 and PAI-1 protein in forebrain and degree of brain injury among gerbils after cerebral ischemia.DESIGN:A randomized controlled animal study.SEITING:Department of Neurology,the Second Xiangya Hospital of Central South University;Department of Neurology,Mawangdui Hospital of Hunan Province.MATERIALS:A total of 40 healthy gerbils,of both genders,aged 9 months,weighing(90±10)g,were selected in this study.Anti-COX-2 multi-antibody,anti-PAI-1 multi-antibody,SABC immunohistochemical kit and DAB kit were provided by Wuhan Boster Biological Engineering Co.,Ltd.;and flunarizine capsule was provided by Xi'an Yangsen Pharmaceutical Company(batch number:041018726,dosage:5 mg/pill).METHODS:The experiment was Carried out in Laboratory of Mental Disease,Hunan Provincial Gedatdcs Institute affiliated by Hunan Provincial Mawangdui Hospital from January 2004 to March 2005.① All gerbils were randomly divided into cerebral ischemia group,flunarizine intervention group,sham operation group and normal control group with 10 in each group.Gerbils in normal control group were only cut off their heads.Gerbils in sham operation group were only dissected their bilateral common carotid arteries and sacdficad 1 day later.Gerbils in cerebral ischemia group and flunanzine intervention group were anesthetized,centrally cut open skin of neck,bluntly dissected bilateral common carotid arteries,clipped bilateral common carotid artedes with microvascular clamp for 10 minutes,then reperfused and sacrificed 3 in each group on the 1st-,3rd- and 7th-day reperfusion.One day before experiment,gerbils in flunarizine intervention group were given 20 mg/kg mixed flunanzine solution.②All gerbils were sacrificed in different periods.In addition,forebrain was fixed with 100 g/L formaldehyde,routinely embedded with paraffin,and cut into seeral sections with the thickness of 5 μm.Neighboring brain tissues were done with routine HE staining and pathological changes were observed under optic microscope to evaluate the degree of brain injury.Expressions of COX-2 and PAI-1 protein in forebrain were detected with immunohistochemical technique,I.e.,numbers of immune positive cells were accounted.③Averages of samples were compared with SNK-q test.MAIN OUTCOME MEASURES:Pathological changes of forebrain tissue and expressions of COX-2 and PAI-1 protein in forebrain of gerbils.RESULTS:All 40 gerbils were involved in the final analysis.①Pathological changes of forebrain tissue:Morphological structure of codex tissue was normalin normal control group and sham operation group.At 1 day after ischemia/reperfusion,nerve Cells in cortex and hippocampus of forebrain were focal necrosis in cerebral ischemia group,and proliferation of glial cell was slight.In addition,at 3 days after ischemia/reporfusion,nerve cells were sheet-like necrosis and proliferation of glial cell was obvious.Meanwhile,at 7 days after ischemia/raperfusion,nerve cells were necrosis in a large area and a lot of glial cells were proliferated.Degrees of brain injury were severer in cerebral ischemia group than those in flunadzine intervention group at various time points.② Expressions of COX-2 and PAI-1 protein in forebrain:Numbers of COX-2 positive cells in flunadzine intervention group were 62.38±3.38,33.84±2.48 and 13.82±2.23 at 1.3 and 7 days after ischemia/reperfusion,respectively.which were less than those in cerebral ischemia group(73.08±2.11,51.76±2.38,23.96±2.86;p＜0.01).Numbers of COX-2 positive cells in these two groups were decreased with the lasting time of ischemia/reperfusion.Expressions of PAI-1 in flunadzine intervention group were 14.48±2.0,27.44±3.06 and 41.84±2.48 at 1,3 and 7 days after ischemia/reperfusion,respectively,which were higher than those in cerebral ischemia group(8.48±1.53,20.93±2.16,31.12±2.89;P＜0.01).Expressions of PAI-1 protein in these two groups were increased with the lasting time of ischemia/reperfusion. CONCLUSION:Ischemia/reperfusion can increase the expressions of COX-2 and PAI-1 protein in forebrain of gerbils;however, pretreatment of flunarizine can down-regulate the expression of COX-2 but up-regulate the expression of PAI-1 protein so as to prevent brain injury after ischemia/reperfusion.Moreover,the preventive effect is time-dependent.