Protective Effect And Its Mechanisms Of Flurbiprofen Axetil(FA) Against Focal Cerebral Ischemia Injury In Rats Subjected To Middle Cerebral Artery Occlusion

Cerebral ischemia results from a transient or permanent local reduction of cerebral blood flow. With a mortality rate of 30%,stroke is the first one of the leading causes of death and adult disability worldwide. The poor prognosis of patients suffering from stroke results from the lack of effective therapies.In search of an effective treatment of stroke,numerous studies have been conducted to understand the cerebral ischemia pathophysiological mechanisms that lead to neuronal death.Recent experimental evidences have shown that brain damage occurring after focal cerebral ischemia (FCI) develops over a relatively long period of time. One of the processes that play a pivotal role in the delayed progression of brain damage is postischemic inflammation. Cerebral ischemia is followed by infiltration of neutrophils in the injured brain, blood brain barrier disruption,an event initiated by the expression of pro-inflammatory cytokines,chemokines,and adhesion molecules."Attenuating cerebral injury after cerebral ischemia"or"inhibiting postischemic inflammation"is an interesting prospect for targeting the late phase of the damage. The inflammation-related enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase- 2 (COX-2) plays an important role in the secondary events that amplify the damage after cerebral ischemia.So more and more scientists have attented the neuroprotecrion of COX or COX-2 inhibitor,recently. Although administration of selective COX-2 inhibitors has been proven to reduce brain damage following cerebral ischemia. Certain severe side-effects of COX-2 inhibitors have limited them to be used in clinic ,due to disorder the balance of PGI2 and TXA2. So the neuroprotection of non-select COX inhibitors, maintenancing the balance of PGI2 and TXA2 ,was focused recently. As one of the non-select COX inhibitor,flurbiprofen axetil (FA) has treated some inflammatory diseases,fever and pain. we haven't known if it has neuroprotection in a rat transient focal cerebral ischemia model. Hence, the aim of the present study was to investigate the effectiveness of FA on the rat model of middle cerebral artery occlusion (MCAO) when treatment started after rat ischemia/ reperfusion(I/R) and the possible mechanisms after cerebral ischemia/reperfusion were investigated .PartⅠThe effect of flurbiprofen axetil and therapeutic time window against focal cerebral ischemia injury in rats subjected to middle cerebral artery occlusionExperiment one To investigate the protective effect of FA on transient focal cerebral ischemic injury in rats.Methods 48 male SD rats were divided into 6 groups randomly(n=8):Group C(ischemia/ reperfusion,I/R) ;Animals in group S (shame) experienced the identical surgery apart from the insertion of the nylon filament;Animals in group F1 (I/R+FA 5mg/kg),group F2 (I/R+FA 10mg/kg),group F3 (I/R+FA),group L(I/R+lipo-microballoons) were administrated FA5mg/kg,10mg/kg, 20mg/kg or lipo-microballoons 1ml/kg(vehicle of FA) by caudal vein just after MCAO 120min,respectively. The neurological outcome was evaluated at 24 ,48,72h after reperfusion by the method of Garcia .The infarct volume percentage was then assessed after 10g/l TTC staining following the last neurological outcome evaluation.Results The neurologic deficit score (NDS) were significantly increased in group F1 [10.0(7-13)],group F2 [12.0(10-15)]and group F3[11.5(13-16)] at 72 h after reperfusion compared with those of Control group I/R[ 7.0 (6-10) ]( p < 0.05). The infarct volume percentage of Control group I/R (47.07±10.36)% was significantly greater than those of group F1 (34.94±3.52) % ,group F2 (23.52±9.90)% and group F3(25.53±9.46)% at 72 h after reperfusion (p< 0.01). There were no significantly differences of NDS among FA treated groups at 24h,48h,72 h after reperfusion, yet the infarct volume percentage of group F1 is significantly different from the other two treated groups ( p<0.05). there were no significantly different of NDS and the infarct volume percentage between group L and group C.Experiment two Therapeutic time window for FA neuroprotection in a rat transient focal cerebral ischemia modelMethods 32 male SD rats were divided into 4 groups randomly(n=8): group C(ischemia/ reperfusion,I/R);group F6h,group F12h and group F24h,with the rat MCAO model for 120 minutes,the effect of FA(10mg/kg;i.v.) was studied in a situation in which its first administration was delayed for 6,12,24 h by caudal vein after reperfusion,respectively. The neurologic deficit score (NDS) were performed at 72 h after reperfusion. The brain infarct volume percentage were determined with 10g/l TTC staining at 72 h after reperfusion.Results The neurologic deficit score (NDS) were significantly increased in group F6h[10.5(7-13)],group F12h[9.5(7-14)] at 72 h after reperfusion compared with group I/R [7.0 (6-10)] ( p < 0.05). The infarct volume percentage of Control group I/R (47.07±10.36)% was significantly greater than those of group F6h (25.56±10.69)%,group F12h (33.69±14.42)% at 72 h after reperfusion (p< 0.01).PartⅡThe mechanism of flurbiprofen axetil(FA) against focal cerebral ischemia injury in rats subjected to middle cerebral artery occlusionExperiment three PPAR-γcontributes the neuroprotection of FA against focal cerebral ischemia injury in rats.Methods 48 male SD rats were divided into 6 groups randomly(n=8): Animals in group I/R only received 120min MCAO;the animals in group I/R +FA were administrated FA10mg/kg by caudal vein just after 120min MCAO;the animals in group I/R +FA+GW9662 were administrated GW9662(a PPAR-γinhibitor) 4mg/kg intraperitoneally 30min before cerebral ischemia onset and FA10mg/kg by caudal vein just after 120min MCAO;the animals in group I/R +GW9662 were administrated GW9662 4mg/kg intraperitoneally 30min before cerebral ischemia onset; the animals in group I/R +DMSO were administrated 3% DMSO (vehicle of GW9662)1ml/kg intraperitoneally 30min before cerebral ischemia onset;the animals in the sham group experienced the identical surgery apart from the insertion of the nylon filament. The neurologic deficit score (NDS) were performed at 72 h after reperfusion,then the brain infarct volume percentage was determined with10g/l TTC staining.Result The neurologic deficit score (NDS) were significantly increased in group I/R+FA[12.0(10-15)],group I/R+FA+GW9662[10.0(7-13) at 72 h after reperfusion compared with those in group I/R[7.0(6-10)] ( p < 0.05).Yet ,they were not significantly different among group I/R+FA, group I/R+FA+GW9662 and group I/R+FA+DMSO at 72 h after reperfusion ( p > 0.05). The infarct volume percentage of group I/R(47.07±3.664)% is significantly greater than those of group I/R +FA (23.52±3.502)%,of group I/R+FA+GW9662 (33.17±2.527)% (p<0.01), The infarct volume percentage of group I/R +FA(23.52±3.502)% is significantly smaller than those of group I/R +FA+GW9662(33.17±2.527)% (p<0.05) , yet it is no significantly different between group I/R +FA and I/R +FA +DMSO.Experiment four Postischemic treatment with FA reduces blood brain barrier disruption following MCAO in rats partly by activating PPAR-γ.Methods 48 male SD rats were divided into 6 groups randomly(n=8): Animals in group I/R only received 120min MCAO;the animals in group I/R +FA were administrated FA10mg/kg by caudal vein just after 120min MCAO;the animals in group I/R +FA+GW9662 were administrated GW9662 4mg/kg intraperitoneally 30min before cerebral ischemia onset and FA10mg/kg by caudal vein just after 120min MCAO;the animals in group I/R +GW9662 were administrated GW9662 4mg/kg intraperitoneally 30min before MCAO onset; the animals in group I/R +DMSO were administrated 3% DMSO (vehicle of GW9662) 1ml/kg intraperitoneally 30min before cerebral ischemia onset; the animals in the sham group experienced the identical surgery apart from the insertion of the nylon filament. 2% Evans blue 4 ml/kg was injected i.v. at 46 h after reperfusion in groups. Two hours later, the chest was opened.Rats were perfused with saline 400ml through the left ventricle until colorless perfusion fluid was obtained from the right atrium. After decapitation, the hippocampus and cortex was taken out . Samples were weighed and placed in 50% trichloroacetic acid solution. Following homogenization and centrifugation, the extracted dye was diluted with ethanol and its fluorescence was determined. Calculations were based on external standards in the same solvent. The tissue content of EB was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue.Results EB content of the ischemic ipsilateral hippocampus were significantly increased in group I/R(95.08±3.67μg/g tissue)compared with those in group I/R+FA(36.54±2.10μg/g tissue),group I/R+FA+GW9662(44.03±2.11μg/g tissue) I/R+ GW9662组(106.70±3.59μg/g tissue)and Sham group(14.74±0.91μg/g tissue)(p<0.000),but there were significantly decreased compared with those in group I/R+GW9662(106.70±3.59μg/g tissue)(p<0.05). EB content of the ischemic ipsilateral cortex were also significantly increased in group I/R(37.99±2.95μg/g tissue)compared with those in group I/R+FA (20.76±2.19μg/g tissue), group I/R+FA+GW9662 (29.61±2.03μg/g tissue) and Sham group (8.55±0.93μg/g tissue)(p < 0.000). EB content of the right cortex were significantly different between those group I/R+FA and group I/R+FA+GW9662 (p=0.001). EB content of the right cortex were no significantly different between those group I/R+FA and group I/R +GW9662.EB content of the ischemic contralateral hippocampus and cortex were not significantly different among each group(p>0.05).Conclusions1. FA can significantly reduce neurologic injury induced by MCAO in rats with the dose- dependent mode, which appears a ceiling-effectiveness. The best dose of FA is 10mg/kg.2. Therapeutic time window for FA neuroprotection in a rat transient focal cerebral ischemia model is not beyond 12 hours after reperfusion.3. GW9662 (PPAR-γinhibitor) will attenuate the neuroprotection of FA against focal cerebral ischemia injury in rats partly. It is indicated that realized the neuroprotection of FA was by activating PPAR-γ.4. Postischemic treatment with FA would greatly reduce blood brain barrier disruption following MCAO in rats ,however, this effect will be decreased by using GW9662.