Protective Effect And Its Mechanisms Of Flurbiprofen Axetil (FA) Preconditioning Against Focal Cerebral Ischemia-reperfusion Injury In Rats

Now there are 5,700,000 patients dead from stroke annually in the world and the total of its cases is increased by the rate of 10% year-to-year. However, the rate of ischemic stroke was occupied near 83% percent in general . Cerebral ischemic injury always cause the neurological deficits which seriously influence the quality of stroke patient's life, fatally duce to dead. Therefore, it is the main and hot spot in neuroscience reseach how to prevent and cure cerebral ischemic injury, how to look for the effective drugs and measures, and how to promot the neurologic functional recovery after stroke.Cerebral ischemia always is resulted by a transient or permanent local reduction of cerebral blood flow. Recent experimental evidences have been shown that brain damage occurring after focal cerebral ischemia (FCI) would continuse for a relative long period of time, its reason was thought to post-ischemia cerebral inflammative response (PICIR), PICIR appeares a significant disruption of the blood-brain barrier (BBB), and a followed massive infiltration of polymorphonuclear (PMN) leukocytes after several hours of the onset of ischemia, which caused brain oedema , triggered microglial activation and upregulate the high expression of pro-inflammatory cytokines, chemokines, and adhesion molecules. All these neuroinflammative responce mechanisms have been demonstrated to contribute to cerebral ischemic injury.Now attenuating cerebral ischemic injury through inhibiting post-ischemic inflammative responce has been an interesting prospect for targeting the late phase of the damage. The inflammation-related enzymes such as cyclooxygenase (COX) plays an important role in the cerebral ischemic injury. Recently the neuroprotection of COX or COX-2 inhibitors for cerebral ischemic injury have been obtained for more and more attention. Administration of highly selective COX-2 inhibitors has been proven to have the neuroprotective effect. Howevere, Some high selective COX-2 inhibitors have some fatal side-effect, which were coused by the imbalance of PGI2 and TXA2.Non-selective COX inhibitors can still maintenance the balance of PGI2 and TXA2. Moreover recent evidences suggest that selective COX-1 inhibitors possess anti-inflammatory effect and potent antioxidant properties. This compound is able to reduce neuronal injury in vitro and in vivo. So the neuroprotection of non-selective COX inhibitors has been attented again.Flurbiprofen axetil (FA), one of the non-selective COX inhibitors, has treated for some inflammatory diseases, fever and pain. FA can obviously inhibit inflammative reaction and degrade inflammatory damage. Our last research had confirmed that FA had obvious neuroprotection against focal cerebral ischemia / reperfusion injury, Houever,it didn't known FA has same neural-protection.under preconditioning .Therefore, the aim of the present study is to investigate the effect of FA preconditioning against rat focal cerebral ischemia/reperfusion injury and detail its possible mechanisms after focal cerebral ischemia/reperfusion injury .PartⅠThe effect of flurbiprofen axetil (FA) by single preconditioning against focal cerebral ischemia-reperfusion injury in ratsExperiment one To investigate the neuroprotection of FA single preconditioning on transient focal cerebral ischemic-reperfusion injury in rats. Methods Healthy 56 male Sprague-Dawley (SD) rats, 9～10weeks, body weight 280～320g, were divided into 7 groups randomly (n=8): There were group I/R; group FA5, group FA10, group FA20, group FA40 and group Lip. Rats in above goups were administrated FA as dosage as 5mg/kg, 10mg/kg, 20mg/kg, 40mg/kg or lipo-microballoons 1ml/kg (vehicle of FA) by rat caudal vein just 6h (FA T1/2β=5.8h) before middle cerebral artery occlusion (MCAO), respectively. In Sham group, animals were experienced the identical surgery apart from the insertion of the nylon filament. The neurological outcome was evaluated by the method of Garcia at 24h after MCAO. The cerebral infarct volume percentage was then assessed after 1% TTC staining following the neurological outcome evaluation and sacrificing animals.Results Data of the neurologic behavior score was shown that NBS in group FA5, group FA10 and group FA20 were significantly increased when being compared with those of group I/R at 24h after reperfusion [8.5(8-9.5)VS7.0(7-9), 8.5(8-9) VS7.0(7-9) and 9.5(9-12)VS7.0(7-9), P<0.05]. However, when the dosage of FA preconditioning was 40mg/kg, the neuroprotective effect of FA preconditioning was nearly disappeared [7.5(7-8)VS7.0(7-9), P>0.05]. The results of cerebral infarct volume percentage were shown that data in group I/R (46.55±3.27)% was significantly greater than those in group FA5 (30.87±6.01)%, group FA10 (32.32±7.33) % and group FA20 (23.48±4.23)% at 24h after reperfusion (P<0.05). Furthermore, the neuroprotective effect of group FA20 was the best one (P<0.05).Experiment two To investigate the time window of neuroprotection of FA by single preconditioning on transient focal cerebral ischemic-reperfusion injury in rats.Methods Healthy 48 male SD rats, 9～10 weeks, body weight 280～320g, were divided into 6 groups randomly (n=8): group I/R (ischemia/reperfusion); group PC-6h, group PC-12h, group PC-24h, group PC-48h and group PC-72h, the effect of FA (20mg/kg; i.v.) was studied in a situation in which its first administration was ahead of schedule for 6, 12, 24, 48 and 72h by caudal vein before MCAO, respectively. The neurologic deficit score (NDS) were performed at 24h after reperfusion. The cerebral infarct volume percentage was then assessed after 1% TTC staining following the neurological outcome evaluation and sacrificing animals.Results Data of the neurologic behavior score was shown in that data was significantly increased in group PC-6h [9.5(9-12)], group PC-12h [9.0(8-10.5)], group PC-24h [9.0(9-10.5)] and group PC-48h [9.5(8-10)] at 24h after reperfusion being compared with those of group I/R [7.0(7-9)] (P<0.05). The cerebral infarct volume percentage results show: group I/R (46.55±3.27)% was significantly greater than those of group PC-6h (23.48±4.23)%, group PC-12h (24.28±2.17)%, group PC-24h (24.70±3.90)%and group PC-48h (31.58±5.38)% at 24h after reperfusion (P<0.05). However, difference from group PC-72h and group I/R was not statistically significant (P>0.05).PartⅡThe comparison of neuroprotection of flurbiprofen axetil (FA) by repetitional preconditioning and single preconditioning against focal cerebral ischemia-reperfusion injury in ratsExperiment three To investigate the comparison of neuroprotection of FA repetitional preconditioning and single preconditioning on transient focal cerebral ischemia-reperfusion injury in rats.Methods Healthy 32 male SD rats, 9～10 weeks, body weight 280～320g, were divided into 4 groups randomly (n=8): group I/R (ischemia/reperfusion); animals in group PC-1d, group PC-3d and group PC-5d experienced FA 1d single preconditioning or FA 3d and 5d repetitional preconditioning by rat caudal vein before MCAO, respectively. With the rat MCAO model for 2h, the neurologic deficit score (NDS) were performed at 24h after reperfusion. The infarct volume percentage was then assessed after 1% TTC staining following the neurological outcome evaluation and sacrificing animals.Results The results of NBS in group PC-1d, group PC-3d and group PC-5d were 9.0(9-10.5), 10(8.5-12) and 9.5(9-12.5), which were significantly increased at 24h after reperfusion compared with those of group I/R [7.0(7-9)] (P<0.05). The results of cerebral infarct volume percentage were shown that Group PC-1d (24.70±3.90)%, group PC-3d (14.82±4.45)%, and group PC-5d (15.11±4.16)%, which were significantly smaller than those of group I/R (46.55±3.27)% at 24h after reperfusion (P<0.05). Furthermore, the neuroprotection of group PC-3d and group PC-5d was better than group PC-1d (P<0.05). PartШThe partial mechanism of flurbiprofen axetil (FA) preconditioning against focal cerebral ischemia-reperfusion injury in ratsExperiment four To investigate the effect of FA preconditioning on COX-2 in cerebral cortex belonged to perimeter of ischemic region after transient focal cerebral ischemia-reperfusion injury in rats.Methods Healthy 25 male SD rats, 9～10 weeks, body weight 280～320g, were divided into 5 groups randomly (n=5): group normal; group FA20'(simple FA20mg/kg administration); group I/R (ischemia/reperfusion); group FA20 (simple FA20mg/kg+I/R); group PC-3d (repetitional FA20mg/kg+I/R). The dose of FA20mg/kg were administered by rat caudal vein just 6h before MCAO in group FA20'and group FA20. Animals in group PC-3d experienced FA 3d repetitional preconditioning by rat caudal vein before MCAO. At 24h after reperfusion, the rats were perfused with polyoxymethylene, and then the brains were made into frozen sections and immunohistochemistry stained.Result The expression results of COX-2 positive cells in cerebral cortex belonged to perimeter of ischemic region show: The response intensity in group I/R was significantly higher than that in group normal and group FA20'(P<0.05). The immune response intensity and the number of COX-2 positive cells in group FA20 and group PC-3d were significantly smaller than that in group I/R (P<0.05), moreover the immune response intensity and the number of COX-2 positive cells in group PC-3d were few than in group FA20. Conclusions1. FA single preconditioning significantly reduced focal cerebral ischemia- reperfusion injury induced by MCAO in rats, and the best protective effect was also observed as dosage as FA20mg/kg.2. The neuroprotection conferred by single FA was observed even if treatment was ahead of schedule for 48h before MCAO.3. The neuroprotection effect of FA repetitional preconditioning was better than that of FA single preconditioning, and a modest protective effect was also observed with FA 3d repetitional preconditioning.4. FA preconditioning reduced expression of COX-2 in cerebral cortex belonged to perimeter of ischemic region after focal cerebral ischemia-reperfusion injury in rats.