Polydatin Ameliorates Blood-brain Barrier Disruption After Transient Focal Cerebral Ischemia In Mice

Objective: Recently,stroke has been one of the most common diseases threatening public life and health.Reperfusion strategies have proven to be the most effective therapies for stroke treatment.However,the good strategies brings risk as well,including the disorder of the blood-brain barrier,which can lead to cerebral edema even intracranial hemorrhage.The pathological process of ischemic stroke is complicated,involving excitotoxicity mechanisms,inflammatory pathways,oxidative damage,ionic imbalances,apoptosis and so on,these secondary injuries can destroy the BBB further and deteriorate edema.Polydatin(PD)has been reported to be protective against myocardial and intestinal ischemia/reperfusion(I/R)injury.Mounting research proved that PD has lots of effect,including anti-inflammatory,anti-oxidant,anti-apoptosis,etc.However,the effect of PD on BBB is not clear,Our study aim to investigate whether polydatin has an effect on the breakdown of BBB after ischemia-reperfusion and the possible underlying mechenism.Methods: Male and healthy CD-1 mice(25-30g)were used and subjected to modified transient middle cerebral artery occlusion(tMCAO)model.CD-1 Mice were divided into 4 groups randomly: Sham group: mice received sham operation only,intraperitoneal injection with equal volume of normal saline after sham operation;tMCAO group: mice received tMCAO surgery and intraperitoneal injection with equal volume of normal saline;PD-L group: mice received tMCAO and intraperitoneal injection with polydatin 30mg/kg after surgery;PD-H group: mice received tMCAO and intraperitoneal injection with polydatin 60mg/kg after surgery.Neurological behavior was evaluated by neurological deficit scores;infarct volume was determined by 2,3,5-triphenyltetrazolium chloride(TTC);cerebral blood flow was detect by the Laser Speckle Contrast Analysis(LASCA);brain watercontent was measured using the standard wet-dry method;evans blue exudation was used to evaluated the permeability of BBB;western blot were employed to detect the expression of CAV-1,ZO-1,Occludin,and Claudin-524 h after tMCAO;the distribution of CD-31,ZO-1,and Claudin-5 was observed by immunofluorescence microscope 24 h after tMCAO.Results:1 Neurological deficit scores: 24 h after t MCAO,the neurological deficit was examined and scored.Compared with Sham group,mice in tMCAO group,PD-L group and PD-H group show a left palsy.There was no statistical difference between tMCAO group and PD-L group.Scores were significantly ameliorated in PD-H group than tMCAO group(PD 60 mg/kg vs.tMCAO P<0.05).Though lower scores of PD-L group than tMCAO group,there was no significant difference(PD 30 mg/kg vs.t MCAO P>0.05).Thus,our subsequent study chose the PD-H group to evaluate the effect of PD.2 Infarct volume was measured by TTC 24 h after tMCAO.In Sham group,there is no infarction was observed.An extensive lesion was detected in tMCAO and PD group,and a significant difference between the two groups,the infarct volume was significantly reduced from 57.95% ± 9.86% in tMCAO group to 32.93% ± 15.16% in PD group.(P < 0.05)3 Cerebral blood flow: 1h after reperfusion,ipsilateral CBF increased obviously,24 h later further adding close to the baseline.Compared with the tMCAO group,the CBF of PD group is higher,but with no significant defference.(PD vs.tMCAO:78.7% ± 14.8% vs.65.5 ± 7.2%,P > 0.05)4 PD declined the brain edema: compared with the tMCAO group,the brain water content of ipsilateral hemispheres was significantly reduced in PD group 24 h after tMCAO(P < 0.05).5 PD ameliorated the leakage of evans blue: there were no leakage of evans blue in sham group;leakage of evans blue was very obvious in both tMCAO group and PD group at 24 h after tMCAO.However,compared with tMCAO group,there was a statistically significant decreased of the permeability of BBB in PD group.6 The effect of PD on the expression of ZO-1?Occludin?Claudin-5?Cav-1: as is shown by the results of western blot: the level of ZO-1?Occludin?Claudin-5,was up-regulated significantly in the PD group 24 h after operation,compared with tMCAO group(P < 0.05).The levels of CAV-1 was increased in both tMCAO group and PD group,and CAV-1 in the ischemic cortex tended to be lower in PD group,with a significant defference(P < 0.05).7 The effect of PD on the distribution of CD-31,ZO-1,Claudin-5 in the ischemic penumbra.Immunofluorescence method showed that: The expression of CD-31 was up-regulated in the cortex ischemic penumbra,and the tube shape of microvascular was smooth and integral.However,we can see the rough and broken shape of microvascular in the tMCAO group.Meanwhile,the disruption of ZO-1 and Claudin-5 staining was reduced in the PD group,compared with t MCAO group.Conclusions: Polydatin administration alleviated the neurological deficit,declined the infarct size,ameliorated the brain edema,reduced the permeability of BBB 24 h after tMCAO,meanwhile it could up-regulated the expression of ZO-1,Occludin,Claudin-5 and CD31,down-regulated Cav-1level in the cortex ischemic penumbra,suggesting that it could ameliorate the breakdown of BBB caused by ischemia-reperfusion.