Inhibition Of RhoA/ROCK Signaling Pathway And Its Role In Hemorrhagic Transformation After Cerebral Ischemia/Reperfusion

Part I Inhibition of Rho kinase by fasudil attenuateshemorrhagic transformation after ischemia/reperfusion indiabetic and non-diabetic MCAO RatsObjective: In the present study, we investigated whether fasudil attenuatedhemorrhagic transformation after ischemia/reperfusion in diabetic andnon-diabetic MCAO Rats.Methods:140Male Sprague-Dawley rats were randomly divided intoexperimental group(120) and control group（20）. Then, the experimentalgroup were randomly divided into4groups:（1） Cerebralischemia/reperfusion+saline（CIRS），（2）Cerebral ischemia/reperfusion+Fasudil（CIRF），（3）Cerebral ischemia/reperfusion with type1diabetesmellitus+saline（DMCIRS），（4）Cerebral ischemia/reperfusion with type1diabetes mellitus+Fasudi（lDMCIRF）. All the rat in experimental groupwere subjected to occlusion of the right middle cerebral artery (MCAO) for2h.5mg/kg Fasudil was intraperitonealy injected immediately after reperfusion twice a day for1day to block ROCK. The neurologicalseverity score were performed to evaluate functional outcome andblood-brain barrier (BBB) permeability was assessed by Evan’s blue dyeextravasation (EB) at26hours after stroke. The intracerebral hemorrhageafter ischemia/reperfusion were quantificated by QuantiChromHemoglobin Assay Kit at the expected time point. ROCK activities weremeasured by ROCK activitiy kits. The expression of occludin，MMP9inischemic cortex and oxidative stress levels were measured byimmunofluorescence staining， Western blotting or oxidative stressreaction assay Kit26hours after MCAO respectively.Results：Fasudil significantly reduced the infarct sizes，extravasation ofEB and intracerebral hemorrhage at a dose of5mg/kg twice a day innon-diabetic and diabetic MCAO/R rat respectively (P<0.05). There werelarger infarct sizes， much more EB extravasation and intracerebralhemorrhage， higher ROCK activity and oxidative stress， increasedexpression of MMP9levels，significant decrease endothelial occludinexpression in diabetic MCAO/R rat compared to the non-diabetic groups(P<0.05). Furthermore, as an ROCK inhibitior， fasudil significantlyattenuated endothelial occludin degradation and decreased MMP9expression26hours after MCAO.Conclusions: The hemorrhagic transformations after ischemia/reperfusionin diabetic rats were obviously serious compared to the non-diabetic groups. Fasudil significantly attenuated blood-brain barrier permeability andreduced intracerebral hemorrhage volumes via blocking RhoA/ROCKsignaling pathway. Part II Peroxisome proliferator-activated receptor γagonist attenuates hemorrhagic transformation viaRhoA/ROCK signaling pathway after ischemia/reperfusion inMCAO RatsObjective: In the present study, we investigated whether peroxisomeproliferator-activated receptor γ agonist pioglitazone attenuatedhemorrhagic transformation after ischemia/reperfusion in MCAO Rats.Methods:140Male Sprague-Dawley (SD) rats were randomly divided intoexperimental group(120) and control group（20）. Then, the experimentalgroup were randomly divided into6groups:（1）MCAO+placebo/DMSO（CIRP）；（2） MCAO+Vehicle （CIRV）；（3） MCAO+Vehicle+placebo/DMSO（CIRVP）；（4）MCAO+PPARγ RNA interference（CIRI）；（5）MCAO+PPARγ RNA interference+PPARγ agonist（CIRIA），（6）MCAO+PPARγ agonis（tCIRA）. All of rats in the experimental group weresubjected to occlusion of the right middle cerebral artery (MCAO) for2h.Specific small interference RNA (siRNA) lentivirus vectors were infectedinto the rat’s brain to interference PPARγ expression.5mg/kg PPARγagonist pioglitazone was intraperitonealy injected immediately afterreperfusion twice a day for1day. The neurological severity score wereperformed to evaluate blood-brain barrier permeability was assessed byEvan’s blue dye extravasation (EB) at26hours after stroke.The intracerebral hemorrhage after ischemia/reperfusion were quantificatedby QuantiChrom Hemoglobin Assay Kit at the expected time point.ROCK activities were measured by ROCK activitiy kits. The expression ofoccludin，MMP9，active RhoA,total RhoA in ischemic cortex and oxidativestress levels were measured by immunofluorescence staining，Westernblotting or oxidative stress reaction assay Kit26hours after MCAOrespectively.Results：RhoA activities after ischemia/reperfusion were down-regulatedby PPARγ agonist pioglitazone, and up-regulated by PPARγ RNAinterference. PPARγ RNA interference decreased RhoA and ROCKactivities, and worsen hemorrhagic transformation and increasedblood-brain barrier permeability. Pioglitazone could significantly reducethe infarct sizes，extravasation of EB and intracerebral hemorrhage at adose of5mg/kg twice a day in normal MCAO/R rat (P<0.05)，but not inMCAO/R groups with PPARγ RNA interference(P>0.05). There weremuch more EB extravasation and intracerebral hemorrhage， higherRhoA/ROCK activity and oxidative stress，increased MMP9expression，and decreased endothelial occludin expression in CIRP，CIRV and CIRVPcompared to CIA(P<0.05). Furthermore, hemorrhagic transformation,blood-brain barrier permeability, RhoA/ROCK activity, expression ofMMP9and occludin oxidative stress were not significantly different amongCIRP，CIRV and CIRVP(P>0.05). Those difference between CIRI and CIRIA were not significant too(P>0.05).Conclusions: The hemorrhagic transformation after ischemia/reperfusionin PPARγRNA interference MCAO/R rat was obviously serious comparedto the control. Pioglitazone significantly attenuated blood-brain barrierpermeability and reduced intracerebral hemorrhage volumes via blockingRhoA/ROCK signaling pathway.