The Role Of Protease-activated Receptor-1in Thrombin-induced Brain Injury

Part1PAR-1mediated brain injury induced by thrombin.Increasing evidences suggest that thrombin play a key role in ischemic,hemorrhagic and traumatic brain injury. The neurotoxic effects of thrombincould be mediated by protease-activated receptors (PARs). In this study, weexamined the role of PAR-1in brain injury induced by thrombin.Male wildtype (WT) and PAR-1knockout (KO) mice received an infusion of0.5unit ofthrombin in10μl saline into the right basal ganglia.(1) Mice had corner turntest, and were killed at days1,3and7for Fluoro-Jade C and PANT stainings,and brain edema.(2) Mice were killed at day28for brain atrophy measurement.(3) Mice were killed at6,12,24,48and72hours later for IL-1β contentmeasurement.(4) Mice were killed at day1for MPO assay.(5) Mice werekilled at days1,3and7for F4/80western blot and immunohistochemistry. InWT mice there were more Fluoro-Jade C positive cells at day-1and PANTpositive cells at day-3after thrombin injection, compared with KO mice. Thethrombin induced brain edema was reduced in KO mice at day-3. Neurologicaldeficits were less in KO mice than in WT mice at day-1, but not days3and7.Brain atrophy induced by thrombin was much more severe in WT mice thanthat in KO mice. The IL-1β protein levels upregulated in WT, but not in KOmice at12hours. There were more neutrophils infiltration and higher levels ofMPO in the ipsilateral basal ganglia of the WT mice at1day after thrombininjection. The number of activated microglia cells and the levels of F4/80weresignificantly higher in WT mice at3days later. PAR-1plays important role inthrombin-induced neuronal death, brain edema formation and brain atrophy viainflammatory responses. Part2PAR-1mediated thrombin-induced hemorrhagictransformationAs shown in part1, PAR-1plays important role in thrombin induced braininjuries. In this study, we examined the role of PAR-1in thrombin inducedhemorrhagic transformation. Male WT and KO mice had an injection of0.5Uthrombin in10l saline into the right basal ganglia. Mice were killed at days1,3,7after performed MRI images and used for western blots, hemoglobinmeasurement, Perls' staining and immunohistochemistry. Compared with thePAR-1knockout group, the wild-type group showed a marked increase in thevolume of T2lesion, especially at3days after surgery. Albumin levels in theipsilateral basal ganglia increase at3days after thrombin injection and thendecreased at day-7. Compared with KO group, albumin levels in the ipsilateralbasal ganglia of WT group were strongly increased at day-3. In KO group,hemoglobin could also be detected in the ipsilateral hemisphere, but less thanWT group. Perls' positive cells were much more in WT group, compared withKO group at day-3. The expression of HO-1were increased as early as1dayafter surgery in WT group, peaked at3days and still be detected at7days afterthrombin injection. There was less expression of HO-1in KO group3daysafter thrombin injection. In WT group the expression of ferritin were increased7days after thrombin injection, and significantly higher than KO group.Thrombin-induced BBB disruption, iron overload and hemorrhagictransformation in mice may be mediated by PAR-1pathway. Part3Deferoxamine treatment reduce thrombin-inducedhemorrhagic transformationHemorrhagic transformation is the major limitation in treat of stroke.There is currently no effective treatment to limit the effect of hemorrhagictransformation after stroke. Deferoxamine (DFX), an iron chelator, reducedbrain injury after intracerebral hemorrhage or ischemic brain damage. In thisstudy, we examined the effect of deferoxamine on thrombin inducedhemorrhagic transformation. Wild-type mice received an injection of0.5Uthrombin into the right basal ganglia and had DFX (100mg/kg) or vehicletreatment at2and6hours, and then every12hours for3days. Mice werekilled at days3after MRI T2*scans and used for hemoglobin content, westernblots and water content determination. DFX treatment reduces T2*lesioncompared with vehicle treated group3days after thrombin injection. Thecontent of hemoglobin and albumin were also decreased in DFX treated groupat day-3. But DFX treatment could not reduce brain water content.Deferoxamine could reduce thrombin-induced hemorrhagic transformation byreduce BBB disruption, hemoglobin and iron overload in mice.