Expression Of MRNA And Protein Of MCP-1 And CCR2 And Change Of Activated Microglia In Newborn Rat Brain With Experimental Hypoxic-Ischemic Brain Damage

ObjectiveTo observe the expression of mRNA and protein of MCP-1 and CCR2 and the alteration of active microglia in cerebral tissue after experimentalhypoxic- ischemic brain damage (HIBD ) in newborn rat.MethodsThe HIBD model of SD newborn rat was setup by ligating the right carotidartery and exposing the animals to 8% oxygen for 2.5 hours. The dynamic change of expression of MCP-1 and CCR2 mRNA were studied by using quantitative, real-time, fluorogenic PCR (TaqMan) assay. The change of expresson of MCP-1 and CCR2 protein were studied by using immuno-fluorescin histochemistry and Western Blotting assay. To observe the change of active microglia in the HIBD model , EDI monoclonal antibody and immunofluorescence histochemistry were used. Results1, Peak expression of MCP-1 mRNA was shown at 6 hours after HIBD and it's level in the HI cerebral hemisphere was significantly higher than that of the control group (p< 0.05) and the level at 12 hours after HIBD was still greater than that of the control group (p < 0.05) , The expression of MCP-1 mRNA was declined at 3d after HIBD in the HI cerebral hemisphere and there was no difference compared with that in the control group (p > 0.05) . 2, From 6h to 24h after HIBD, MCP-1 immunocytochemistry assays indicated that the value of MCP-1 -immunoreactive cells in the HI cerebral hemisphere was greater than the control group (p < 0.05) . Peak expression was shown at 12h and the high level persisted for 24h after HIBD (p < 0.05). MCP-1-immunoreactive cells was detected in the right hippocampus as early as 3h after HIBD.3, Peak expression of CCR2 mRNA was shown at 24h after HIBD and it's level of the HI cerebral hemisphere was significantly higher than that of the control group (p < 0.05) . The CCR2 mRNA level of 72 hours after HIBD was still greater than that of the control group (p < 0.05) and was dropped at 7d after HIBD and there was no difference compared with the control (p >0.05) .4, Increased expression of CCR2 protein was detected at 12h after HIBD andit's content peaked at 24h after HIBD compared with the control (p < 0.05) .Significant increase of CCR2 protein content persisted in HI hemisphere upto 3d after HIBD.5, The number of activated microglia began to increase at 12h after HIBD inHI cerebral hemisphere and right hippocampus .It persisted in high level at24h to 3d and peaked at 3d. ED1-immunoreactive cells were apparent in theright hippocampus as early as 12h after HIBD.There was dense distributionof EDI- immunoreactive cells in the focal infarction region.Conclusion1 , In the HIBD new-born rat, the expression level of mRNA and protein ofMCP-1 and it's receptor CCR2 increased significantly compared with thecontrol group,which suggests that as inflammatory mediators MCP-1 andCCR2 mRNA expression after HIBD may play an important role in themechanism of brain damage.2, In the HIBD new-born rat, the dynamic change of microglia response wasin accordance with that of the MCP-1 mRNA and protein level,but it'schange was approximately 12h~24h later than MCP-1's change. Thedistribution of ED1-immunoreactive cells in the brain was in accordancewith that of the MCP-1 -immunoreactive cells ,and both positive signal werefocused in right hippocampus, thalamus and focal infarction regions in rightcortex. The change of CCR2 protein was also in accordance with themicroglia response. Above results suggest that MCP-1 and CCR2 expressionafter HIBD may participate in the mechanism of brain damage by inducingand regulating the microglia response. As an inflammatory mediator ,chemokines may play a pivotal role in the pathogenesis of HIBD.