| Background and Objective:Clot lysis with recombinant tissue plasminogen activator (rt-PA) is effective in acute ischemic stroke, it is however associated with a risk of hemorrhagic transformation. Previous studies have reported that blood-brain barrier (BBB) disruption is associated with hemorrhagic transformation. Our previous study indicated that the ratio of hemorrhagic transformation was reduced in the rats by transforming growth factor-betal (TGF-β1) treatment. Thus, we hypothesized that TGF-β1 could protect BBB integrity to prevent hemorrhagic transformation after rt-PA treatment. The purpose of this study was to investigate the effects and protective mechanisms of TGF-β1 on the BBB integrity in thrombolytic therapy.Methods:Adult male Sprague-Dawley rats (n=78) weighing 300-350g were subjected to a model of thromboembolic middle cerebral artery occlusion (MCAO). The rats were randomly divided into four groups:the sham-operated group (n=13), neither injects clots nor specific treatment; the control group (n=23), underwent MCAO and received intravenous saline immediately after MCAO 3h; the thrombolysis group (n=21), underwent MCAO and intravenous thrombolysis with rt-PA 3h after MCAO; the rt-PA+TGF-β1 group (n=21), underwent MCAO and intravenous thrombolysis with rt-PA and TGF-β1 3h after MCAO. Neurological examination was performed at 2h,6h,12h and 24h after MCAO. Twenty-four hours after surgery, rats were sacrificed. Brain infarct volume was determined by 2,3, 5-triphenyl tetrazolium chloride (TTC) staining. Edema index and hemorrhage score was calculated. Blood-brain barrier permeability was measured by Evans Blue dye leakage and the integrity of BBB by transmission electron microscopy. The expression of collagen Ⅳ and laminin were performed by immunocytochemistry. The activity of matrix metalloproteinases (MMP)-2 and MMP-9 in tissue extracts were detected by zymography. The expression of MMP-2 and MMP-9 in tissue extracts were detected by Western bolt. The concentration of MMP-2, MMP-9, plasminogen activator inhibitor type-1 (PAI-1), IL-6, IL-10, IL-1β and monocyte chemoattractant protein-1 (MCP-1) in tissue extracts were determined by ELISA.Results:Neurological function was improved and the infarct volume was decreased in the thrombolysis and the TGF-β1 group. Compared with the control group, BBB permeability and the hemorrhage after thrombolysis score were significantly increased by rt-PA treatment. However they were reduced by TGF-β1 treatment. Electron micrograph of BBB ultrastructure:in the sham-operated group, we can see the complete basement membrane and tight junctions; in the control group, the basement membrane was disrupted; in the thrombolysis group, the basement membrane disruption was more serious and tight junctions disappeared; in the TGF-β1 group, the basement membrane was basically complete and tight junctions were visible. In the sham-operated group, positive collagen Ⅳ and laminin immunoreactivity was easily identified in extracellular matrix. Compared with the sham-operated group, there was a significant decrease in the control group. And it was reduced further by rt-PA treatment. However, the collagen Ⅳ and laminin immunoreactivity was obviously increased by TGF-β1 treatment. In the thrombolysis group, the activity, expression and concentration of MMP-2 and MMP-9 were significantly increased compared with the control group. However they were obviously reduced by TGF-β1 treatment. A significant decrease of PAI-1 was detected by rt-PA treatment when compared to control group. This high level of protein expression was significantly improved by TGF-β1 treatment. The concentration of IL-6, IL-10, IL-1β and MCP-1 were significantly increased in the control group compared with the sham-operated group. They were reduced by rt-PA treatment. Particularly, the concentration of IL-6, IL-1β and MCP-1 were further reduced by TGF-β1 treatment.Conclusion:TGF-β1 exerted protection against the integrity of BBB disruption by reduced the breakdown of the collagen IV and laminin, by decreased MMP-2 and MMP-9 levels, by diminished the concentration of inflammatory factors after rt-PA thrombolysis. Moreover, TGF-β1-mediated increase in PAI-1 concentration was correlated with reduced rt-PA activity. Thus, we firmly believe that TGF-β1 might prevent hemorrhagic transformation after rt-PA thrombolysis. |
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