RNA was dissolved in 1 mM EDTA and treated with TURBO DNA-freeTM Kit prior to RTPCR

ility of BBB was caused by aberrant higher activity and expression of matrix metalloproteinases such as MMP-9 and MMP-2. However, accumulating evidences revealed that MMP-9 played a crucial role in modulating the permeability of BBB under various pathological conditions such as head trauma, acute liver failure, focal or global ischemia and reperfusion. Particularly, it was reported that MMP-9 activity elevated significantly in humans after stroke. By contrast, inhibition of MMP-9 either by synthetic inhibitor or by specific monoclonal antibodies was proved to be an effective way to reverse BBB breakdown and attenuate brain extravasation and edema. By now, evidences showed that MMP-9 modulating BBB permeability is via degradation of BBB constituents, such as tight junction proteins occludin and claudin-5, and basal lamina proteins fibronectin, laminin and 21152046 heparan sulfate. Therefore, we focused on MMP-9, occludin and claudin-5 in this study to investigate the mechanism of BBB disruption caused by early relief of carotid stenosis. Ischemic postconditioning is emerging as an effective method to protect injury due to ischemia and reperfusion in many organs, such as heart, lung, kidney, liver and intestine. It is defined as a series of rapid intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of blood flow. In the clinical study, it was found that ischemic postconditioning could decrease heart edema caused by revascularization of stenotic artery and protect endothelial function. In the rat experiment, ischemic postconditioning could decrease brain edema and attenuate the disruption of BBB by inhibiting expression of MMP-9 and attenuating loss of the extracellular matrix proteins. 9721015 These results indicate that ischemic postconditioning might be effective in preventing vasogenic edema due to cerebral ischemia and reperfusion. Particularly, ischemic postconditioning could be performed before re-establishment of blood supply to brain, which makes it become a feasible method that could be used potentially in future clinical practice. Moreover, no reports proved that ischemic postconditioning had any negative effects by now. Despite we have demonstrated in previous study that ischemic postconditioning could rescue neuronal death following early relief of carotid stenosis, the effects of ischemic postconditioning on brain edema and the permeability of BBB is still unclear, we thus investigated as well this issue in this study via using a rat model of cerebral hypoperfusion. Materials and Methods Animals Adult male Wistar rats supplied by Jilin University Experimental Animal Center were housed in plastic cages with soft bedding and free access to food and water at controlled room temperature under a 12:12 h day/night cycle, and they were kept for 7 d before the experiments. All animal procedures were approved by the ethical committee for animal experiments, Jilin University, Changchun, China. Efforts were made to minimize animal suffering and to keep the number of animals used at a minimum. Surgical procedure and postconditioning protocol A method described previously was used in this experiment to make severe carotid stenosis with a slight modification. Briefly, the rats were fasted overnight and AIC316 web freely accessed to water. Anesthesia was induced with 5% halothane and maintained with 2% halothane in oxygen/nitrous oxide gas mixture. Through a midline cervical incision, the skin and muscle