Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND： Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE： To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS： A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 （Santa Cruz, USA） and Evans blue （Sigma, USA） were used to analyze the tissue. METHODS： The rats were randomized into sham-operated （n = 16） and ischemia/reperfusion （n = 96） groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES： Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS： Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Evans blue content significantly increased on the ischemic side at 4 hours after ischemia/reperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group （P 〈 0.05）. Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion （r1 = 0.68, r2 = 0.81, P 〈 0.05）. CONCLUSION： Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.