Alterations In The Time Course Of Expression Of The Nox Family And ROS In The Brain In A Rat Experimental Cerebral Ischemia And Reperfusion Model: Protective Effects Of Nox2- And Nox4-specific Inhibitors

PART 1: Alterations in the time course of expression of the Nox family in the brain in a rat experimental cerebral ischemia and reperfusion model.Background: Ischemia–reperfusion(I/R) injury induces the generation of reactive oxygen species(ROS), which results in a poor prognosis for ischemic stroke patients. Methods: This study was designed to evaluate the time course of expression of the Nox family, a major source of ROS, and whether melatonin, a potent scavenger of ROS, influences these parameters in a rat model of cerebral I/R caused by middle cerebral artery occlusion(MCAO). After 2-hr occlusion, the filament was withdrawn to allow reperfusion. At 0, 3, 6, 12, 24, and 48 hr after reperfusion, brain tissue samples were obtained for assays.Results: Among the Nox family, the m RNA and protein levels of Nox2 and Nox4 were increased both in the ischemic hemisphere and contralateral counterpart in the experimental I/R rats at 0 hr after reperfusion, peaked at 3 hr, and then returned to the basal level at 24 hr. Double-immunofluorescence staining further confirmed the expressions of Nox2 and Nox4 in three major types of brain cells, including neurons, astrocytes, and endothelial cells.Conclusion: Firstly, TTC staining displayed the stability and reproducibility of the ischemia model used in this study. Then, a time course study for the expression of the Nox family after I/R was performed by RT-PCR and Western blot. Among the Nox family, the expressions of Nox2 and Nox4 in the ischemic hemisphere were significantly enhanced from 0 hr after reperfusion and reached a peak level at 3 hr, decreased at 24 hr, and then were similar to the levels in the sham group. Interestingly, the expressions of Nox2 and Nox4 were also increased in the contralateral nonischemic brain. As well, a similar trend was detected in the expression of Duox1, but only in the ischemic hemisphere of the brain. As alterations in the expressions of Nox2 and Nox4 were more significant than that of Duox1, we focused on Nox2 and Nox4 in the following study. To determine the cell identity for Nox2 and Nox4 expressions, double-immunofluorescence staining with the corresponding cell-type-specific marker antibodies was performed at 3 hr after reperfusion. The results revealed that both Nox2 and Nox4 expressions were significantly increased in three major types of brain cells, including neurons, astrocytes, and endothelial cells. Similarly, ROS production in the ischemic hemisphere was obviously higher than that of sham group at 3 hr after reperfusion and remained so to 48 hr or longer, while in the contralateral counterpart of the brain, ROS production was also significantly enhanced at 12 and 24 hr after reperfusion.PART 2: Protective Effects of Nox2- and Nox4-Specific Inhibitors on Cerebral Ischemia and Reperfusion-Induced Brain Damage in RatsBackground: Studies have shown that non-phagocytic cell oxidases(Nox) members, Nox2 and Nox4, participate in brain damage caused by ischemia and reperfusion. The aim of this study was to investigate the effects of Nox2 and Nox4-specific inhibitors on cerebral ischemia and reperfusion-induced brain injury in rats.Methods: 32 adult male Sprague Dawley(SD) rats were randomly divided into two experimental groups: sham operation group(Sham group, n = 8) and middle cerebral artery occlusion group(MCAO group, n = 24). MCAO was prepared by thread-thrombus method. The artery was obstructed for 2h, followed by reperfusion for 24 h. MCAO group was then subdivided into three groups: At 0.5h before MCAO surgery, the rats were pretreated with vehicle control(n = 8), Nox2 inhibitor(gp91ds-tat, n = 8), Nox4 inhibitor(GKT37831, n = 8). After reperfusion for 24 h, rats were euthanized, and the infarct size was determined. The penumbra(ischemic) was taken to measure ROS levels, apoptosis and necrosis, as well as the integrity of the blood-brain barrier, in brain tissue of the rats.Results: Nox2 inhibitor(gp91ds-tat) and Nox4 inhibitor(GKT37831) pre-treatment significantly reduced the infarct size, and could effectively suppress ischemia-reperfusion-induced increase in ROS levels, neuronal apoptosis and necrosis, and alleviated blood-brain barrier damage.Conclusion: Under ischemia-reperfusion conditions, Nox2 inhibitor(gp91ds-tat) and Nox4 inhibitor(GKT37831) can effectively play a protective role in the brain.