Alterations in the time course of expression of the Nox family in the brain in a rat experimental cerebral ischemia and reperfusion model: effects of melatonin

Abstract

Ischemia–reperfusion (I/R) injury induces the generation of reactive oxygen species (ROS), which results in a poor prognosis for ischemic stroke patients. 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 After 2‐hr occlusion, the filament was withdrawn to allow reperfusion.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. Among the Nox family, the mRNA 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. In addition, melatonin (5 mg/kg) or its vehicle was injected intraperitoneally at 0.5 hr before MCAO. Compared with I/R + vehicle group, melatonin pretreatment diminished the increased expression of Nox2 and Nox4, reduced ROS levels, and inhibited cell apoptosis. Our findings suggested that the inhibition of Nox2 and Nox4 expressions by melatonin may essentially contribute to its antioxidant and anti‐apoptotic effects during brain I/R.

Hydrogen Sulfide Ameliorates Early Brain Injury Following Subarachnoid Hemorrhage in Rats

“…As described previously [32], we costained brain sections for CBS and NeuN or MST and NeuN to evaluate the expressions of CBS and 3MST in neurons. All the primary antibodies were applied at dilution of 1:100, and all the second antibodies were diluted at 1:500.…”

“…Normal rabbit IgG was used as a negative control (data not shown). The fluorescence images were captured, and the relative fluorescence intensity was analyzed as described in our previous study [32].…”

NADPH oxidase in brain injury and neurodegenerative disorders

“…Additional studies revealed that NOX2 is primarily localized in neurons and endothelial cells at early time-points (3–6 h) after tGCI, followed by expression in microglial cells at later time-points (72 hr) [132, 134]. Likewise, studies in focal cerebral ischemia animal models also found that NOX2 and NOX4 mRNA and protein, as well as NOX activity are elevated in the peri-infarct region of the cerebral cortex from 2–48 h after reperfusion [135–138]. Few studies have examined later time-points, but one study found NOX2 protein expression was markedly elevated in the peri-infarct region of the cortex up to 2 weeks after focal cerebral ischemia [139].…”

Detrimental role of pericyte Nox4 in the acute phase of brain ischemia

“…34 Unexpectedly, the upregulation of Nox4 was gradually induced and reached maximal levels at day 4 after brain ischemia, and was higher in pMCAO than in tMCAO (Figures 1b and c). Although there is a paper demonstrating the immediate upregulation of Nox4 after brain ischemia, 35 some papers have shown that Nox4 is significantly upregulated at 24 hours after MCAO in mice [36][37][38] . In particular, Kleinschnitz et al 36 demonstrated the significant upregulation of Nox4 in the cortex at later than 24 hours after brain ischemia.…”