Normobaric hyperoxia inhibits NADPH oxidase‐mediated matrix metalloproteinase‐9 induction in cerebral microvessels in experimental stroke

Abstract

Abbreviations used: BBB, blood-brain barrier; Gd-DTPA, gadolinium-diethylenetriamine pentaacetic acid; MCAO, middle cerebral artery occlusion; MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; NBO, normobaric hyperoxia; PBS, phosphate-buffered saline; TTC, 2,3,5-triphenyltetrazolium chloride. AbstractMatrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O 2 ) or normoxia (21% O 2 ) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91 phox and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91 phox expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91 phox containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.

NADPH oxidases as therapeutic targets in ischemic stroke

“…In the transient MCAO model, Nox4 protein content increased in the ischemic hemisphere within hours of reperfusion and was elevated in murine cerebral vessels and human brain endothelial cells following ischemic stroke. Likewise, Nox2 protein was found to increase after cerebral ischemia in rodents, with significant amounts observed in microvessels [120] and endothelial cells at 24 and 72 h following stroke onset [118]. At the same time Nox activity was enhanced in cerebral arteries of the penumbra for 3 days [123].…”

“…In rodent cerebral blood vessels, Nox1, Nox2, Nox4, p22, p47, p67, NOXO1, and NOXA1 [56,107,[109][110][111] have been identified at the mRNA level, and Nox1, Nox2, Nox4, p22, and p47 proteins [112][113][114][115][116][117][118][119][120][121][122] have been described. Remarkably, Nox4 protein was reported to be expressed tenfold higher in the basilar artery than in the aorta [116].…”

Effect of Normobaric Oxygen Therapy in a Rat Model of Intracerebral Hemorrhage

“…NBO is beneficial in experimental models of cerebral ischemia. [7][8][9][10][11][12][13] However, to our knowledge, NBO has never been tested in ICH. Because there are many overlapping mechanisms in ischemia and hemorrhage, it was theoretically possible that NBO could also be protective after hemorrhage.…”

Synergetic Neuroprotection of Normobaric Oxygenation and Ethanol in Ischemic Stroke Through Improved Oxidative Mechanism

“…16 Brain homogenized samples containing phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Thermo; 20 μL) were added to a 96-well luminescence plate containing 6.25 μmol/L of lucigenin. The reaction was initiated by the addition of nicotinamide adenine dinucleotide phosphate (100 μmol/L).…”