Normobaric hyperoxia inhibits NADPH oxidase-mediated matrix metalloproteinase-9 induction in cerebral microvessels in experimental stroke - PubMed (original) (raw)

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

Wenlan Liu et al. J Neurochem. 2008 Dec.

Abstract

Matrix 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% O2) or normoxia (21% O2) 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.

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Figures

Figure 1

Figure 1

Representative fluorescent micrograph of cerebral microvessl preparation after immunostaining of tight junction protein claudin-5.

Figure 2

Figure 2

TTC staining verified successful MCAO in normoxic and NBO-treated rats. Typical TTC staining of the 1-mm thick brain slice 6 mm away from the tip of frontal lobe after 90-min MCAO with 22.5 (A) or 3 (B) hrs of reperfusion. NBO was delivered during 90-min ischemia.

Figure 3

Figure 3

Gp91phox protein expression in cerebral microvessels after 90-min MCAO with 3- or 22.5-hr reperfusion. Cerebral microvessel lysates (50 μg) were analyzed for gp91phox protein by western blot. As a loading control, the blots were stripped and re-blotted with β-actin antibody. A) Representative blots of gp91phox and corresponding β-actin are shown. Non-I: nonischemic hemispheric microvessels; I: ischemic hemispheric microvessels. B) The relative quantity of protein was calculated after normalization to β-actin. The gp91phox protein expression was significantly increased in the ischemic hemispheric microvessels in those rats reperfused for 22.5 hrs (n = 6, *p < 0.05 versus nonischemic hemispheric microvessels). Only a slight increase of gp91phox was observed for those rats reperfused for 3 hrs (n =5, _P_ > 0.05). Data are expressed as mean ± SEM.

Figure 4

Figure 4

Effect of apocynin on MMP-9 induction in ischemic cerebral microvessels after 90-min MCAO with 22.5-hr reperfusion. Apocynin (30 mg/kg body weight) or vehicle (0.15 ml/kg body weight) was intraperitoneally injected to rats 1 hr before the onset of MCAO. Cerebral microvessel lysates (50 μg) were analyzed for MMP-9 expression with gelatin zymography. A) Representative gelatin zymogram showing MMP-9 expression in the nonischemic (Non-I) and ischemic (I) hemispheric cerebral microvessels. MMP-9 was clearly visualized on the gelatin zymogram, while MMP-2 was barely visible on the gel. STD is a mixture of standard MMP-2 and MMP-9. B) The relative band intensity of MMP-9 was quantified. A significant increase was observed for MMP-9 expression in the ischemic hemispheric microvessels in both vehicle- and apocynin-treated rats (*p < 0.05 versus nonischemic hemispheric microvessels). Apocynin significantly reduced MMP-9 expression in both nonischemic and ischemic hemispheric microvessels (#p < 0.05 versus vehicle-treated rats). Data are expressed as mean ± SEM, n = 3 in the vehicle-treated group, n = 6 in apocynin-treated group.

Figure 5

Figure 5

The effect of NBO on gp91phox-containing NADPH oxidase in ischemic hemispheric microvessels after 90-min MCAO with 22.5-hr reperfusion. A) Representative blots of gp91phoxand corresponding β-actin are shown. Cerebral microvessel lysates (50 μg protein) were analyzed for gp91phox protein by western blot. As a loading control, the blots were stripped and reblotted with β-actin antibody. Non-I: nonischemic hemispheric microvessels; I: ischemic hemispheric microvessels. B) The relative quantity of protein was calculated after normalization to β-actin. Ischemia and reperfusion significantly increased gp91phox protein expression in the ischemic hemispheric microvessels (*p < 0.05 versus nonischemic hemispheric microvessels), which was significantly inhibited by NBO treatment (*p < 0.05 versus normoxic rats). Data are expressed as mean ±SEM, n = 6 in each group. C) NADPH oxidase activity in ischemic hemispheric microvessels was assayed using lucigenin-enhanced chemiluminescence. NBO orapocynin treatment significantly reduced NADPH oxidase activity (*p < 0.05). Data are expressed as mean ±SEM, n = 6 in each of normoxic and NBO-treated group, n = 5 in apocynin-treated group.

Figure 6

Figure 6

Effect of NBO on MMP-9 induction in ischemic cerebral microvessels after 90-min MCAO with 22.5-hr reperfusion. Cerebral microvessel lysates (50 μg) were analyzed for MMP-9 expression with gelatin zymography. A) Representative gelatin zymogram showing MMP-9 expression in the nonischemic (Non-I) and ischemic (I) hemispheric cerebral microvessels. STD is a mixture of standard MMP-2 and MMP-9. B) The relative band intensity of MMP-9 was quantified. A significant increase was observed for MMP-9 expression in the ischemic hemispheric microvessels in both normoxic and NBO-treated rats (*P < 0.05 versus nonischemic hemispheric microvessels). NBO significantly reduced MMP-9 expression in the ischemic hemispheric microvessels (#P < 0.05 versus vehicle-treated rats). Data are expressed as mean ±SEM, n = 6 in each group.

Figure 7

Figure 7

Effect of NBO on brain edema formation and BBB permeability after 90-min MCAO with 22.5-hr reperfusion. A) Representative brain sections of the normoxic, NBO-, vehicle- and apocynin-treated rats. Significant right (ischemic) hemispheric enlargement is seen in rats of all groups. B) Quantitative image analysis showed a significant reduction of hemispheric enlargement in the NBO- or apocynin-treated rats when compared to normoxic rats. Data are expressed as mean ±SEM, n = 4 in the vehicle-treated group, n = 7 in each of other three groups. *P < 0.05 versus normoxic rats. C) Effect of NBO on BBB permeability coefficient assessed by MRI based technique. A significant increase was observed for BBB permeability coefficient in the ischemic hemisphere of both normoxic and NBO-treated rats (*P < 0.05 versus nonischemic hemisphere). NBO significantly reduced BBB permeability in the ischemic hemisphere (#P < 0.05 versus normoxic rats). Data are expressed as mean ± SEM, n = 6 in each group.

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