Cyclooxygenase (COX)-2 and
matrix metalloproteinase (MMP)-9 are two crucial mediators contributing to blood-brain barrier (BBB) damage during
cerebral ischemia. However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in
ischemic stroke. In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of
ischemic stroke. Male COX-2 knockout (COX-2-/-) and wild-type (WT) mice were subjected to 60 min of
middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Genetic deletion of COX-2 or post-ischemic treatment with CAY10404, a highly selective
COX-2 inhibitor, significantly reduced BBB damage and hemorrhagic transformation, as assessed by
immunoglobulin G (
IgG) extravasation and brain
hemoglobin (Hb) levels, respectively. Immunoblotting analysis showed that
tight junction proteins (TJPs) zonula occludens (ZO)-1 and
occludin as well as
junctional adhesion molecule-A (JAM-A) and the basal lamina
protein collagen IV were dramatically reduced in the ischemic brain.
Stroke-induced loss of these BBB structural
proteins was significantly attenuated in COX-2-/- mice. Similarly,
stroke-induced loss of ZO-1 and
occludin was significantly attenuated by CAY10404 treatment.
Ischemia-induced increase in MMP-9
protein levels in the ipsilateral cerebral cortex was significantly reduced in COX-2-/- mice.
Stroke induced a dramatic increase in MMP-9 enzymatic activity in the ischemic cortex, which was markedly reduced by COX-2 gene deficiency or pharmacological inhibition with CAY10404. Levels of
myeloperoxidase (MPO, an
indicator of neutrophil infiltration into the brain parenchyma),
neutrophil elastase (NE), and
lipocalin-2 (LCN2, also known as
neutrophil gelatinase-associated lipocalin), measured by western blot and specific ELISA kits, respectively, were markedly increased in the ischemic brain. Increased levels of markers for neutrophil infiltration were significantly reduced in COX-2-/- mice compared with WT controls following
stroke. Altogether, neurovascular protective effects of COX-2 blockade are associated with reduced BBB damage, MMP-9 expression/activity and neutrophil infiltration. Our study shows for the first time that MMP-9 is an important downstream effector contributing to COX-2-mediated neurovascular damage in
ischemic stroke. Targeting the COX-2/
MMP-9 pathway could represent a promising strategy to reduce neuroinflammatory events in order to preserve the BBB integrity and ameliorate
ischemic stroke injury.