Published Online
on April 1, 2004

A more recent version of this article appeared on May 1, 2004

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Submitted on December 15, 2003
Revised on January 20, 2004
Accepted on January 22, 2004

Matrix Metalloproteinase-9 and Myeloperoxidase Expression. Quantitative Analysis by Antigen Immunohistochemistry in a Model of Transient Focal Cerebral Ischemia

Carolina M. Maier PhD; Lily Hsieh BA; Fengshan Yu MD; Paige Bracci PhD; and Pak H. Chan PhD^*

From the Department of Neurosurgery (C.M.M., L.H., F.Y., P.H.C.), Department of Neurology and Neurological Sciences and the Program in Neurosciences, Stanford University School of Medicine, Stanford, Calif; and the Department of Neurology (P.B.), University of California at San Francisco, Calif.

^* To whom correspondence should be addressed. E-mail: phchan{at}stanford.edu.

Background and Purpose--Expression of matrix metalloproteinases (MMPs), proteolytic enzymes that degrade extracellular proteins, is altered after ischemia/reperfusion injury and may contribute to blood-brain barrier (BBB) breakdown. Neutrophils, a source of reactive oxygen species and MMP-9, infiltrate damaged tissue 6 to 24 hours after ischemia and have also been implicated in delayed secondary tissue damage. Here we examined the spatial-temporal relation between MMP-9 expression and neutrophil infiltration after stroke.

Methods--Knockout mice containing 50% manganese superoxide dismutase activity (SOD2-KOs), which are more susceptible to ischemic damage than wild-type (WT) littermates, underwent quantitative antigen (MMP-9, myeloperoxidase) immunohistochemistry (24 and 72 hours) analysis and protein expression by Western blotting (6, 12, 24, 48, and 72 hours) after transient focal cerebral ischemia. BBB breakdown was determined by Evans blue extravasation.

Results--There was a clear spatial relation between MMP-9 expression and Evans blue extravasation. MMP-9-positive cell and vessel counts for SOD2-KOs (72 hours) were significantly different from SOD2-KO (24 hours, P=0.004), WT (24 hours, P=0.01), and WT (72 hours, P=0.007) mice. In contrast, MMP-9-positive neutrophil counts were comparatively low and did not differ by time or animal type. MMP-9 expression was biphasic in SOD2-KOs but not in WT littermates, with a significant increase observed 6 to 12 hours after ischemic insult and again at 48 to 72 hours. SOD2-KOs showed increased MMP-9 expression compared with WT littermates at all time points studied (P0.05).

Conclusions--In this model, neutrophils are not the primary source of MMP-9 protein and thus are unlikely the key contributor to BBB breakdown observed in SOD2-KOs.

Key words: metalloproteinases • inflammation • cerebral ischemia, focal • blood-brain barrier • oxygen radical

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