Matrix metalloproteinase-9 and myeloperoxidase expression: quantitative analysis by antigen immunohistochemistry in a model of transient focal cerebral ischemia.
OBJECTIVE
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 (P< or =0.05).


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.
Maier
Carolina M
CM
Department of Neurosurgery, University School of Medicine, Stanford, Calif 94305-5487, USA.
Hsieh
Lily
L
Yu
Fengshan
F
Bracci
Paige
P
Chan
Pak H
PH
D000818:Animals; D015153:Blotting, Western; D002545:Brain Ischemia / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement / Q000276:immunology; D005070:Evans Blue; D005119:Extravasation of Diagnostic and Therapeutic Materials; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020556:Neutrophil Infiltration / Q000276:immunology; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D020521:Stroke / Q000276:immunology / Q000378:metabolism*
D005070:Evans Blue; D009195:Peroxidase; D020780:Matrix Metalloproteinase 9