Immunohistochemistry of matrix metalloproteinases in reperfusion injury torat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures

Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinase s (MMPs) are associated with the opening of the BBB, but their cellular loc alization and activation mechanisms are uncertain. We used immunohistochemi stry to determine the cellular localization of the MMPs in reperfused rat b rain, and cell cultures to study their activation. Spontaneously hypertensi ve rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunos tained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostainin g in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunorea ctivity for MMP-3 and MMP-3 appeared. MMP-3 co-localized with activated mic roglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days i ntense MMP-2 staining was seen in reactive astrocytes around the ischemic c ore. Studies of activation of the MMP were done in lipopolysaccharide (LPS) -stimulated astrocyte and microglia cultures. Stimulated astrocytes produce d an activated form of MMP-2. When microglia were stimulated, they activate d MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cel ls. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is n ormally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP -9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the pr oteolytic disruption of the BBB. (C) 2001 Elsevier Science B.V. All rights reserved.