Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia

Abstract

Mechanisms of selective neuronal death in the hippocampus after global cerebral ischemia remain to be clarified. Here, we explored a possible role for matrix metalloproteinases (MMPs) in this phenomenon. Although many studies have demonstrated detrimental roles for the gelatinase MMP-9 in focal cerebral ischemia, how dysregulated MMP proteolysis influences global cerebral ischemia is less well understood. In this study, CD-1 mice were subjected to transient global ischemia. Transient occlusions of common carotid arteries for periods between 20 and 40 min led to increasing hippocampal neuronal death after 3 d. Gel zymography showed elevations in gelatinase (MMP-2 and MMP-9) activity. In situ zymography showed that gelatinase activity was mostly colocalized with neuron-specific nuclear protein-stained pyramidal neurons. Mice treated with the broad-spectrum metalloproteinase inhibitor BB-94 (50 mg/kg, i.p.) showed reduced hippocampal gelatinase activity after transient global cerebral ischemia and suffered significantly reduced hippocampal neuronal damage compared with vehicle-treated controls (p < 0.01). Additionally, hippocampal gelatinase activity and neuronal damage after transient global ischemia were also significantly reduced in MMP-9 knock-out mice compared with wild-type mice (p < 0.05). These data indicate a potential deleterious role for MMP-9 in the pathogenesis of delayed neuronal damage in the hippocampus after global cerebral ischemia.

Figure 1.

Photomicrographs of Nissl staining in hippocampus after transient global cerebral ischemia. A, Sham-operated brain shows clear delineation of pyramidal layer and dentate gyrus. B, Three days after 40 min transient cerebral ischemia, pyramidal neurons are severely damaged. C, Increasing hippocampal damage (mean + SD score) after increasing duration of transient cerebral ischemia; Sham (n = 3); all other groups (n = 6 per group). Scale bars, 300 μm. DG, Dentate gyrus; CA1, cornus ammonis sector 1; CA3, cornus ammonis sector 3.

Figure 2.

Gel zymography of hippocampal brain homogenates after 40 min transient cerebral ischemia. A, Representative zymogram gel showing elevation of MMP-9 and MMP-2. Murine MMP-9 and human MMP-2 were loaded as standards. B, Quantitative increases in relative optical density for active 97 kDa MMP-9 (mean + SD; n = 6). C, Quantitative increases in relative optical density for 72 kDa proforms of MMP-2 (mean + SD; n = 6). ^*p < 0.05; ^**p < 0.01.

Figure 3.

A-C, Representative in situ gelatin zymograms in hippocampus at 3 d after 40 min transient global cerebral ischemia. D-F, Suppression of gelatinolytic activity in postischemic hippocampus after coincubation with the metalloproteinase inhibitor GM6001. Scale bars: A, 300 μm; B, 100 μm.

Figure 4.

Cellular localizations of gelatinolytic activity in postischemic hippocampus (3 d after 40 min transient global cerebral ischemia). Increased gelatinolytic activity (green fluorescence) broadly colocalizes with NeuN staining of neurons (red fluorescence). A-C, CA1 sector. D-F, Dentate gyrus region. Scale bar, 100 μm.

Figure 5.

MMP-9 immunohistochemistry (green fluorescence) in postischemic hippocampus shows colocalization with NeuN staining of neurons (red fluorescence). Signals appear to suggest intracellular and extracellular localizations. A-D, CA1 sector. E-H, Dentate gyrus regions. G, H, Insets, Negative controls for immunohistochemistry data (FITC or TRITC secondary antibodies alone without primary antibodies). Scale bars: A, 100 μm; C, 33 μm.

Figure 6.

MMP-9 immunohistochemistry (green fluorescence) in postischemic hippocampus shows colocalization with GFAP staining of astrocytes (red fluorescence). A-D, CA1 sector. E-H, Dentate gyrus regions. G, H, Insets, Negative controls for immunohistochemistry data (FITC or TRITC secondary antibodies alone without primary antibodies). Scale bars: A, 100 μm; C, 33 μm.

Figure 7.

Neuroprotection with pharmacologic MMP inhibition. A, Representative sections show that in situ gelatinolytic activity within postischemic hippocampus is reduced in BB-94-treated mice compared with vehicle controls at 3 d after 40 min transient global cerebral ischemia. B, Nissl-stained sections show improved neuronal survival after MMP inhibition. C, Quantified scores (mean + SD) of hippocampal neuronal damage are significantly reduced in BB-94 treated mice (n = 7) compared with vehicle controls (n = 10). ^**p < 0.01. Scale bars: A, 200 μm; B, 100 μm.

Figure 8.

Neuroprotection in MMP-9 knock-out mice. A, Representative sections show that in situ gelatinolytic activity within postischemic hippocampus is reduced in MMP-9 knock-out mice compared with matching wild-types at 3 d after 40 min transient global cerebral ischemia. B, Nissl-stained sections show improved neuronal survival in MMP-9 knock-outs. C, Quantified score (mean + SD) of hippocampal neuronal damage is significantly reduced in MMP-9 knock-out mice (n = 7) compared with wild-type controls (n = 5). ^*p < 0.05. Scale bars: A, 200 μm; B, 100 μm.