Reduction in levels of matrix metalloproteinases and increased expression of tissue inhibitor of metalloproteinase—2 in response to mild hypothermia therapy in experimental stroke

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Object. Mild hypothermia is a robust neuroprotectant, and the results of prospective clinical trials have indicated that it may improve neurological outcome in certain instances. One aspect of this protection has been associated with the prevention of blood—brain barrier (BBB) disruption. Matrix metalloproteinases (MMPs) have been implicated in BBB disruption because they can degrade the extracellular matrix. In this study the authors explored the relationship between hypothermia and MMPs and whether BBB preservation resulting from mild hypothermia therapy is due to alterations in MMP expression.

Methods. Rats were subjected to middle cerebral artery occlusion for 2 hours; the animals were maintained in a state of normothermia or mild hypothermia (33°C) immediately after the onset of ischemia. The animals' brains were collected 2, 6, and 24 hours after ischemia began. Contrast-enhanced T1-weighted magnetic resonance imaging was performed at 24 hours to assess the extent of BBB disruption.

Consistent with prior reports, areas of BBB disruption detected on T1-weighted images were smaller in the brains of rats maintained in a state of hypothermia (normothermia group 8.6 ± 3% of the brain; hypothermia group 0.2 ± 0.1% of the brain; p < 0.01). Expression of both MMP-2 and MMP-9 at the transcriptional and translational levels was reduced in hypothermic brains at 6 hours and 24 hours after ischemic injury. Matrix metalloproteinase—9 was primarily localized to cells of monocytic origin but was also observed in neurons and astrocytes. Matrix metalloproteinase—2 was found in some neurons and astrocytes but not in inflammatory cells. In addition, hypothermia increased the levels of the endogenous MMP inhibitor, tissue inhibitor of metalloproteinases—2.

Conclusions. The authors conclude that mild hypothermia attenuates BBB disruption, decreases MMP expression, and suppresses MMP activity.

Article Information

Address reprint requests to: Midori A. Yenari, M.D., Department of Neurology (127), VAMC, 4150 Clement Street, San Francisco, California 94121. email: yenari@alum.mit.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Mild hypothermia reducing ischemic injury and BBB disruption. A: A T1-weighted MR image revealing regions of contrast enhancement within the ipsilateral cortex (arrows) of a normothermic animal subjected to 2 hours of MCA occlusion followed by 22 hours of reperfusion. B: In contrast, virtually no enhancement is seen in an animal exposed to 2 hours of mild hypothermia. C: Ischemic areas from diffusion-weighted MR images (DWI) and areas of contrast enhancement from T1-weighted images (T1W) were measured and computed as the percentage of the ipsilateral hemisphere. Mild hypothermia (33°C) significantly reduced the size of the ischemic lesion on diffusion-weighted MR images and areas of contrast enhancement on T1-weighted images compared with normothermia (38°C) (six hypothermic and seven normothermic animals; *p < 0.05, **p < 0.01).

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    Matrix metalloproteinase-2 and -9 observed within the brain 24 hours after onset of ischemia. Markers for neurons (MAP2), astrocytes (GFAP), and microglia/macrophages (ED1) were used to determine which cells expressed MMP-2 (A–C) and MMP-9 (D–F). Merged, double immunohistochemical staining of a normothermic ischemic cortex demonstrating that MMP-2 is found in some reactive astrocytes (A) and neurons (B), but not in microglia/macrophages (C). In contrast, MMP-9 is found mostly in microglia/macrophages (E) and to a lesser extent in rare astrocytes (D) and a few neurons (F). Both MMP-2 and MMP-9 are shown in red and the cell type markers (MAP2 to identify neurons, GFAP to identify astrocytes, and ED1 to identify microglia and macrophages) are shown in green. Yellow areas indicate cells in which both markers are present.

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    Immunostaining of MMP-2 (A and C) and MMP-9 (B and D) showing increased expression within the ipsilateral cortex 24 hours after onset of ischemia. The extent and intensity of expression is markedly reduced in the hypothermic brain (C and D) compared with the normothermic brain (A and B).

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    Mild hypothermia reducing MMP-2 protein expression after experimental stroke. A: Representative Western blots of MMP-2 under hypothermic (33°C) and normothermic (38°C) conditions. Mild hypothermia reduces MMP-2 expression at all time points within the ischemic cortex (CI), but only after 6 and 24 hours within the ischemic striatum (SI). Within the contralateral nonischemic hemisphere (Con), MMP-2 expression is similar regardless of temperature. Actin is shown as a loading control. B: Bar graphs representing the optical densities of the Western blots showing that mild hypothermia significantly reduced MMP-2 protein levels in both the ischemic cortex and the ischemic striatum (three experiments/group; *p < 0.001). (The Western blots depict pro—MMP-2 with a molecular weight of 72 kD.)

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    Mild hypothermia reducing MMP-9 protein expression in the brain after ischemic injury. A: Representative Western blots of MMP-9 under hypothermic (33°C) and normothermic (38°C) conditions. Mild hypothermia reduces MMP-9 expression 6 and 24 hours after stroke onset within the ischemic cortex and striatum. Within the contralateral nonischemic hemisphere, MMP-9 expression is also reduced by hypothermia. Actin is shown as a loading control. B: Bar graphs representing optical densities of the Western blots showing that mild hypothermia significantly reduced MMP-9 protein levels in both the ischemic cortex and the ischemic striatum (three—four experiments/group; *p < 0.001). (The Western blots depict pro-MMP-9 with a molecular weight of 92 kD.)

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    A: Western blots of TIMP-2 protein in hypothermic (33°C) and normothermic (38°C) cortices after ischemic injury. B: Bar graph showing that the level of TIMP-2 in normothermic brains was not significantly different from that in hypothermic brains 2 and 6 hours after ischemia onset; however, by 24 hours the TIMP-2 was increased in hypothermic brains (three—four experiments/group; *p < 0.001).

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    A: Zymograms of brain extracts from the ischemic cortex demonstrating gelatinolytic activities after ischemia onset. The bands shown represent the active forms of the proteins. Hypothermia reduced gelatinolytic activity of MMP-9 6 and 24 hours after ischemia onset; however, little gelatinolytic MMP-2 activity was observed, and hypothermia did not influence this. B and C: Northern blots showing MMP-2 (B) and MMP-9 (C) mRNA in hypothermic (hypo) and normothermic (normo) brains after ischemic injury. The MMP-2 mRNA levels were unchanged by hypothermia at all time points within the ischemic cortex (arrow); however, MMP-9 mRNA was reduced in this region by hypothermia at 12 and 24 hours (arrow).

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