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. 2001 Oct 1;21(19):7724-32.
doi: 10.1523/JNEUROSCI.21-19-07724.2001.

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia

M Asahi  1 , X Wang, T Mori, T Sumii, J C Jung, M A Moskowitz, M E Fini, E H Lo
Affiliations

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia

M Asahi et al. J Neurosci. .

Abstract

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood-brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood-brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood-brain barrier-associated protein, occludin. Correspondingly, blood-brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood-brain barrier and white matter components.

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Figures

Fig. 1.
Fig. 1.
Latex–carbon black perfusions of MMP-9 knock-out (KO), wild-type (WT), and CD-1 (CD) brains show a similar distribution of dorsal vessels and boundary zone for the anterior and middle cerebral arteries.
Fig. 2.
Fig. 2.
A, Western blots of MMP-9 demonstrate the upregulation of protein expression after transient focal cerebral ischemia. MMP-9 expression was mainly limited to the l05 kDa zymogen. Extracts from sham-operated brain (S) did not show MMP-9 upregulation. Thelanes labeled 24h contra and 24h KO represent samples at 24 hr from contralateral brain and ischemic brain from MMP-9 knock-out mice, respectively. Murine MMP-9 was used as a standard. B, Gelatin zymograms demonstrate the upregulation of MMP-9 after transient focal cerebral ischemia. Mostly, the 105 kDa zymogen was detected, although these zymograms also showed some gelatinolytic activity at 97 kDa, corresponding with the cleaved–activated enzyme. Sham-operated brain (S) did not show MMP-9 upregulation. Thelanes labeled 24h contra and 24h KO represent samples at 24 hr from contralateral brain and ischemic brain from MMP-9 knock-out mice, respectively. Subtle gelatinolytic activity was also present close to the level for MMP-2. Murine MMP-9 and human MMP-2 were used as standards.
Fig. 3.
Fig. 3.
Immunohistochemistry with anti-MMP-9. A, B, Sham-operated brains showed no MMP-9 expression.C–F, MMP-9 staining increased in wild-type mouse brain after transient focal ischemia. Diffuse MMP-9 immunoreactivity was observed within both the cortical and striatal areas of ischemic hemispheres. Immunoreactive MMP-9 mainly appeared in endothelial cells. In addition, MMP-9 expression was also detectable in parenchymal cells of ischemic regions. G, H, No immunoreactive cells were observed in ischemic brains of knock-out mice (KO).I, J, Negative controls incubated without primary antibody showed no staining. Scale bar, 50 μm.
Fig. 4.
Fig. 4.
A, Western blots showed a time-dependent degradation of the BBB-associated protein ZO-1 after ischemic onset. Densitometric analysis demonstrated that ZO-1 degradation was significantly ameliorated in MMP-9 knock-out mice (KO). n = 5 animals per time point; *p < 0.05. B, Western blots showed no significant change in another BBB-associated protein, occludin.n = 5 animals per time point.
Fig. 5.
Fig. 5.
A, Representative brains showing Evans blue leakage in wild-type mice (WT) and reduced leakage in MMP-9 knock-outs (KO).B, Fluorescent quantitation of Evans blue showed that BBB leakage was significantly reduced in knock-out mice compared with wild-type mice. n = 7 per group; *p < 0.05.
Fig. 6.
Fig. 6.
A, Western blots of MBP detected all four isoforms (21.5, 18.5, 17, and 14 kDa). After ischemia, a degraded band (deg) appeared at ∼10 kDa.B, Quantitative densitometric analysis showed that the 21.5, 18.5, and 14 kDa MBP bands were significantly degraded compared with sham controls by 24 hr after ischemia; *p < 0.05. In MMP-9 knock-out mice (KO), the band intensities for 18.5 and 14 kDa MBP were significantly higher compared with wild-type mice at 24 hr after ischemia; †p < 0.05; n = 5 per time point. C, Densitometric analysis showed that degraded MBP (deg) increased over time after ischemic onset in wild-type mice. This accumulation of degraded MBP was significantly reduced in MMP-9 knock-out mice (KO).n = 5 per time point; *p < 0.05. A.U., Arbitrary units.
Fig. 7.
Fig. 7.
A, Western blots of PLP and DM20 showed no detectable degradation after ischemia. B, Densitometric quantitation demonstrated that there were statistically significant effects in wild-type or MMP-9 knock-out mice (KO). n = 5 per time point.
Fig. 8.
Fig. 8.
A, Western blots of the extracellular matrix component laminin showed no detectable degradation after ischemia in wild-type or MMP-9 knock-out mice (KO). n = 5 per time point.B, Western blots of the ubiquitous intracellular protein actin showed no detectable degradation after ischemia in wild-type or MMP-9 knock-out mice. n = 5 per time point.
Fig. 9.
Fig. 9.
Twenty-four hour ischemic lesion volumes (A) and areas (B) showed that knock-out mice (KO; n = 8) had significantly reduced ischemic injury compared with wild-type mice (WT; n = 7). *p< 0.05.
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