Plasma and brain matrix metalloproteinase-9 after acute focal cerebral ischemia in rats

Abstract

Background and purpose: Plasma levels of matrix metalloproteinase-9 (MMP-9) have been proposed to be a useful biomarker for assessing pathological events in brain. Here, we examined the temporal profiles of MMP-9 in blood and brain using a rat model of acute focal cerebral ischemia.

Methods: Plasma and brain levels of MMP-2 and MMP-9 were quantified at 3, 6, 12, and 24 hours after permanent middle cerebral artery occlusion in male Sprague-Dawley rats. Infarct volumes at 24 hours were confirmed with 2,3,5-triphenyl-tetrazolium-chloride staining.

Results: In plasma, zymographic bands were detected between 70 and 95 kDa corresponding to pro-MMP-2, pro-MMP-9, and activated MMP-9. A higher 135-kDa band was also seen that is likely to be NGAL-conjugated MMP-9. After ischemia, there were no significant changes in pro-MMP-2, but plasma levels of pro-MMP-9 steadily increased over the course of 24 hours. Activated MMP-9 levels in plasma were significantly elevated only at 24 hours. Plasma NGAL-MMP-9 complexes showed a transient elevation between 3 to 6 hours, after which levels decreased back down to pre-ischemic baselines. In brain homogenates, pro-MMP-2, pro-MMP-9, and activated MMP-9 were seen but no NGAL-MMP-9 bands were detected. Compared to the contralateral hemisphere, MMP-2 and MMP-9 levels in ischemic brain progressively increased over the course of 24 hours. Overall levels of MMP-9 in plasma and brain were significantly correlated, especially at 24 hours. Plasma levels of pro-MMP-9 at 24 hours were correlated with final infarct volumes.

Conclusions: Elevated plasma levels of MMP-9 appear to be correlated with brain levels within 24 hours of acute cerebral ischemia in rats. Further investigation into clinical profiles of MMP-9 in acute stroke patients may be useful.

Figure 1

Temporal profiles of MMP-9 in plasma. A, Representative zymogram shows bands of NGAL–MMP-9 complexes, pro-MMP-9, activated MMP-9, and pro-MMP-2 before ischemia and at 3 hours, 6 hours, 12 hours, and 24 hours after permanent focal cerebral ischemia in rats. B, Western blotting against MMP-9 confirms that pro-MMP-9 and activated MMP-9 are detected in the rat plasma samples. C, Immunoblotting against NGAL and MMP-9 protein supports the existence of higher-molecular-weight bands of NGAL–MMP-9 complexes in the rat plasma samples.

Figure 2

Quantified temporal profile of plasma MMP levels via densitometric analysis of zymograms. A, Plasma pro-MMP-9 levels are significantly increased at all time points after ischemia. B, Plasma-activated MMP-9 levels are only significantly elevated at 24 hours. C, Plasma levels of total (activated plus pro) MMP-9 levels progressively increase over time. Note, however, that stable pro-MMP-9 and relatively unstable activated MMP-9 densitometry may not be comparable when summed. D, Plasma NGAL–MMP-9 complexes shows statistically significant changes at 3 and 6 hours. *P<0.05 vs values before ischemia.

Figure 3

Temporal profile of MMP in brain homogenates. A, Representative zymogram shows bands of brain pro-MMP-9, activated MMP-9, and pro-MMP-2, but no NGAL-MMP-9 complexes are detected. Compared to contralateral hemisphere, MMP-9 levels are increased in ischemic ipsilateral tissue over time. B, Quantitative densitometry shows that brain pro-MMP-9 is elevated at all time points. C, Activated MMP-9 is increased at 12 and 24 hours. D, Total (activated plus pro) MMP-9 is increased at all time points. E, Brain MMP-2 appears to be increased at 12 and 24 hours. *P<0.05 and **P<0.01 between ipsilateral and contralateral levels.

Figure 4

Overall correlation between plasma and brain MMP-9 regardless of time. A, Total MMP-9: r=0.643, P<0.001. B, Pro-MMP-9: r=0.561, P=0.001. C, Activated MMP-9: r=0.540, P=0.001.