Research reportEarly appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion
Introduction
Matrix metalloproteinases (MMPs) are a family of zinc-binding proteolytic enzymes which are capable of degrading components of the extracellular matrix in a variety of physiological and pathophysiological conditions. Among MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining blood–brain barrier (BBB) impermeability, by regulating tight junctions leading to the opening of the BBB. In the pathological condition of ischemia/reperfusion, digestion of the endothelial basal lamina is reported to occur as early as 2 h after ischemia [14], which may result in BBB permeability a few hours after ischemia 4, 18. Recently, MMP-9 and/or MMP-2 have been implicated in focal cerebral ischemia (FCI) 9, 23, 24, 27, as well as other central nervous system disorders such as multiple sclerosis [25], intracerebral hemorrhage [26]and cerebral aneurysm 5, 15.
A previous report by Rosenberg and his colleagues on FCI showed the marked increase of pro-MMP-9 after both permanent and transient ischemia, suggesting the possible involvement of MMP-9 in FCI 24, 27. However, it has still not been established whether the 88-kDa activated MMP-9, which is considered to be the fully active form to digest basal lamina [32], could appear after transient FCI. In the present study, we sought to clarify this point by measuring gelatinase activity in the brain before and after transient FCI by using the protein extraction method with detergent extraction and affinity-support purification, which allowed us to recover gelatinase activity over a 4- to 10-fold range using zymography [36]. The results showed that 88-kDa activated MMP-9 is absent from the control specimens, while it appeared 3 h after 60 min of transient ischemia, as shown by zymography and type IV collagenase activity assay. At this time point, BBB permeability alteration was detected in the ischemic brain by the Evans blue extravasation method. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were detected in the control specimens and increased after transient FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis after transient FCI. These results, which are consistent with a previous report showing the prevention of BBB impermeability with the MMP-9 inhibitor BB-1101 3 h after reperfusion [27], suggest that the activation of MMP-9 during reperfusion may contribute to BBB disruption and the formation of vasogenic edema after transient FCI.
Section snippets
Focal cerebral ischemia and histological assessment
Adult male CD-1 mice (35–40 g, n=33) were subjected to transient FCI by intraluminal middle cerebral artery (MCA) blockade with a nylon suture as described [33]. The mice were anesthetized with 2.0% isoflurane in 30% oxygen and 70% nitrous oxide using a face mask. The rectal temperature was controlled at 37°C with a homeothermic blanket. Cannulation of a femoral artery allowed the monitoring of blood pressure and arterial blood gases, samples for analysis being taken immediately after
Physiological data and cerebral infarction
Physiological parameters showed no significant differences in mean arterial blood pressure (MABP) and arterial blood gas analysis between each time point. The pre-ischemic physiological variables were as follows: MABP, 71.0±3.8 mmHg; PaO2, 146.4±9.9 mmHg; PaCO2, 28.9±7.8 mmHg; pH, 7.35±0.04 (variables are mean±S.D., n=4). There was no deviation from these values over the period of assessment. An ischemic lesion of the core of the caudate putamen was visible as a pale, slightly stained area in
Discussion
The current study provides evidence that activated MMP-9 appears as early as 3 h after 60 min of transient FCI, and that a significant increase of pro-MMP-9 occurred in a time dependent manner during reperfusion. These conclusions are derived from the following observations. First, a constitutive expression of pro-MMP-9 in the control specimens was evident as a 96-kDa characteristic band by both zymography (Fig. 1) and Western blot analysis (Fig. 4), and markedly increased during reperfusion
Acknowledgements
The authors are grateful to Ms. Cheryl Christensen for her editorial assistance and Ms. Liza Reola, Mr. Bernard Calagui and Ms. Jane O. Kim for their technical assistance. This study was supported by National Institutes of Health grants NS25372, NS14543, NS36147, NS38653 and NO1 NS82386. P.H.C. is a recipient of the Jacob Javits Neuroscience Investigator Award.
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