Evidence to implicate matrix metalloproteinases (MMPs) in the fast IL-1β elevation following transient focal brain ischemia in rat

Amantea D.1, Gliozzi M.2, Russo R.1, Morrone L.A.1, Bagetta G.1 and Corasaniti M.T.2, 1Department of Pharmacobiology, University of Calabria, Cosenza, Italy; 2Department of Phamacobiological Science, University of Catanzaro “Magna Graecia, Catanzaro, Italy.

The proinflammatory cytokine interleukin(IL)-1β is an important mediator of neurodegeneration induced by experimental cerebral ischemia in rodents (Rothwell, 2003). Matrix metalloproteinases (MMPs) cleave protein components of the extracellular matrix, but also process a number of cell surface and soluble proteins including receptors, cytokines and chemokine (Sternlicht and Werb, 2001). Here we investigate the putative involvement of IL-1β processing in the detrimental effects exerted by the early upregulation of MMPs in ischemic stroke.

Brain ischemia was induced in male Wistar rats (280- 320 g) by middle cerebral artery occlusion (MCAo) via the intraluminal filament technique (Longa et al., 1989). Two hours after occlusion, the filament was withdrawn to allow reperfusion. Cerebral infarct volume was evaluated 24 h after MCAo by staining 2 mm-thick consecutive coronal brain slices with 2,3,5-triphenyltetrazolium chloride and measuring the infarct area (unstained) using computer assisted image analysis. GM6001, a potent broad-range inhibitor of MMPs, and its negative control (GMneg) were dissolved in DMSO and administered in a volume of 2 μl through the external carotid artery (i.a.), 15 min prior to MCAo. Two hours after reperfusion, cortical tissue was dissected to measure pro-IL-1β immunoreactivity by western blotting and mature IL-1β levels by an established, rat specific, sandwich ELISA (Corasaniti et al., 2001). Gelatin and in situ zymography were performed, respectively, on cortical homogenates and fresh cryostat-cut sections of rat brains harvested after 2 h reperfusion (Gu et al., 2002). Data were expressed means ± S.E.M. and analysed by ANOVA followed by Tukey’s post-hoc test.

In situ zymography revealed a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2 h MCAo followed by 2 h reperfusion. Accordingly, expression and activity of MMP-2 and MMP-9, as assessed by gelatine zymography, were enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter effect appears to be instrumental for development of delayed brain damage, since administration of GM6001 (0.5 μg, i.a.), but not its negative control, resulted in a significant reduction of ischemic brain volume (GMneg: 503±18, GM6001: 252±9 mm3, P<0.001). Increased gelatinase activity in the ischemic cortex was coincident with elevation of mature IL-1β (contralateral cortex: 5.6±1.0, ipsilateral cortex: 8.4±0.2 pg mg protein-1, P<0.05) after 2 h reperfusion and this did not appear to implicate a caspase-1-dependent processing of pro(31 KDa)-IL-1β to yield mature (17 KDa) IL-1β . Quite surprisingly, GM6001 (0.5 μg, i.a.) abolished the early IL-1β increase in the ischemic cortex and reduced the cleavage of the cytokine pro-form supporting the deduction that MMPs may initiate IL-1β processing in ischemic brain injury.

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