Cathepsin and calpain inhibitor E64d attenuates matrix metalloproteinase-9 activity after focal cerebral ischemia in rats

Background and Purpose-Matrix metalloprotemases (MMPs) and cysteine proteases (calpain and cathepsin B) play an important role in cell death and are upregulated after focal cerebral ischemia. Because there is a significant interaction between MMP-9 with calpain and cathepsin B, we investigated the role of E64d (a calpain and cathepsin B inhibitor) on MMP-9 activation in the rat focal ischemia model. Methods-Male Sprague-Dawley rats were subjected to 2 hours of middle cerebral artery occlusion by using the suture insertion method followed by 22 hours of reperfusion. In the treatment group, a single dose of E64d (5 mg/kg IP) was administrated 30 minutes before the induction of focal ischemia, whereas the nontreatment group received dimethyl sulfoxide only. The neurological deficits, infarct volumes, Evans blue extravasation, brain edema, and MMP-9 activation in the brain were determined. Results-Pretreatment with E64d produced a significant reduction in the cerebral infarction volume (353.1 +/- 19.8 versus 210.3 +/- 23.7 mm(3)) and the neurological deficits. Immunofluorescence studies showed MMP-9, calpain, and cathepsin B activation colocalized to both neurons and the neurovascular endothelial cells after ischemia, which was reduced by E64d. Conclusion-These results suggest that E64d treatment provides a neuroprotective effect to rats after transient focal cerebral ischemia by inhibiting the upregulation of MMP-9.