Dynamic platelet accumulation at the site of the occluded middle cerebral artery and in downstream microvessels is associated with loss of microvascular integrity after embolic middle cerebral artery occlusion

Information is lacking regarding dynamic platelet accumulation at the site of the occluded middle cerebral artery (MCA) and the relationship between p latelet aggregation in downstream cerebral microvessels and loss of perfusi on and vascular integrity of these microvessels. In the present study, we e mployed a model of embolic MCA occlusion in the rat to simultaneously measu re temporal and spatial profiles of platelet accumulation at the site of th e embolus occluding the MCA and within downstream cerebral microvessels. We also measured the integrity of microvessels and matrix metalloproteinase ( MMP) activity in ischemic brain, Rats (n = 36) were subjected to embolic MC A occlusion. Immunohistochemistry was used to detect microvascular integrit y, plasminogen activator inhibitor 1 (PAI-1) and the deposition of fibrin. SDS-PAGE zymography was used to measure MMP2 and MMP9 activities. Accumulat ion of platelets and increases in PAI-1 immunoreactivity at the site of the embolus occluding the MCA were detected 1 h (n = 7) and 4 h (n = 7) after ischemia, respectively, and numbers of GPIIb/IIIa immunoreactive downstream cerebral microvessels increased significantly (209+/-59; n=7; P<0.05) 4 h after ischemia, suggesting dynamic platelet aggregation. A significant (n = 7; P<0.01) diffuse loss of type TV collagen immunoreactivity in microvesse ls was temporally associated with platelet GPIIb/IIIa immunoreactivity with in the vessels. Triple immunostaining revealed that microvessels containing platelet aggregates exhibited loss of type IV collagen immunoreactivity an d both intra- and extra-vascular fibrin deposition, suggesting that intrava scular platelet aggregation is associated with decreases in the integrity o f the microvascular basal lamina and blood-brain barrier leakage. A signifi cant increase (P<0.05) in MMP9 was detected at 4 h (n=3) and 24 h (n = 3) a fter ischemia but levels of MMP2 were not significantly changed in ischemic brain. Our data suggest that dynamic platelet aggregation in ischemic brai n may contribute to time-dependent resistance to fibrinolysis. In addition, platelet deposition and increased MMP9 coincided with degradation of type IV collagen and loss of vascular integrity. These data suggest an important role for post-occlusive distal platelet deposition in the pathophysiology of stroke. (C) 2001 Elsevier Science B.V. All rights reserved.