Reperfusion-Induced Oxidative/Nitrative Injury to Neurovascular Unit After Focal Cerebral Ischemia

Abstract

Background and Purpose-Use of thrombolysis in stroke is limited by a short therapeutic window because delayed reperfusion may cause brain hemorrhage and edema. Available evidence suggests a role for superoxide, NO, and peroxynitrite in reperfusion-induced injury. However, depending on their cellular origin and interactions between them, these molecules may exert protective or deleterious actions, neither of which is characterized in the intact brain. Methods-Using fluorescent probes, we determined superoxide and peroxynitrite formation within neurons, astrocytes, and endothelium, and the association between oxidative/nitrative stress and vascular injury in mice brains subjected to 2-hour middle cerebral artery occlusion and 3 or 5 hours of reperfusion. Results-Both signals were colocalized, suggesting that the main source of peroxynitrite in the reperfused brain was a reaction between superoxide and NO. Superoxide and peroxynitrite formation was particularly intense in microvessels and astrocytic end-feet surrounding them, and overlapped with dense mitochondrial labeling. Sites of oxidative/nitrative stress on microvessels were colocalized with markers of vascular injury such as Evans blue (EB) leakage and matrix metalloproteinase-9 (MMP-9) expression, suggesting an association between peroxynitrite and microvascular injury. Supporting this idea, partial inhibition of endothelial NO synthesis at reperfusion with a low dose of L-nitroarginine (1 mg/kg IP) reduced 3-nitrotyrosine formation in microvessels and EB extravasation. Conclusion-During reperfusion, intense superoxide, NO, and peroxynitrite formation on microvessels and surrounding end-feet may lead to cerebral hemorrhage and edema by disrupting microvascular integrity. Combination of thrombolysis with agents diminishing oxidative/nitrative stress may reduce reperfusion-induced injury and extend the therapeutic window for thrombolysis.

Hemorrhagic Transformation after Ischemic Stroke in Animals and Humans

“…After 2 hours of MCAO and 3 hours of reperfusion in a rat, both superoxide and peroxynitrite are increased in microvessels and astrocytic end feet. 46 In this model, disruption of the BBB correlated with ROS levels, and was prevented by inhibiting nitric oxide. Levels of ROS are increased in reperfusion compared with permanent MCAO models.…”

“…BBB, blood-brain barrier; MMP, matrix metalloproteinase; ROS, reactive oxygen species; SMAD, mothers against decapentaplegic homolog; TGFb1, transforming growth factor b1; tPA, tissue plasminogen activator; VEGF, vascular endothelial growth factor; HMGB1, high-mobility-group-box-1. 29,46 ( Figure 3). Increased ROS produced by ischemia-reperfusion can disrupt the neurovascular unit through damage to endothelial cells, pericytes, smooth muscle cells, and astrocytes.…”

Can Restoring Incomplete Microcirculatory Reperfusion Improve Stroke Outcome after Thrombolysis?

“…Pericyte cytoplasm and mitochondria were shown to swell soon after ischemia with electron microscopy. 9,62 The pericyte dysfunction may be caused by oxygen and nitrogen radicals that are intensely generated on the microvascular wall during ischemia/reperfusion 63,64 because the unregulated pericyte contraction can be reversed by inhibiting oxidative and nitrative mechanisms. 50 Reversing pericyte contraction with these agents administered during recanalization restored microcirculatory patency and promoted tissue survival, reinforcing the idea that microcirculatory obstructions may unfavorably affect recovery after recanalization as discussed above.…”

Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion

“…Although the recovery of structure with reperfusion was remarkable, initial studies only used an average of 7 min of occlusion , or used photothrombotic occlusion in which reperfusion was undefined (Zhang et al, 2005), thus limiting their power as models of human focal stroke that typically involve the occlusion of major arteries for hours. Although reperfusion permitted recovery of dendritic structure after short-term occlusion (Zhang et al, 2005;Murphy et al, 2008), it is conceivable that reperfusion may lead to exacerbation of injury (Aronowski et al, 1997;Gürsoy-Ozdemir et al, 2004) when combined with longer periods of occlusion. Out of these limitations, several important questions arose: (1) if occlusion is maintained for hours, will dendritic structure recover during reperfusion; and (2) if structural recovery occurs, is it limited to penumbra regions near the stroke border, or does it include the ischemic core; (3) will the penumbra be subject to reperfusion damage after prolonged occlusion?…”