Reperfusion-induced oxidative/nitrative injury to neurovascular unit after focal cerebral ischemia.
OBJECTIVE
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.


CONCLUSIONS
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.
Gürsoy-Ozdemir
Yasemin
Y
Department of Neurology, Faculty of Medicine, Institute of Neurological Sciences & Psychiatry, Hacettepe University, Ankara, Turkey.
Can
Alp
A
Dalkara
Turgay
T
D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002545:Brain Ischemia / Q000201:enzymology / Q000378:metabolism*; D004396:Coloring Agents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005070:Evans Blue; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000096:biosynthesis*; D015427:Reperfusion Injury / Q000201:enzymology / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D004396:Coloring Agents; D004791:Enzyme Inhibitors; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D005070:Evans Blue; D019001:Nitric Oxide Synthase; D020780:Matrix Metalloproteinase 9