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(Stroke. 2000;31:1744.)
© 2000 American Heart Association, Inc.

Original Contributions

Oxygen-Glucose Deprivation Induces Inducible Nitric Oxide Synthase and Nitrotyrosine Expression in Cerebral Endothelial Cells

Jan Xu, PhD; Luming He, PhD; S. Hinan Ahmed, MD; Sha-Wei Chen, MD; Mark P. Goldberg, MD; Joseph S. Beckman, PhD Chung Y. Hsu, MD, PhD

From the Department of Neurology, Washington University School of Medicine, St. Louis, Mo, and the Department of Anesthesiology and Biochemistry (J.S.B.), School of Medicine, University of Alabama, Birmingham.

Correspondence to Chung Y Hsu, MD, PhD, Department of Neurology, Box 8111, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110. E-mail hsuc{at}neuro.wustl.edu

Background and Purpose—The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro.

Methods—OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription–polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining.

Results—Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N^G-(1-iminoethyl)ornithine and nitro-L-arginine, and an NO scavenger, the Fe²⁺-N-methyl-D-glucamine dithiocarbamate complex.

Conclusions—OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

Editorial Comment

Costantino Iadecola, MD, Guest Editor

Center for Clinical and Molecular Neurobiology, Departments of Neurology and Neuroscience, University of Minnesota, Minneapolis, Minnesota

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