Background--Inducible NO synthase (NOS)-derived peroxynitrite (ONOO^-) during ischemia/reperfusion contributes to ischemic brain injury. However, inducible NOS (iNOS) regulation in ischemic stroke remains unknown. Tetrahydrobiopterin (BH₄) is an essential cofactor for NOS activity. The present study tested the hypothesis that inhibition of endogenous BH₄ rate-limiting enzyme GTP cyclohydrolase I (GTPCH I), and thus BH₄ synthesis, reduces cerebral infarction via inhibiting iNOS and ONOO^- in transient focal ischemia.

Methods--Focal ischemia (2 hours) was created in adult male Sprague-Dawley rats (250 to 300 g) by middle cerebral artery occlusion (MCAO). Rats were treated 12 hours before MCAO with vehicle or diamino-6-hydroxypyrimidine (DAHP; 0.5 g/kg IP), a selective GTPCH I inhibitor. Brains were harvested 24 hours after reperfusion for assays of infarct volume, blood-brain barrier (BBB) permeability, GTPCH I activity, BH₄ levels, GTPCH I and NOS mRNA, protein expression, and superoxide anion (O₂·^-) and ONOO^- levels.

Results--Endogenous GTPCH I activity, BH₄ levels, iNOS activity, and (O₂·^- and ONOO^- levels were all augmented after ischemia/reperfusion. DAHP treatment significantly reduced GTPCH I activity, resulting in decreased BH₄ levels, iNOS activity, and ONOO^- levels. Consequently, DAHP treatment significantly reduced the infarct size compared with the nontreated group (22.3±5.6 versus 38.3±7.4%; n=6; P<0.05). Similarly, BBB permeability was significantly reduced after DAHP pretreatment compared with the control group (4.11±0.22 versus 7.78±0.44 µg/g tissue; n=5; P<0.05).

Conclusion--These results demonstrate that blockade of endogenous brain BH₄ synthesis attenuates cerebral infarction via inhibiting iNOS and ONOO^-, which may provide a mechanistic basis of novel therapeutic strategies for ischemic stroke.