The Akt-Endothelial Nitric Oxide Synthase Pathway in Lipopolysaccharide Preconditioning-Induced Hypoxic-Ischemic Tolerance in the Neonatal Rat Brain

Background and Purpose-Low-dose lipopolysaccharide (LPS) preconditioning provides neonatal rats long-term neuroprotection against hypoxic ischemia (HI). Upregulating endothelial nitric oxide synthase (eNOS) protects against cerebral ischemia; however, whether eNOS is required for LPS preconditioning-induced protection in neonatal rats is unknown. We hypothesized that Akt activation, which upregulates eNOS in neurons and endothelial cells, is required for LPS preconditioning-induced tolerance against HI in the neonatal brain. Methods-Six-day-old rat pups were intraperitoneally injected with LPS (0.05 mg/kg) or normal saline 24 hours before HI. Immunoblotting and immunohistochemistry were used to determine the phospho-Akt (pAkt Ser473), phospho-eNOS (peNOS Ser1177), and eNOS levels and immunofluorescence to determine the cellular distribution of eNOS and pAkt Ser473. Pharmacological and genetic approaches were used to regulate Akt and eNOS, and the weight loss of cerebral hemispheres on postnatal Day 21 was used to assess outcomes. Results-eNOS, peNOS (Ser1177), and pAkt (Ser473) levels were significantly higher in LPS-than in normal saline-treated rats 24 hours postinjection. LPS-induced eNOS was expressed primarily in neurons and vascular endothelial cells. N-omega(omega)-nitro-L-arginine and antisense oligodeoxynucleotide treatment significantly reduced eNOS expression in neurons and endothelial cells and inhibited LPS-induced protection against HI in rat pups. L-arginine and adenovirus eNOS transfection upregulated eNOS and protected the rat pups against HI. Wortmannin treatment before LPS preconditioning significantly reduced eNOS expression in neurons and endothelial cells, which inhibited LPS-induced protection against HI. Conclusions-Akt-mediated eNOS upregulation in neurons and vascular endothelial cells is required for LPS-induced tolerance against HI in the neonatal rat brain. (Stroke. 2010; 41: 1543-1551.)