Abstract 37: Inducible Nitric Oxide Synthase In Infiltrating Neutrophils Mediates Neurotoxicity After Ischemic Brain Injury In Mice

Abstract

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes to brain damage after cerebral ischemia and iNOS-deficient mice have smaller infarcts after middle cerebral artery occlusion (MCAO) (J Neurosci, 17:9157, 1997). However, the cell type(s) expressing iNOS and mediating tissue damage remain to be defined. We tested the hypothesis that iNOS expressed in neutrophils infiltrating the ischemic brain contributes to tissue injury. To this end, we examined the outcome of cerebral ischemia in iNOS -/- mice reconstituted with iNOS+/+ leukocytes. Lethally irradiated iNOS -/- mice were transplanted with iNOS+/+ bone marrow (BM) to produce chimeric mice expressing iNOS in peripheral leukocytes. Control chimeras were also obtained by transplanting iNOS+/+ or -/- BM in iNOS+/+ or -/- mice, respectively. Five weeks later, mice underwent transient MCAO (n=10/group) and injury volume was assessed after 3 days in cresyl violet-stained brain sections. Chimerism was 89%, indicating successful BM engraftment. iNOS -/- mice transplanted with iNOS+/+ BM had larger infarcts (40±3 mm3; Mean±SE) than iNOS -/- mice receiving iNOS -/- BM (25±3 mm3; p<0.05; ANOVA), and not different from those of iNOS+/+ mice transplanted with iNOS+/+ BM (36±5 mm3; p>0.05). Cell sorting demonstrated iNOS expression in neutrophils infiltrating the infarct of iNOS -/- mice transplanted with iNOS+/+ BM. To confirm that iNOS in neutrophils contributes to tissue damage, adoptive transfer experiments were performed. Neutrophils (Ly6G positive) from iNOS+/+ or -/- mice were isolated by negative selection and 5x105 cells were administered i.v. to iNOS+/+ or -/- mice 24h after MCAO (n=10/group). Consistent with the result in chimeric mice, transfer of iNOS+/+ neutrophils into iNOS -/- mice increased the infarct 3 days after MCAO (vehicle: 35±6; iNOS+/+ neutrophils: 47±6 mm3; p>0.05 from iNOS+/+ mice receiving vehicle), but not when iNOS -/- neutrophils were transferred (38±8 mm3; p>0.05 from iNOS -/- mice). The findings identify iNOS in infiltrating leukocytes, particularly neutrophils, as a mediator of neurotoxicity in the post-ischemic brain and establish iNOS in circulating leukocytes as a therapeutic target for ischemic brain injury. Supported by NIH grant NS34179