Cerebral Aneurysm Formation in Nitric Oxide Synthase-3 Knockout Mice

Todd Abruzzo; Ady Kendler; Robert Apkarian; Michael Workman; Jane C. Khoury; Harry J. Cloft

Abstract

We sought to evaluate the influence of specific vasoactive gene knockouts on the process of intracranial aneurysm formation in mice. Thirty wild type, 7 nitric oxide synthase (NOS)-2 knockout, 6 NOS-3 knockout, and 8 plasminogen activator inhibitor (PAI)-1 knockout female mice underwent left common carotid artery ligation at 2 to 6 months of age. After a survival period (average 20.4 months ± 1.5 months), the brains were perfusion fixed with 10% buffered formalin for 10 minutes and then perfused with India ink. Brain and intact cerebral circulation were surgically removed and further fixed in 10% buffered formalin for 4 additional days. The basal cerebral circulation of each brain was examined for the presence of intracranial aneurysms under a surgical microscope (3x-21x). Suspected aneurysms were further dissected for histological analysis. Specimens were embedded in epoxy resin, cut into 0.5 and 1.0 micron sections, and stained with Toluidine blue. A neuropathologist blinded to genotype and surgical microscopy results examined the slides for evidence of aneurysmal pathology. Two intracranial aneurysms in 2 NOS-3 knockout mice were confirmed by histology. No intracranial aneurysms were confirmed in any wild type, NOS-2 knockout, or PAI-1 knockout mice. Histological analysis of aneurysms revealed loss of elastica, subendothelial collagen deposition, and perivascular lymphocytic infiltration. Our results suggest that NOS-3 knockout, but not PAI-1 or NOS-2 knockout, predisposes to the formation of intracranial aneurysms in mice subjected to unilateral carotid artery ligation. Due to small sample sizes however, selection bias cannot be excluded and further investigation is necessary to confirm our results.

Keywords: Intracranial aneurysm, animal models, NOS, PAI, single gene knockout mice, carotid ligation, hypertension, vascular remodeling