Abstract Body: Background & Objectives: It remains unclear why unilateral proximal carotid artery occlusion (UCAO) causes benign oligemia, without progressing to cerebral infarction, in mice, yet leads to a wide variety of outcomes (asymptomatic-to-death) in humans. We hypothesized that inhibition of nitric oxide synthase (NOS) both transforms UCAO-mediated oligemia into full infarction and expands preexisting infarction.
Methods: Using 900 mice, we i) investigated stroke-related effects of a single intraperitoneal dose of the NOS inhibitor N_ω-nitro-L-arginine methyl ester (L-NAME, 400 mg/kg) + UCAO; ii) examined the rescue effect of the NO donor, molsidomine (200 mg/kg, at 30 minutes); iii) tested the impact of antiplatelet medications; iv) queried if UCAO without L-NAME administration could induce infarction when mice had hyperglycemia and hyperlipidemia; and v) measured blood levels of endogenous NOS inhibitors (asymmetric and symmetric dimethylarginines; ADMA and SDMA, respectively). Furthermore, we conducted i) a multi-center study (n=438 UCAO patients) to identify predictors of infarct volume and ii) Mendelian randomization analysis to estimate the causal effect of the endogenous NOS inhibitors on human ischemic stroke.
Results: UCAO alone induced infarction rarely (~2%) or occasionally (~14%) in C57BL/6 and BALB/c mice, respectively. However, L-NAME+UCAO induced large-arterial infarction in ~75% of C57BL/6 and BALB/c mice. Laser speckle imaging for 6 hours detected spreading ischemia in ~40% of C57BL/6 and BALB/c mice with infarction (vs. none without), as assessed at 24 hours. In agreement with vasoconstriction and microthrombus formation shown by intravital microscopy, molsidomine and the endothelial NOS-activating antiplatelet cilostazol attenuated or prevented progression to infarction. Moreover, UCAO without L-NAME caused infarction in ~22% C57BL/6 and ~31% ApoE knock-out mice with hyperglycemia and hyperlipidemia, which, in turn, associated with ~60% greater SDMA levels. Further, increased levels of glucose and cholesterol associated with significantly larger infarct volumes in UCAO patients. Lastly, Mendelian randomization identified a causative role of NOS inhibition, particularly in elevated SDMA concentration, in ischemic stroke risk (OR=1.24; 95% CI, 1.11–1.38; P=7.69×10^-5).
Conclusions: NOS activity is a key factor determining the fate of hypoperfused brain following acute carotid occlusion or clamping, where SDMA could be a potential risk predictor.