Activation of the redox-sensitive
transcription factor NF-E2 related factor 2 (Nrf2) affords protection against cerebral ischaemia-
reperfusion injury via the upregulation of
antioxidant defence genes. We have quantified for the first time Nrf2 content in brains from rats subjected to
stroke and from cultured bEnd.3 brain endothelial cells using a novel immunohistochemical technique. Male Sprague-Dawley rats were subjected to
middle cerebral artery occlusion for 70 min followed by reperfusion for 4, 24 or 72 h. Coronal brain sections were incubated with anti-Nrf2 primary and biotinylated-
horseradish peroxidase-conjugated secondary antibody, after which sections were reacted with
3,3-diaminobenzidine (DAB) in the presence of
hydrogen peroxide. The initial rates of DAB
polymer formation were directly proportional to the Nrf2
protein concentration. Image processing was used to determine the temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in
stroke-affected and contralateral hemispheres. Nuclear to cytoplasmic Nrf2 ratios were increased in the
stroke region after 24 h reperfusion and declined after 72 h reperfusion. Pretreatment with the Nrf2 inducer
sulforaphane reduced total cellular Nrf2 levels in peri-
infarct and core regions of the
stroke hemisphere after 24 h reperfusion. Treatment of cultured murine brain endothelial cells with
sulforaphane (2.5 μm) increased nuclear accumulation of Nrf2 over 1-4 h. We report the first quantitative measurements of spatial and temporal nuclear Nrf2 expression in rat brains following
stroke, and show that
sulforaphane pretreatment affects Nrf2 distribution in the brain of naïve rats and animals subjected to cerebral ischaemia. Our findings provide novel insights for targeting endogenous redox-sensitive
antioxidant pathways to ameliorate the damaging consequences of
stroke.