Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique
Authors: Srivastava Salil , Alfieri Alessio , Siow Richard C. M. , Mann Giovanni E. , Fraser Paul A. 
Published: May 2013

Key points
The redox‐sensitive transcription factor NF‐E2 related factor 2 (Nrf2) plays a key role in regulating adaptive cellular antioxidant defences, and activation of Nrf2 in stroke protects the brain against oxidative stress following ischaemia‐reperfusion injury.
We report the first measurements of temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in cells in the ischaemic core, peri‐infarct regions and contralateral hemisphere of rat brain following cerebral ischaemia‐reperfusion injury for 4, 24 or 72 h using a novel quantitative immunohistochemical technique, which was further validated in cultured bEnd.3 murine brain endothelial cells.
Nrf2 expression in brain sections was increased in core and peri‐infarct regions after 24 h reperfusion, with levels remaining elevated only in peri‐infarct regions after 72 h. Pretreatment of rats with the Nrf2 inducer sulforaphane reduced core and peri‐infarct Nrf2 levels after 24 h reperfusion.
The time course of stroke‐induced changes in nuclear to cytoplasmic Nrf2 content and its modulation by pretreatment with sulforaphane provide novel insights for targeting endogenous redox sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.
Abstract  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.