title: Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique.
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.
authors.affiliation: Cardiovascular Division, BHF Centre of Research Excellence, School of Medicine, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK.
authors.forename: Salil
authors.initials: S
authors.lastname: Srivastava
authors.forename: Alessio
authors.initials: A
authors.lastname: Alfieri
authors.forename: Richard C M
authors.initials: RC
authors.lastname: Siow
authors.forename: Giovanni E
authors.initials: GE
authors.lastname: Mann
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fraser
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism*; D017879:Isothiocyanates / Q000494:pharmacology; D008297:Male; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013454:Sulfoxides
chemical_list: D017879:Isothiocyanates; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; C495637:Nfe2l2 protein, rat; D013454:Sulfoxides; C016766:sulforaphane