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The Journal of Physiology logoLink to The Journal of Physiology
. 2013 May 20;591(Pt 14):3525–3538. doi: 10.1113/jphysiol.2013.257964

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique

Salil Srivastava 1, Alessio Alfieri 1, Richard C M Siow 1, Giovanni E Mann 1, Paul A Fraser 1
PMCID: PMC3731611  PMID: 23690560

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.

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.

Introduction

Brain damage following ischaemic stroke is the result of a series of pathophysiological mechanisms (Dirnagl et al. 1999; Candelario-Jalil, 2009), including an excess production of reactive oxygen species and reactive nitrogen species, with severe consequences for the viability of cells critical for brain function and cerebrovascular permeability (Alfieri et al. 2011; Chen et al. 2011; Fraser, 2011; Woodfin et al. 2011). The brain is at an increased risk of oxidative damage due its high demand for oxygen, high metabolic activity, increased content of unsaturated fatty acids and low intracellular antioxidant capacity (Shohami et al. 1997; Ozkul et al. 2007; Ikonomidou & Kaindl, 2011). The adverse neurological consequences following ischaemic stroke are initiated in the early hours after the onset of ischaemia (Thompson et al. 1999; Kolominsky-Rabas et al. 2006). Treatment strategies targeting endogenous repair mechanisms in the brain are now a prime focus of stroke research (Alfieri et al. 2011; Iadecola & Anrather, 2011).

The redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) orchestrates endogenous antioxidant defences against oxidative and nitrosative stress via the upregulation of phase II detoxifying enzymes and antioxidant stress proteins (Ishii et al. 2000). Under physiological conditions Nrf2 is bound by its cytoplasmic repressor Kelch-like associated protein 1 (Keap1) and targeted for proteasomal degradation (Motohashi & Yamamoto, 2004; Itoh et al. 2010; Taguchi et al. 2011). Oxidative and electrophilic stress induce nuclear translocation and binding of Nrf2 to the antioxidant response element (ARE) in the promoter of protective genes such as heme oxygenase 1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO1), peroxiredoxin 1 (Prx1) and γ-glutamyl cysteine ligase (Ishii et al. 2000, 2004; Motohashi & Yamamoto, 2004; Taguchi et al. 2011; Chapple et al. 2012). Activation of this pathway increases total protein expression and nuclear levels of Nrf2 (Kwak et al. 2002).

Although activation of Nrf2 has been reported to attenuate brain damage and neurological deficits following stroke (Shah et al. 2007; Yang et al. 2009; Alfieri et al. 2011; Kam et al. 2011; Tanaka et al. 2011), there are no reports that have quantified temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments of cells in the ischaemic core, peri-infarct regions and contralateral hemisphere following transient ischaemia-reperfusion injury. Moreover, the effects of pretreatment of rats in vivo with sulforaphane, a known Nrf2 inducer contained in cruciferous vegetables (Zhang et al. 1992; Dinkova-Kostova & Kostov, 2012), on intracellular distribution of Nrf2 following stroke has to our knowledge not been reported.

Immunohistochemistry using 3,3′-diaminobenzidine (DAB) is a widely employed technique for studying the expression of proteins in tissues (Graham & Karnovsky, 1966). To date, application of this technique has only provided a semi-quantitative, pixel-by-pixel measure of the intensity of staining (Toyokuni et al. 1997; Matkowskyj et al. 2000). Formation of the coloured DAB polymer is dependent upon the number of horseradish peroxidase (HRP) active sites available to enzymatically oxidise DAB in the presence of hydrogen peroxide (Nakane & Pierce, 1967). Notably, the number of HRP active sites available to catalyse the oxidation of DAB is dependent upon the concentration of a given protein within the sample (Gauden et al. 2007). Thus, evaluation of the rate of DAB polymer formation allows for a more accurate quantification of protein content within nuclear and cytoplasmic compartments of cells in a given tissue sample.

In this study we have developed and validated a novel DAB-based immunohistochemical technique to quantify the temporal and spatial distribution of Nrf2 in nuclei and cytoplasmic compartments in brain sections ex vivo obtained from rats subjected to transient middle cerebral artery occlusion (MCAo) and reperfusion injury. Simultaneous application of DAB and fluorescence immunohistochemical techniques enabled us to define nuclear to cytoplasmic distribution of Nrf2 and total cellular Nrf2 levels in core and peri-infarct regions of stroke-affected and contralateral brain hemispheres after 4, 24 and 72 h reperfusion. We further quantified the time course of nuclear to cytoplasmic distribution of Nrf2 in the mouse-derived brain endothelial cell line b.End3 challenged acutely with sulforaphane. We report the first quantitative measurements of temporal and spatial Nrf2 distribution in the brain following experimental stroke and further demonstrate that treatment of naïve animals with sulforaphane elevates Nrf2 levels in the brain, whilst pretreatment with sulforaphane prior to stroke reduces Nrf2 content in peri-infarct regions after 24 h reperfusion.

Methods

Materials

Porcine gelatine, bovine serum albumin (BSA), rabbit anti-BSA antibody, ascorbic acid and d,l-sulforaphane were purchased from Sigma (Gillingham, Dorset, UK). Rabbit anti-Nrf2 and donkey anti-goat Cy5 were purchased from Abcam (Cambridge, UK); donkey anti-mouse Alexafluor 555 (Invitrogen, Paisley, UK); 4′,6-diamidino-2-phenylindole (DAPI; Roche, Welwyn Garden City, UK); Bloxall endogenous peroxidase solution, Avidin and Biotin blocking solution, goat anti-rabbit biotinylated secondary antibody (Vectastain ABC elite kit), DAB and hydrogen peroxide (H2O2) substrate kit were purchased from Vector Laboratories Ltd (Peterborough, UK). Dulbecco's modified Eagle's medium (DMEM) containing 5.5 mm d-glucose and supplemented with 10% fetal calf serum, 4 mm l-glutamine and penicillin/streptomycin (100 IU ml−1) was from Sigma.

Animals

Male Sprague-Dawley rats (weighing 250–300 g; Harlan, UK) were acclimatized for at least 1 week before surgery and maintained on a 12 h light/dark cycle. All procedures were performed under the authority of Project Licence No. PPL70/6579, in accordance with the UK Animal (Scientific Procedures) Act 1986 and rigorous ethical review process by the UK Home Office and King's College London.

Middle cerebral artery occlusion

Cerebral ischaemia was induced by transient intraluminal occlusion of the right middle cerebral artery (MCAo) under 4% isofluorane anaesthesia (Modo et al. 2000). A silicone-coated filament was inserted from the carotid bifurcation and left in place for 70 min. At the end of the occlusion period, the intraluminal filament was removed to allow reperfusion. MCAo was confirmed by examining flexion of the contralateral forelimb by lifting of the tail and spontaneous asymmetric turning behaviour. To examine the effects of activating Nrf2 in vivo, rats were pretreated with the Nrf2 inducer d,l-sulforaphane (5 mg kg−1i.p., dissolved in 1% corn oil; Zhao et al. 2007; Benedict et al. 2012) 1 h before 70 min MCAo followed by 24 h reperfusion.

Removal of rat brains and coronal sectioning

Rats were killed with an overdose of pentobarbitone (120 mg kg−1i.p.) following 4, 24 or 72 h reperfusion, and brains were then perfused with cold saline followed by 40 g l−1 paraformaldehyde in 0.1 m phosphate-buffered saline (PBS). After collection, fixed brains were left overnight in paraformaldehyde before being transferred to 300 g l−1 sucrose and stored at 4°C for subsequent sectioning. Coronal brain sections (10 μm) were cut on a cryostat (Bright, UK) onto Superfrost Plus glass slides (Thermo Scientific, UK; formal saline pretreated) and left to air dry for 48 h at room temperature. Sections were stored at −20°C for subsequent immunohistochemical and/or immunofluorescence analyses.

Immunohistochemical quantification of HRP covalently linked antibodies

The novel technique applied in this study extends our previous report, where HRP was used as a fluid phase marker to estimate blood–brain barrier permeability following ischaemia-reperfusion injury (Gauden et al. 2007). The principle that HRP can be measured in tissue sections from the initial rate of DAB polymer formation has been extended to quantify HRP covalently linked antibodies in ex vivo brain sections. In proof-of-principle experiments, we established that the relationship between conjugated secondary and primary antibodies and a target protein is essentially linear (Fig. 1). Known concentrations of BSA were dissolved in warm porcine gelatine 200 g l−1 solution and allowed to set at room temperature. The blocks, when set, were frozen, sectioned onto glass slides and fixed similarly to brain tissue (see below). Sections (10 μm) were incubated with rabbit anti-BSA at 4°C for 16 h, and then with a goat anti-rabbit biotinylated secondary antibody for 30 min at room temperature, followed by a further 30 min with the ABC reagent to amplify the DAB reaction product signal via the formation of the avidin and biotin complex. One section was taken from four replicate blocks for each albumin concentration, and regression coefficients calculated for the linear portion of each of the optical density curves (see Fig. 1A). Figure 1B shows the relationship between the initial rate of DAB product development versus the BSA concentration in the gelatine blocks.

Figure 1. Proof-of-principle experiment that protein content in frozen sections can be quantified from initial rates of DAB–H2O2 reaction product formation.

Figure 1

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Frozen sections (10 μm) of porcine gelatine containing known concentrations of bovine serum albumin (BSA) were incubated with anti-BSA primary antibody, complementary HRP-conjugated secondary antibody and treated with ABC solution. A, sequential images of the DAB–H2O2 reaction product were captured, optical densities plotted and a regression calculated for the initial linear portion for each BSA concentration. Data denote mean ± SEM of measurements in four frozen gelatine sections for each BSA concentration. B, regression coefficients for initial DAB development rate plotted against BSA concentration; mean ± SEM of measurements from four sections for each BSA concentration, noting that standard errors of the regression analyses fall within the symbols.

Preparation of brain sections for immunohistochemistry and antibody incubation

Brain sections were defrosted at room temperature following removal from −20°C and quenched for endogenous peroxidase activity using Bloxall (Vector Laboratories Ltd) for 10 min at room temperature. Sections were gently washed with PBS and incubated with avidin D for 15 min at room temperature, excess avidin D solution gently blotted off, and the section then incubated for a further 15 min at room temperature with biotin blocking solution (Vector Laboratories Ltd) according to the manufacturer's instructions. Brain sections were permeabilised with 1 g l−1 Triton-X 100 (Sigma) in PBS with 10% normal donkey serum and then incubated with primary antibodies goat anti-glial fibrillary acidic protein (GFAP, 1:400) and rabbit anti-Nrf2 (1:100) overnight at 4°C. GFAP was also identified by fluorescence, and sections incubated with secondary antibodies (donkey anti-goat Cy5, 1:500) for 1 h at room temperature.

To quantify Nrf2 by immunohistochemistry, brain sections were incubated with goat anti-rabbit biotinylated secondary antibody and ABC reagent to amplify the DAB reaction product signal. Brain sections were washed with doubly distilled water and stained with DAPI (2 μg ml−1) for 5 min. Quantification using our immunohistochemical technique is critically dependent on the stability of the tissue in the first few seconds after application of the DAB–H2O2 reaction mix. We found that preincubation of sections with ascorbic acid (1 mm) and DAB for 30 min, before application of the reaction mix, eliminated tissue movement.

Quantification of immunohistochemistry and image processing

Hydrated brain sections were placed in a specially designed microscope stage on a Nikon Diaphot fluorescence microscope. Images of nuclei and GFAP staining were captured via a cooled CCD camera (ORCA-03G, Hamamatsu, Japan) and ImageHopper (Samsara Research, Dorking, UK). Water was removed and replaced with the DAB–H2O2 reaction mixture and images in brightfield were captured at 1 s−1 over 100 s.

Figure 2A shows selected images obtained during the rapid time course of DAB–H2O2 reaction product development in ex vivo brain section from rats subjected to MCAo (70 min) and 24 h reperfusion. Images of DAB polymer density were subsequently generated by applying Beer–Lambert's Law to the sequence, i.e. the log ratio of the first image to subsequent images, thereby generating an optical density map (Fig. 2B). The optical densities through the stack for different regions (red square in Fig. 2B) were corrected for DAB–H2O2 reaction product development over the lumen of a cerebral microvessel free of tissue (green square in Fig. 2B). Time courses for DAB–H2O2 product development in tissue, the tissue-free region (blank) and resulting net tissue are shown in Fig. 2C. The dotted line represents the regression analysis of the linear portion for net tissue product development over the first 25 s. Optical density values in the stack that fell outside the linear portion were excluded from further analysis. The Nrf2 spatial map shown in Fig. 2D was calculated by carrying out a linear regression on the stack of optical density images after subtracting blank values.

Figure 2. Method for estimating Nrf2 content in a brain tissue section.

Figure 2

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A, selection of a series of images captured at 1 s−1 during incubation with DAB–H2O2. B, the optical density (OD) calculated on a pixel by pixel basis using the Beer–Lambert law (ln(Io/It)). A region of interest through the image stack over a portion free of tissue (green square) was subtracted from that over the tissue (red square) leaving the HRP-dependent reaction product development. C, time courses for tissue, blank (cerebral microvessel lumen) and net tissue DAB–H2O2 product formation. The initial rate of DAB–H2O2 reaction (dotted line) was assessed from the first 25 s, and the image in D formed from a pixel by pixel regression of that initial portion of the stack shown in B after subtraction of the blank values. This image was taken to represent levels of Nrf2. Scale bars = 50 μm.

The time course of Nrf2 activation after ischaemia-reperfusion injury was examined in core and peri-infarct regions of the stroke-affected hemisphere as well as the contralateral hemisphere. The image in Fig. 3A is a composite of the upper left-hand region of the section shown in Fig. 2A. GFAP fluorescence staining was used to identify activated astrocytes and to discriminate the infarct core and peri-infarct region in the stroke-affected hemisphere (Fig. 3A). Total Nrf2 levels are shown in pseudo-colour in Fig. 3B. To quantify the nuclear to cytoplasmic ratios of Nrf2 distribution, we used the DAPI image of nuclei (Fig. 3C) and applied a binary mask to segment the Nrf2 image to obtain nuclear Nrf2 content (Fig. 3D). A similar binary mask of a 4 μm wide ring around each nucleus (taken to be its cytoplasm) was also applied (Fig. 3E), and ratios were then obtained for each nucleus–cytoplasm pair. The cellular Nrf2 content was determined from the weighted mean content of each nucleus–cytoplasm pair and expressed as optical density (OD) increase rate per 1000 s, i.e. (s ×103)−1. Stroke regions of the brain were separated into core and periphery based on the intensity of GFAP staining, used as a marker for astrocytic activation.

Figure 3. Segmentation to identify Nrf2 content in nuclear and cytoplasmic cellular compartments in the brain following MCAo and 24 h reperfusion.

Figure 3

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A, composite image of the left-hand portion of the section in Fig. 2A, with a fluorescence overlay of nuclei stained with DAPI (blue) and astrocytes with GFAP (green). B, Nrf2 image is scaled to a pseudo-colour map. C, DAPI image of the nuclei was used to generate a mask that segmented the Nrf2 concentration in nuclei, as shown in D. E, cytoplasm was taken to be a ring of 4 μm around each nucleus. The combined nucleus and its cytoplasm were taken to represent a cell.

Culture of murine brain endothelial cell line bEnd.3

The immortalised murine-derived brain endothelial cell line bEnd.3 was purchased from ATCC-LGC (Teddington, UK) and grown in DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum, 4 mm l-glutamine, 100 U ml−1 penicillin G and 100 μg ml−1 streptomycin. bEnd.3 cells (passage 9–15) were maintained in a humidified incubator at 37°C and 5% CO2 and 95% air atmosphere. Cell suspensions were diluted in DMEM and plated onto Superfrost Plus slides for DAB immunohistochemistry. An endothelial phenotype was confirmed based on immunostaining monolayers for endothelial nitric oxide synthase and von Willebrand factor (Rowlands et al. 2011).

Nrf2 content in bEnd.3 cells determined by quantitative immunohistochemistry

bEnd.3 cells were treated for 1–4 h with DMEM containing vehicle (DMSO, 0.01% v/v) and or 2.5 μm d,l-sulforaphane. Cells were fixed, permeabilised and quenched for endogenous peroxidase, avidin and biotin as described previously. Cells were incubated with a rabbit anti-Nrf2 primary antibody for 16 h at 4°C and then with the biotinylated secondary antibody and ABC reagent. DAPI in PBS was used for the identification of cellular nuclei. Stained cells remained hydrated in PBS on the Nikon Diaphot microscope stage and were imaged for nuclei by DAPI fluorescence. PBS was removed and replaced with the DAB–H2O2 reaction mix in PBS. Images of the reaction were captured under brightfield at 0.5 s intervals over 50 s and processed to determine nuclear to cytoplasmic distribution of Nrf2 following treatment with sulforaphane.

Statistical analysis

Data denote mean ± SEM of measurements in n= 3–5 different animals, with a minimum of three sections analysed per brain in each animal. Data in bEnd.3 cells were obtained from five independent cultures. Analysis of variance (ANOVA) followed by Tukey's post hoc test or Student's paired t test was applied as appropriate with P < 0.05 considered significant.

Results

Initial rate of DAB–H2O2 reaction product formation as an index of Nrf2 content in brain tissue sections

When rat brain sections (10 μm), obtained after MCAo (70 min) and 24 h reperfusion, were incubated with an anti-Nrf2 antibody, HRP-conjugated secondary and treated with ABC solution, the DAB–H2O2 reaction product formation was linear over the first 25 s (Fig. 2C), confirming the initial linear time course of DAB polymer formation in BSA gelatine sections (Fig. 1A and B). As illustrated in Fig. 3, nuclear to cytoplasmic ratios of Nrf2 distribution were quantified by using the DAPI image of nuclei (Fig. 3C) to segment the Nrf2 image (Fig. 3B) into nuclear (Fig. 3D) and cytoplasmic (Fig. 3E) compartments.

Temporal changes in nuclear to cytoplasmic Nrf2 distribution in rat brains following MCAo

The ratio between nuclear to cytoplasmic Nrf2 content after 4 h reperfusion was lower in the stroke-affected than contralateral hemisphere (Fig. 4D) and values observed in control brains (Fig 4A and D). After 24 h reperfusion, there was a significant increase in the mean nuclear to cytoplasmic Nrf2 ratio (>1.0) in the stroke-affected versus contralateral hemisphere (Fig. 4B and D) or control brains (Fig. 4A and D). Following 72 h reperfusion, Nrf2 ratios in both the stroke-affected and the contralateral hemispheres decreased below values in control brains, and notably Nrf2 ratios were similar in stroke-affected and contralateral regions (Fig. 4D).

Figure 4. Temporal changes in nuclear to cytoplasmic Nrf2 levels in rat brain following stroke and reperfusion injury.

Figure 4

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The ratio of Nrf2 content in each nucleus relative to its surrounding cytoplasm was calculated and is displayed in the histograms. A, nuclear to cytoplasmic Nrf2 ratios in 3199 cells from five control animals compared to 1869 cells from three animals 24 h after sulforaphane (SFN) treatment. B, data from three animals after 24 h reperfusion in stroke (1194 cells) and contralateral hemispheres (1675 cells). C, pretreatment with sulforaphane for 1 h followed by MCAo and 24 h reperfusion: stroke hemisphere (2420 cells) versus contralateral hemisphere (1273 cells) in three animals. D, summary of nuclear to cytoplasmic Nrf2 ratios after MCAo and ischaemia-reperfusion, with additional mean data shown for 4 h reperfusion (2633 cells in stroke versus 1580 cells in contralateral hemisphere, n= 3 animals) and 72 h reperfusion (1725 cells in stroke versus 1168 cells in contralateral hemisphere, n= 3 animals). Data denote mean ± SEM. **P < 0.01, ***P < 0.001, #P < 0.001 between contralateral and stroke hemispheres in untreated and SFN treated groups; ‡P < 0.01 and †P < 0.001 versus control brains.

Temporal changes in total cellular Nrf2 content in stroke and contralateral hemispheres

Immunopositive staining for GFAP, an index of astrocyte activation in the brain, was only just detectable after 4 h reperfusion and increased further after 24 and 72 h reperfusion (data not shown). Figure 5 summarises paired measurements in individual animals of the mean Nrf2 content per cell in stroke and contralateral brain hemispheres following 4, 24 and 72 h reperfusion. Increased GFAP-immunopositive staining detected after 24 and 72 h reperfusion enabled us to define core and peri-infarct regions in the stroke hemisphere. After 4 h reperfusion, the total Nrf2 content per cell was similar in the contralateral hemisphere in control brains, although a significant increase in Nrf2 content was observed in the stroke-affected regions (Fig. 5A and E). After 24 and 72 h reperfusion, total Nrf2 levels were significantly increased in peri-infarct compared to core regions (Fig. 5B, C and E) of the stroke hemisphere. Notably, the elevated Nrf2 content in the core after 24 h reperfusion (Fig. 5B) returned to levels detected in the contralateral hemisphere after 72 h reperfusion (Fig. 5C and E).

Figure 5. Temporal and spatial changes in total cellular Nrf2 content in brain cells following stroke and reperfusion injury.

Figure 5

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Total Nrf2 content was calculated on a cellular basis for stroke-affected and contralateral hemispheres in four sets of animals, with additional data showing Nrf2 content in control animals (diamonds in A) or sulforaphane-treated animals not undergoing MCAo (diamonds in D). Paired comparisons are shown for each animal following 4 h (A), 24 h (B) and 72 h (C) reperfusion, respectively. Total cellular Nfr2 content in core and peri-infarct regions of the stroke hemisphere could only be resolved following 24 and 72 h reperfusion. D, effects of sulforaphane pretreatment on Nrf2 content in core and peri-infarct regions of the stroke hemisphere and contralateral hemisphere following 24 h reperfusion. In A–D data denote mean ± SEM. *P < 0.05, **P < 0.01 and standard errors lie within the symbols. E, summary of mean Nrf2 content per cell pooled from different brain regions in all animals following reperfusion. Data denote mean ± SEM, n= 3 animals per group. #P < 0.001 for respective regions of sulforaphane pretreated versus untreated animals. In the MCAo groups, values for contralateral, core and peri-infarct regions are significantly different from each other, ***P < 0.001.

Sulforaphane pretreatment modulates nuclear and total cellular Nrf2 content in the brain after 24 h ischaemia-reperfusion

Treatment of naïve rats with sulforaphane (5 mg kg−1i.p.) had no significant effect on nuclear to cytoplasmic distribution of Nrf2 (Fig 4A and D) or total cellular Nrf2 content (data not shown) when measured 24 h later. However, pretreatment of animals with sulforaphane 1 h before MCAo and 24 h reperfusion led to a significant decrease in nuclear to cytoplasmic Nrf2 ratios (Fig. 4C and E) and total Nrf2 content (Fig. 5D and E) in the stroke-affected and contralateral hemispheres.

Sulforaphane induces nuclear accumulation of Nrf2 in bEnd.3 brain endothelial cells

To further validate the application of our quantitative DAB–H2O2 immunohistochemical technique for cultured brain endothelial cells, we treated bEnd.3 cells with sulforaphane (2.5 μm) for 1–4 h and determined the time course of changes in nuclear to cytoplasmic Nrf2 distribution. Figure 6A illustrates representative images of DAB–H2O2 reaction product formation in bEnd.3 cells treated for 2 h with sulforaphane, with nuclear to cytoplasmic Nrf2 content in response to vehicle and sulforaphane summarised in Fig. 6B. Notably, sulforaphane significantly increased Nrf2 content in nuclear structures after 1–4 h treatment (Fig. 6C).

Figure 6. Nuclear distribution of Nrf2 in bEnd.3 endothelial cells following sulforaphane treatment.

Figure 6

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Confluent bEnd.3 endothelial cells on glass slides were treated for 1–4 h with sulforaphane (SFN, 2.5 μm) and nuclear to cytoplasmic distribution of Nrf2 was analysed by quantitative immunohistochemistry. A, images showing DAB–H2O2 reactivity map for Nrf2, nuclei identified by DAPI (blue) and a merge for bEnd.3 cells treated with vehicle (upper panel) or sulforaphane (SFN, lower panel) for 2 h. B, histogram of nuclear to cytoplasmic ratio of Nrf2 for each respective bEnd.3 cell analysed (106 cells in three different bEnd.3 cultures). C, mean nuclear to cytoplasmic ratio of Nrf2 for cells treated with vehicle (V) or SFN for 1, 2, or 4 h. Data denote mean ± SEM, n= 4 independent experiments in different bEnd.3 cell cultures. ***P < 0.001 versus vehicle. #P < 0.001 for 1 h versus 4 h SFN treatment and †P < 0.001 2 h versus 4 h SFN treatment.

Discussion

We have investigated whether stroke leads to time-dependent changes in the distribution of Nrf2 between the nucleus and surrounding cytoplasm in cells in the brain, and whether pretreatment with the Nrf2 inducer sulforaphane affects the spatial distribution of Nrf2 following cerebral ischaemia-reperfusion injury. Our novel immunohistochemical technique has enabled us to simultaneously quantify the content of the redox sensitive transcription factor Nrf2 in nuclear and cytoplasmic compartments of cells in brain ex vivo following reperfusion injury. We report that nuclear to cytoplasmic Nrf2 ratios in stroke-affected regions were initially decreased after 4 h, increased after 24 h and then declined after 72 h reperfusion. Moreover, Nrf2 expression was significantly higher in peri-infarct than in infarct core regions of the stroke-affected hemisphere. Pretreatment of animals with sulforaphane decreased nuclear Nrf2 content in the stroke and contralateral hemispheres, and the total Nrf2 content in peri-infarct and core regions of the stroke-affected hemisphere after 24 h reperfusion. These findings suggest that sulforaphane pretreatment affords protection against oxidative stress and thereby reduces the necessity for marked increases in nuclear accumulation of Nrf2 following stroke.

Immunohistochemical techniques involving DAB have predominantly employed the development of DAB polymer for a qualitative analysis of the expression and localisation of proteins of interest in cells using histological stains (Taylor & Levenson, 2006). Immunohistochemical and immunofluorescence techniques currently require sophisticated image analysis software to quantify the degree of staining on a pixel by pixel basis (Matkowskyj et al. 2000; Kirkeby & Thomsen, 2005; Montgomery et al. 2008). Such methods of quantification, however, provide only semi-quantitative read-outs and do not provide an accurate measure of the protein concentration present in cellular compartments. Measurement of protein in tissues has relied on a combination of immunoblotting of whole tissue homogenates, whereby a global change in the tissue is detected, but information on spatial changes is lost. The novel method described in this study is a development of a technique we previously reported (Gauden et al. 2007), where HRP was quantified in brain tissue to obtain permeability–surface area product measurements of cerebrovascular permeability following ischaemia-reperfusion injury. We have advanced this technique and here demonstrate that changes in protein concentration, e.g. the redox sensitive transcription factor Nrf2, can be quantified directly with sufficient temporal and spatial resolution to measure the concentration within nuclear and cytoplasmic compartments of cells in the brain.

Activation and nuclear accumulation of Nrf2 upregulates endogenous antioxidant defences to restore cellular redox homeostasis via the induction of phase II defence enzymes and antioxidant stress proteins (Ishii et al. 2000, 2004; Motohashi & Yamamoto, 2004; Taguchi et al. 2011), yet no information is available on the nuclear to cytoplasmic distribution of Nrf2 in brain tissue following transient ischaemia-reperfusion injury. In our rat MCAo model, we have obtained the first measurements of nuclear to cytoplasmic Nrf2 distribution in both contralateral and stroke-affected hemispheres. We found a decrease in nuclear Nrf2 content in stroke regions after 4 h reperfusion, potentially reflecting an increase in Nrf2 synthesis and greater cytoplasmic content. Nrf2 ratios were increased significantly in stroke regions after 24 h reperfusion, and after 72 h declined in both stroke-affected and contralateral regions to values below those in control brains (Fig. 4D). Notably, after 72 h reperfusion, the nuclear to cytoplasmic Nrf2 distribution was similar in stroke-affected and contralateral hemispheres (Fig. 4D), suggesting possible export of Nrf2 from the nucleus.

Although phosphorylation of Nrf2 has been reported to affect nuclear translocation of Nrf2 and transcriptional activation of its target antioxidant enzymes, (Niture et al. 2011; Chen et al. 2012) others have shown that site-directed mutagenesis of Nrf2 at different mitogen-activated protein kinase consensus sites has only a limited effect on Nrf2-dependent gene expression and/or ARE-dependent luciferase activity (Zipper & Mulcahy, 2003; Shen et al. 2004; Sun et al. 2009). To further validate our polyclonal Nrf2, we compared two different Nrf2 antibodies (see Supplemental Fig. S1) and examined the effects of treating ex vivo brain sections with alkaline phosphatase on nuclear/cytoplasmic Nrf2 ratios and total cellular Nrf2 levels (see Supplemental Fig. S2). Notably, similar total and nuclear/cytoplasmic ratios for Nrf2 were measured using the two different Nrf2 antibodies. Furthermore, treatment with alkaline phosphatase had no significant effect on Nrf2 ratios or total cellular Nrf2 content in ex vivo brain sections.

In our study a significant increase in total cellular Nrf2 content was observed in peri-infarct region and core regions of the stroke hemisphere after 24 h reperfusion (Fig. 5B), which decreased following 72 h reperfusion (Fig. 5C). Based on the significantly lower cellular Nrf2 content in the infarct core compared to peri-infarct regions after reperfusion (Fig. 5E), we suggest that cells in peri-infarct regions have an elevated antioxidant capacity, enabling them to counteract the stress induced by reactive oxygen species following ischaemia-reperfusion. Previous studies in mice subjected to MCAo have only detected Nrf2 immunopositive cells in the peri-infarct region after 8–72 h reperfusion and, unlike our findings, could not observe Nrf2 staining in the infarct core over this time period (Tanaka et al. 2011). More recent studies in rats subjected to MCAo and 24 h reperfusion were also unable to detect Nrf2 immunopositive staining in the infarct core (Dang et al. 2012), highlighting the sensitivity of our quantitative immunohistochemical technique.

We found that pretreatment of animals with the Nrf2 inducer sulforaphane prior to MCAo significantly affected nuclear accumulation of Nrf2 (Fig. 4C and D) and total cellular Nrf2 content (Fig. 5D and E) in both stroke-affected and contralateral regions after 24 h reperfusion. Previous studies in mice have shown that following intraperitoneal sulforaphane administration, this isothiocyanate rapidly crosses the blood–brain barrier, accumulates in the striatum and cortex within 15 min, and increases Nrf2 protein levels within 1–2 h (Jazwa et al. 2011). Furthermore, increased Nrf2 protein levels in the brain induced by sulforaphane are accompanied by an increased expression of its target antioxidant genes HO-1 and NQO1 after 16 h (Jazwa et al. 2011), indicating that pretreatment with sulforaphane activates Nrf2 and cellular antioxidant defences in the brain.

Oxidative stress experienced by infarcted areas of the brain upon reperfusion (Dirnagl et al. 1995; Peters et al. 1998; Yamato et al. 2003) results in increased cerebrovascular permeability (Kaya et al. 2003; Kahles et al. 2007; Woodfin et al. 2011). Protective effects of Nrf2 in cerebral ischaemia-reperfusion injury have been reported (Shih et al. 2005; Yang et al. 2009; Shah et al. 2010; Son et al. 2010), and notably, post-treatment of the ischaemic brain with inducers of Nrf2, such as curcumin, epicatechin, plumbagin and sulforaphane, reduces the volume of infarcted tissue and oedema formation (Yang et al. 2009; Shah et al. 2010; Son et al. 2010), implying a decrease in cerebrovascular permeability. Moreover, administration of sulforaphane to animals following stroke partially restores neurological and behavioural function (Zhao et al. 2007). Activation of Nrf2 in cells in vitro has been shown to protect astrocytes and brain endothelial cells (Bell et al. 2011; Pan et al. 2011; Williamson et al. 2012), with sulforaphane enhancing nuclear accumulation of Nrf2 and induction of its downstream phase II detoxification enzyme NQO1 (Kraft et al. 2004; Danilov et al. 2009). We further validated our immunohistochemical technique for application in vitro by examining the effects of sulforaphane on nuclear to cytoplasmic Nrf2 distribution in bEnd.3 brain endothelial cells. In our study, sulforaphane induced nuclear accumulation of Nrf2 in bEnd.3 cells over 1–4 h (Fig. 6), mirroring the time course of Nrf2 expression in the striatum and cortex of mice treated with sulforaphane in vivo (Jazwa et al. 2011). Thus, nuclear accumulation of Nrf2 is modulated by sulforaphane in the brain in vivo and in brain endothelial cells in vitro.

In Nrf2-deficient mice, experimental stroke is characterised by exacerbated oedema formation and an increased recruitment of pro-inflammatory cells to stroke-affected regions (Shih et al. 2005; Yang et al. 2009). Nrf2 has been implicated in dampening down pro-inflammatory processes (Li et al. 2008), and anti-inflammatory actions of sulforaphane may in part be mediated by inhibition of NF-κB signalling (Heiss et al. 2001; Cheung & Kong, 2010; Benedict et al. 2012). As treatment with sulforaphane after stroke reduces cerebral infarct volume and oedema formation (Zhao et al. 2006), it seems likely that the cytoprotective actions of sulforaphane in the brain involve both activation of Nrf2 and inhibition of pro-inflammatory signalling pathways.

In conclusion, we have developed a novel quantitative DAB–H2O2 immunohistochemical technique to analyse temporal and spatial expression of proteins, and in particular transcription factors such as Nrf2, in the brain following stroke and endothelial cells in vitro. We have quantified the nuclear to cytoplasmic Nrf2 content in stroke-affected regions and observed a significant increase in the Nrf2 ratio in peri-infarct regions after 24 h reperfusion. To our knowledge, our study is the first to investigate the effects of sulforaphane pretreatment on spatial Nrf2 distribution in the brain following cerebral ischaemia-reperfusion. Rapid accumulation of sulforaphane in the brain and subsequent upregulation of Nrf2 and antioxidant enzymes (Jazwa et al. 2011) may reduce the necessity for later adaptive increases in Nrf2 expression following stroke. Importantly, this technique can readily be applied for quantification of nuclear accumulation of other transcription factors, such as NF-κB, HIF-1α, CREB or AP-1, involved in redox signalling in the brain following ischaemic or traumatic insults, in other tissues and in cultured cells in vitro. We propose that upregulation of Nrf2 and its target antioxidant genes prior to stroke using natural hormetic agents such as sulforaphane and/or other Nrf2 inducers to modulate intracellular redox signalling (Siow & Mann, 2010) affords protection against cerebrovascular oxidative damage.

Glossary

ARE

antioxidant response element

BSA

bovine serum albumin

DAB

3,3′-diaminobenzedine

DAPI

4′,6-diamidino-2-phenylindole

GFAP

glial fibrillary acidic protein

HO-1

heme oxygenase-1

HRP

horseradish peroxidise

Keap1

Kelch-like associated protein 1

MCAo

middle cerebral artery occlusion

NQO1

NAD(P)H quinine oxidoreductase 1

Nrf2

NF-E2 related factor 2

PBS

phosphate-buffered saline

Additional information

Competing interests

None.

Author contributions

S.S. conducted the immunohistochemical experiments and analysis of DAB polymer formation in ex vivo brain sections. A.A. conducted all MCAo and pharmacological interventions. G.E.M., S.S., A.A., R.C.M.S. and P.A.F. contributed to the design of experiments and drafting of the manuscript. G.E.M. and P.A.F. are the guarantors of this work and assume full responsibility for the integrity of the experimental data and analysis.

Funding

We gratefully acknowledge the support of the British Heart Foundation (FS/09/056) and The Henry Smith Charity (RG20092511).

Supplementary material

Supplement Figure S1

Supplement Figure S2

tjp0591-3525-SD1.tif (1.5MB, tif)
tjp0591-3525-SD2.tif (1.5MB, tif)

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