Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC186
Novel Immunohistochemical Technique to Quantify Nuclear Translocation of the Redox-Sensitive Transcription Factor Nrf2 Following Middle Cerebral Artery Occlusion In the Mouse
X. Tai1, R. Chen2, G. Mann1, P. Fraser1
1. Cardiovascular Division, King's College London, London, United Kingdom. 2. Experimental Medicine Division, University of Oxford, Oxford, United Kingdom.
Cerebral ischaemia initiates numerous cellular processes that can lead to oedema and loss of viable tissue or repair and recovery. The redox-sensitive nuclear factor NF E2-Related Factor 2 (Nrf2) moves from the cytoplasm to the nucleus following experimental stroke, leading to the induction of antioxidant enzymes such as hemoxygenase-1 (HO-1). Notably, Nrf2 knockout mice are affected much more severely by ischaemia-reperfusion injury. The aim of the present study was to develop a technique by which movement of Nrf2 from cytoplasm to nucleus could be quantified in brain tissue sections from mice that had undergone experimental stroke. Mice (C57Bl/6: 25-30 g) were subjected to 45 min middle cerebral artery occlusion (MCAO) under isoflurane anesthesia (2% initial, 1% to 1.5% maintenance in 30% O2 and 70% N2) and allowed to recover for 24 hours before they were killed. The brains were flushed, fixed with formaldehyde, embedded in paraffin wax and 6 µm sections were obtained. These sections were processed to remove the wax and retrieve epitopes before treatment with a polyclonal anti-Nrf2 antibody, followed by a biotinylated secondary antibody and streptavidin-horseradish peroxidase (HRP) conjugate. Propidium iodide (PI) was used to identify nuclei. Sequential images were obtained at 1 s intervals of diaminobenzidine reaction product (DAB) development, formed in the presence of hydrogen peroxide under a Nikon Diaphot microscope (x40 objective), were captured by a computer by using a cooled CCD camera (Photonic Science, Surrey) and ImageHopper software (Samsara Research, Surrey). These images were processed by generating the log of the ratio of the first image divided by subsequent images, and it was possible to ascertain the initial rate of DAB development from these. The assumption that this represents the concentration of Nrf2 on a pixel by pixel basis was tested by performing a similar operation on sections of gelatine blocks containing a known concentration of bovine serum albumin (BSA) and processed in a similar way (Fig 1A). Nrf2 distribution in infarct and control regions of the brain was assessed by image processing using the PI image as a nuclear mask, and the Nrf2 nucleus to cytoplasm ratio was calculated from the mean concentrations in the nucleus divided by that in a 5 µm wide surrounding annulus. The nuclear : cytoplasm ratio was 1.13 ± 0.178 (mean ± sd; 135 nuclei in 9 sections from 5 mice) in control regions and 1.44 ± 0.155 (90 nuclei in 8 sections from 5 mice) in infarcted regions (Fig 1B).
Where applicable, experiments conform with Society ethical requirements