ORIGINAL ARTICLE In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice Xavier de la Rosa & Tomàs Santalucía & Pierre-Yves Fortin & Jesús Purroy & Maria Calvo & Angélica Salas-Perdomo & Carles Justicia & Franck Couillaud & Anna M. Planas Received: 6 August 2012 / Accepted: 4 October 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract Purpose Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. Methods A genomic DNA sequence of the Hspa1b promot- er was used to generate an Hsp70-mPlum far-red fluores- cence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum trans- genic mice were generated. Focal brain ischaemia was in- duced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Results Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischae- mia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. Conclusion This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core. Electronic supplementary material The online version of this article (doi:10.1007/s00259-012-2277-7) contains supplementary material, which is available to authorized users. X. de la Rosa : T. Santalucía : J. Purroy : A. Salas-Perdomo : C. Justicia : A. M. Planas (*) Department of Brain Ischemia and Neurodegeneration, Institute for Biomedical Research of Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC)( Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Rosselló 161, planta 6, 08036 Barcelona, Spain e-mail: Anna.planas@iibb.csic.es P.-Y. Fortin : F. Couillaud Laboratory for Molecular and Functional Imaging: UMR 5231 CNRS/University Bordeaux Segalen, Bordeaux, France M. Calvo Unitat de Microscòpia Òptica Avançada CCiTUB, Centres Científics i Tecnològics, School of Medicine University of Barcelona, Barcelona, Spain Present Address: J. Purroy Parc Científic Barcelona, Baldiri Reixac 4, Torre R, 08028 Barcelona, Spain Eur J Nucl Med Mol Imaging DOI 10.1007/s00259-012-2277-7