title: 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.
abstract: OBJECTIVE
  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 promoter was used to generate an Hsp70-mPlum far-red fluorescence 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 transgenic mice were generated. Focal brain ischaemia was induced 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 ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Brain Ischemia and Neurodegeneration, Institute for Biomedical Research of Barcelona, Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Rosselló 161, planta 6, 08036, Barcelona, Spain.
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Rosa
authors.forename: Tomàs
authors.initials: T
authors.lastname: Santalucía
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Fortin
authors.forename: Jesús
authors.initials: J
authors.lastname: Purroy
authors.forename: Maria
authors.initials: M
authors.lastname: Calvo
authors.forename: Angélica
authors.initials: A
authors.lastname: Salas-Perdomo
authors.forename: Carles
authors.initials: C
authors.lastname: Justicia
authors.forename: Franck
authors.initials: F
authors.lastname: Couillaud
authors.forename: Anna M
authors.initials: AM
authors.lastname: Planas
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000175:diagnosis* / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D013050:Spectrometry, Fluorescence; D015533:Transcriptional Activation; D000097573:Red Fluorescent Protein
chemical_list: D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein