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Title

Development of an HSP70-regulated far-red fluorescent reporter mouse for imaging the ischemic penumbra

AuthorsRosa, Xavier de la ; Santalucía, Tomàs ; Purroy, Jesús; Calvo, María; Salas-Perdomo, Angélica ; Justicia, Carles ; Planas, Anna M.
Issue DateMay-2011
CitationBRAIN 2011
Abstract[Objectives]: Neurons surrounding the ischemic area express Hsp-70 and this is regarded as a marker of the ischemic penumbra. Hsp-70 is also strongly induced in blood vessels within the ischemic territory. Our aim was to obtain transgenic mice expressing a fluorescent reporter to image the induction of Hsp70. For such applications, the use of a far-red fluorescent protein reporter is desirable so that fluorescence absorption and scattering by the cerebral parenchyma are reduced. [Methods]: An Hsp70 promoter-driven reporter vector for the far-red fluorescent protein mPlum was constructed with Invitrogen's Multisite Gateway Vector Construction kit. NIH3T3 cells were transfected with this vector and stimulated with sodium arsenite (50 uM) to induce Hsp-70 expression mPlum and Hsp70 protein expression was detected with specific antibodies by Western blotting and immunocytochemistry. mPlum fluorescence was detected with a fluorescence inverted-microscope. Transgenic mice were generated at the “Norsk Transgen Senter” (Norway). Transient middle cerebral artery occlusion (MCAO) was performed on adult transgenic or wt mice as previously described. A cranial window sealed with a cover slip was opened on ketamine/xylacine-anesthetised mice over the ipsilateral hemisphere for live observation under high speed confocal microscope (590nm ex, 649nm em) 24h after ischemia. Mice were killed thereafter and the brain was processed for immunohistochemistry. In another set of animals, brain samples were obtained 24h after MCAO and were processed for either quantitative real time RT-PCR or Western blotting. [Results]: Cells transfected with the Hsp-70 reporter vector showed induction of mPlum when Hsp-70 expression was stimulated, showing the usefulness of our construct to track Hsp-70 induction. In transgenic mice, MCAO induced expression of mPlum and hsp70 mRNAs and proteins in the ipsilateral hemisphere. Intravital confocal microscopy showed mPlum fluorescence in superficial blood vessels of the ipsilateral hemisphere. Immunohistochemistry on post-mortem brain sections revealed expression of mPlum in the infarct area, which colocalised with endogenous Hsp70 expression in blood vessels and neurons. Despite mPlum's limited intensity of fluorescence signal, our studies reveal that mPlum fluorescence can be detected in live animals after brain ischemia, mainly in cortical blood vessels that show the strongest induction of Hsp-70 and mPlum. [Conclusions]: Our results show that our Hsp70-mPlum reporter vector in the transgenic mice responds to ischemia in a parallel way to endogenous Hsp70, and that it is possible to track Hsp-70 expression in vivo through mPlum fluorescence.
DescriptionTrabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Function y a la Xth International Conference on Quantification of Brain Function with PET celebrados del 25 al 28 de mayo en Barcelona.
URIhttp://hdl.handle.net/10261/92147
Appears in Collections:(IIBB) Comunicaciones congresos
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