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Xudong Qiu, James R. Johnson, David Piwnica-Worms, Edward M. Barnett; In Vivo Confocal Scanning Laser Ophthalmoscopy Imaging Of Retinal Ganglion Cell Apoptosis Using Caspase-activatable Probes In A Rat Glaucoma Model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3421.
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Peptide-based molecular probes have show promise for in vivo diagnostic imaging of retinal ganglion cell (RGC) physiology. Previously, we validated the ability of effector caspase-activated cell-penetrating peptide probes (CPP), TcapQ and KcapQ, to identify apoptotic RGC in an in vivo glaucoma model via examination of ex vivo retinal flat mounts. These probes are small peptidomimetics consisting of a permeation peptide targeting moiety, an effector caspase recognition sequence (DEVD) flanked by a fluorophore-quencher pair (Qsy21 and Alexa Fluor 488 respectively). Upon cleavage of the recognition sequence, dequenching of the fluorophore occurs resulting in release of fluorescence. Herein we report the results of our in vivo optical imaging of probe activation in that same model using a Heidelberg Retinal Angiograph 2 (HRA2) confocal scanning laser ophthalmoscope (CSLO).
RGC apoptosis was induced in rats by intravitreal injection of NMDA (5-80nmol) followed by intravitreal injection of 0.35nmol CPP probes, TcapQ488 or KcapQ488. RGCs with activated probe were identified in vivo by fluorescent imaging of the fundus using the HRA2 CSLO. To confirm correspondence with fluorescent microscopy, retinal flat mounts were subsequently examined.
In vivo CSLO imaging throughout the fundus in eyes revealed RGC apoptosis via fluorescence detection of CPP probe activation. The number of detectable fluorescent RGCs in vivo increased with exposure to larger amounts of NMDA, consistent with previous flat mount experiments. Subsequent ex vivo examination of retinal flat mounts revealed correspondence of fluorescent labeled cells with in vivo imaging. Control eyes (no NMDA, PBS only) showed no probe activation in RGCs. Additionally, CPP probes showed peaking labeling in vivo within 48 hours.
We have established a method for imaging RGC apoptosis in vivo using caspase-activatable fluorescent CPP probes using CSLO. This approach is currently applicable to animal models of RGC apoptosis and may ultimately have utility in the diagnosis and management of human glaucoma.
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