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X. Zhang, D. Maxwell, D. Piwnica-Worms, E. M. Barnett; Validation of an Effector-Caspase Specific Activatable Fluorescent Peptide Probe in Rodent Models of RGC Degeneration. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5473. doi: https://doi.org/.
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Activatable peptide probes contain enzyme specific cleavage sites which, when cleaved, results in a signal denoting the activity of the specified enzyme. An activatable fluorescent peptide probe incorporating an effector caspase cleavage site has been developed and validated in vitro. Previous in vivo studies utilizing models of retinal ganglion cell degeneration (RGC) have shown correspondence with other markers of apoptosis, such as TUNEL labeling. Additional studies have been undertaken to examine probe activation in the intravitreal NMDA model using a range of NMDA concentrations, as well as in the presence of caspase inhibitor.
The activatable peptide probe (TcapQ647) is a small peptidomimetic consisting of an all D-amino acid modified Tat permeation peptide, an L-amino acid effector caspase recognition sequence, a quencher, and a fluorophore. Upon cleavage of the caspase recognition sequence and subsequent loss of fluorescent quenching, fluorescence from the retained intracellular fluorophore is detectable via fluorescence imaging. RGC apoptosis was induced in vivo in rats either through unilateral optic nerve transaction or pharmacologically using intravitreal injection of NMDA. NMDA concentrations ranging from 4 to 40 mM were used. Retinas were imaged as eyecups or flat mounts with a fluorescence microscope. The number of RGCs with activated probe was quantified in retinal flat mounts, assisted by retrograde labeling with fluorogold in some animals.
The number of retinal cells displaying probe activation followed a dose-dependent pattern based on the concentration of NMDA used for intravitreal injection. As demonstrated previously, TUNEL assay and nucleic DAPI stain showed good correspondence with fluorescent RGCs. Retrograde labeling of RGCs with fluorogold confirmed that probe activation was largely limited to RGCs. Pre- and/or co-injection of an effector caspase inhibitor significantly reduced the number of cells with activated probe in the NMDA model. No evidence of retinal toxicity was seen up to 7 days post-injection of TcapQ647 on retinal histology.
Our findings provide further validation of this activatable fluorescent probe for identifying RGC apoptosis in vivo following intravitreal injection. This probe should be useful for in vivo imaging of RGC apoptosis in animal models of glaucoma and may eventually have applications in the diagnosis and management of human glaucoma
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