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Xudong Qiu, James R. Johnson, David Piwnica-Worms, Edward M. Barnett; In Vivo Imaging Of Retinal Ganglion Cell Apoptosis Using Caspase-activatable Cell-penetrating Probes In The Rat: Optimizing Signal-to-noise And Assessing Potential Toxicity. Invest. Ophthalmol. Vis. Sci. 2012;53(14):281.
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We have developed small peptide-based probes for in vivo imaging of retinal ganglion cell (RGC) apoptosis. These probes consist of a cell-penetrating peptide (CPP) targeting moiety and a fluorophore (Alexa Fluor 488)-quencher (Qsy7) pair flanking an effector caspase consensus sequence (DEVD). Intracellular probe activation consists of DEVD cleavage by activated effector caspases followed by separation of the fluorophore and quencher, resulting in a detectable fluorescent signal. Previously, we validated the capacity of two such probes, TcapQ and KcapQ, to identify apoptotic RGCs in vivo. Herein we report the results of full quantification of probe performance in regards to time-dependent activation, sensitivity, signal-to-noise ratio, and potential toxicity.
RGC apoptosis was induced in rats by intravitreal injection of NMDA (5 - 80 nmol) followed by intravitreal injection of probe (0.137 to 0.775 nmol) for a total of 30 NMDA-probe dosage combinations, including PBS controls. Under each condition, probe activation was monitored immediately after, as well as at 4, 12, 24, 48 and 72 hours post probe injection. RGCs with activated probe were identified in vivo by fluorescence imaging of the fundus using the Heidelberg Retinal Angiograph 2 (HRA2) confocal scanning laser ophthalmoscope (CSLO) and quantified as foci per wide-angle field using Image J software. Electroretinography (ERG) was performed following intravitreal injection of PBS, 0.193 and 0.387 nmol TcapQ488 to determine potential probe toxicity.
In vivo fluorescence imaging of the fundus revealed distinct single-cell probe activations as an indicator of RGC apoptosis. Rigorous 3-dimensional titration curves of probe activation were generated based on NMDA dosage, probe dosage, and time. Peak activation of probe in vivo was detected at 12 hours post probe injection. The number of detectable fluorescent RGCs per field increased with exposure to increasing doses of NMDA. The sensitivity of this detection generally increased with increasing doses of probe, plateauing at 0.387 nmol. Additionally, ERG testing in rats receiving intravitreal injections of TcapQ488 at 0.193 and 0.387 nmol showed no significant difference compared with PBS- only injections.
We have optimized the signal-to-noise ratio of caspase-activatable CPP probes for quantitative detection of RGC apoptosis in vivo using CSLO fluorescence imaging. Probe induced no detectable toxicity by ERG at clinically relevant concentrations. We plan to further assess these probes in advanced rat and non-human primate models of glaucoma.
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