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Kabhilan Mohan, Laura Sanman, Kyung Jung, Jacob Roney, Dingyuan Lou, Jennifer Brown, Matt Bogyo, Mark Ellsworth Kleinman; An Acyloxy-Conjugated Caspase Probe for In Vivo Retinal Bio-imaging. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2338.
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To optimize a non-invasive in vivo caspase detection probe for bio-imaging retinal pigment epithelium cell death in age-related macular degeneration.
Wild type C57BL/6J mice received subretinal injections of Alu RNA (300ng, n=5-6 eyes per group) or PBS as a negative control. At 24 and 72 hours after treatment, intravitreous injections of a near-infrared fluorophore (NIRF, Ex: 780 Em: 800) conjugated pan-caspase probe (VAD-acyloxymethylketone, AOMK) were conducted (100 ng in 1 μl DMSO). Fundus photos and near-infrared fluorescent imaging were acquired (Topcon 50-IX) at 8 and 24 hours. Similarly, intravitreous injections of the pan-caspase probe was conducted in transgenic mice with mutant PRPH2 (Jackson Laboratories) at 2 weeks of age and near infrared fluorescent imaging was conducted at 8 hours. Eyes were then harvested and either prepared as frozen sections or RPE/choroid flatmounts.
Treatment of Wild-type mouse eyes with Alu-derived dsRNAs caused RPE cell death whereas PBS did not. On frozen sections, a strong NIRF signal from the pancaspase probe was observed in the degenerating regions of RPE and retina, and this signal co-localized with TUNEL staining. The flat-mounted tissues also revealed an intense and focal signal in the degenerating area where subretinal Alu RNA injections were performed. Eyes treated with vehicle PBS did not reveal probe signal or staining by TUNEL on frozen sections or flat-mounts. A similar signal from degenerating photoreceptor and RPE layers was observed in the Prph2 transgenic mice.
We have previously developed a NIRF caspase bio-imaging probe with a fluoromethyl ketone (fmk) group. In this study, we have engineered a safer conjugate with aomk (DyLight780-VAD-aomk) that resolves potential toxicity from metabolism of fmk to fluoroacetate. Here, we have shown that the newly engineered caspase probe provides an in vivo imaging strategy to visualize RPE cell in different mouse models of death. Future studies will evaluate alternate delivery routes for the caspase bio-imaging probe and systemic toxicity screening.
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