Abstract
Purpose:
We have previously shown that double-stranded RNAs (dsRNA) longer than 21-nucleotides is toxic to the retinal pigment epithelium (RPE) in vitro and in vivo. RPE cell death was concurrent with upregulation of active caspase-1 and -3, and mice deficient in caspase-1 expression are resistant to dsRNA induced RPE toxicity. Here, we describe the application of near-infrared fluorophore conjugated substrates that irreversibly bind to specific caspases and provide further data on in-situ co-localization of these fluoroprobes in our mouse models.
Methods:
Wild type C57BL/6 mice received subretinal injections of dsRNAs (Alu RNA or poly I:C, 300ng, n=5-6 eyes per group) or PBS vehicle. At 24 and 72 hours after treatment, intravitreous injections of a near-infrared fluorophore (NIRF, Ex: 790 Em: 800) conjugated pan-caspase probe (VAD-fmk) were performed. Fundus photos and NIRF imaging were taken (Topcon 50-IX) at 8 and 24 hours. Eyes were harvested and prepared as frozen sections or RPE/choroid flatmounts. Probe localization on sections was performed with fluorescent microscopy and localized with TUNEL positive apoptotic retinal cells. Flat-mounted tissues were analyzed for probe using an 800nm laser scanner (LICOR Odyssey).
Results:
Treatment of Wild-type mice with Alu-derived dsRNAs and poly I:C both caused RPE cell death and PBS did not. Tissue sections from eyes with strong NIRF caspase signal on bio-imaging demonstrated probe localization in the RPE and outer retina. TUNEL staining revealed defined borders of apoptotic and normal cells which overlapped to the areas of NIRF signal from the pan-caspase probe. The flat-mounted tissues showed an intense focal signal in the degenerating RPE where subretinal dsRNA injections were done. Eyes treated with vehicle PBS did not reveal probe signal or staining by TUNEL on frozen sections or flat-mounts.
Conclusions:
Multimodal imaging techniques show that Alu RNA and poly I:C cause RPE cell death occurs through a caspase dependent mechanism. We show that an in vivo imaging strategy to visualize caspase activation is feasible in a mouse model of RPE apoptosis with probe signal co-localizing to TUNEL positive cells in the RPE and outer retina. Further studies are evaluating probe toxicity and other approaches to cell death visualization aiming to translate bio-imaging to detect RPE apoptosis in diseases of the human retina.
Keywords: 701 retinal pigment epithelium •
551 imaging/image analysis: non-clinical •
412 age-related macular degeneration