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Claudia Mueller, Francesca Mazzoni, Silvia Finnemann; Non-invasive, in vivo assessment of retinal cell death in light-induced retinal degeneration.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5906.
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© ARVO (1962-2015); The Authors (2016-present)
Detecting retinal cell death in animal models for eye disease using non-invasive, non-toxic live imaging will benefit translational vision research. We previously found that eye drops containing PSVue, a fluorescent probe that binds phosphatidylserine (PS) displayed by dying cells, label dying photoreceptors in the mutant Royal College of Surgeons (RCS) rat. As RCS rat RPE lacks the capability for PS-dependent clearance phagocytosis, excessive levels of PS-marked debris accumulate in degenerating RCS retina. Here, we ask if PSVue eye drops detect dying photoreceptors in induced retinal degeneration, in which wild-type RPE has normal capacity to remove PS-marked debris.
Retinal cell death was induced by exposing dark adapted albino rats to white light at 10,000 lux for 1 hour, followed by 6,000 lux for 23 hours. Control rats were dark adapted but not exposed to bright light. Retinal degeneration was followed for up to 6 days after light exposure by marker immunohistochemistry and immunoblotting. 5 days after light damage rats received PSVue or vehicle only as eye drop. Fluorescence imaging was performed 24 h later either ex situ on live dissected neural retina by microscopy or in vivo on live rats using a Kodak FX-Pro imager. All experiments were performed 3 times independently with 3-4 animals per group. Fluorescence levels were quantified, ratios of PSVue to control signals calculated and analyzed by Student’s t-test.
Changes in retinal morphology were obvious starting 5 days after light damage including rosette formation, outer segment thinning and reduction in photoreceptor nuclei. Immunoblotting confirmed ongoing retinal degeneration and partial photoreceptor loss. Tissue dissection revealed that PSVue applied as eye drops labeled dying photoreceptors as early as 3 days after and was robust in all rats 5 days after light exposure. In vivo live imaging detected significantly increased fluorescence in all rats 5 days after light damage compared to control rats.
Our results show that PS-probe applied as eye drops detects PS-marked cell debris in acutely induced photoreceptor degeneration in wild-type rats with intact phagocytic activity of the RPE. We suggest that PS-probe eye drops have utility as non-invasive, efficient screening method to monitor ongoing retinal degeneration in a wide range of animal models.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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