Non-invasive in vivo fluorescence imaging of apoptotic retinal photoreceptors

Abstract

Phosphatidylserine externalization is an early molecular signature for apoptosis. In many retinal degenerative diseases, photoreceptor neurons die by apoptosis. Here, we report utility of the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in transiently labeling apoptotic photoreceptors in living pigmented or albino rats and mice with retinal degeneration. Applying PSVue-550 as eyedrop is non-toxic and eliminates need for intraocular injection. PSVue-550 fluorescence specifically and transiently labeling dying retinal photoreceptors is detectable in anesthetized animals using standard retinal or whole small animal imaging systems. Importantly, prior PSVue-550 eyedrop administration and imaging does not affect repeat testing. Altogether, our results establish PSVue-550 imaging as a completely non-invasive method that provides the opportunity to longitudinally monitor retinal photoreceptor cell death in preclinical studies.

Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency

“…The b-wave indicative of activities of second order retinal neurons was also improved by tmx eye drop treatment ( Figure 5E). RCS rats with ongoing retinal degeneration do not show normal retinal activity in ERG recordings with consistent increases in a-and b-wave amplitudes in response to light flashes of increasing intensity (23). Recording at P33 specifically in our study we observed flat or even moderately declining a-and b-wave amplitudes at highest flash intensities applied indicative of dysfunction of photoreceptors at this age.…”

“…Animals of both sexes were used. Wild-type (WT) and mertk −/− mice in the same 129T2/SvEmsJ genetic background ( 23 ), pink-eyed dystrophic RCS rats (rdy/rdy-p) and Sprague Dawley (SD) WT rats were raised to yield litters at defined age for experiments. For tissue harvest, animals were euthanized by CO 2 asphyxiation before immediate eye enucleation and processing.…”

“…RCS rats were dark-adapted overnight before intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine to induce anesthesia. Scotopic responses were recorded exactly as described previously using a UTAS-E2000 visual electrodiagnostic system (LKC Technologies, Gaithersburg, MD) (23). Stimuli were presented in order of increasing intensity as a series of white flashes of 1.5 cd-s/m2 attenuated with neutral density filters.…”

“…Outer segment renewal including daily clearance phagocytosis in rodents commences after postnatal formation of photoreceptor outer segments and has been assumed to be associated with retinal maturation around eye opening, which in our RCS rats occurs at postnatal day 15 (P15) plus or minus one day. No significant debris buildup is observed before P22, yet rod photoreceptor apoptosis is substantial only shortly thereafter, at ∼P25 (22,23). Retinal function as measured by electroretinography shows normal photoreceptor response at eye opening, and modestly and severely reduced photoreceptor responses at P22 and P34, respectively (11).…”

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia

“…Once activated by zinc, PSVue has fast, calcium-independent binding kinetics; is significantly smaller in size compared to annexin-based probes, and, similarly to ANXV, is cell impermeable, thus labeling only PS that has been externalized (Smith et al, 2011). While PSVue has been used previously to monitor cell death in vivo in several disease and injury models (Chan et al, 2015;Mazzoni et al, 2019;Smith et al, 2012), it remains unknown whether localized, non-apoptotic PS exposure occurs in vivo.…”