Abstract
Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.
Highlights
Molecular imaging utilizing peptide probes provides a noninvasive means for interrogating cells in vivo for a specific intracellular biochemical event [1,2]
We demonstrated highly specific uptake by retinal ganglion cells (RGC) following intravitreal injection of fluorophore conjugated to our modified cell-penetrating peptide sequence [13], and subsequently utilized these probes in a rat model of Nmethyl- D-aspartate (NMDA)-induced RGC degeneration to validate probe activity and localization ex vivo [18,19]
Strong fluorescent signals were observed with in vivo imaging in the RGC layer of eyes of living rats pretreated with NMDA followed by TcapQ488 (Figure 1B)
Summary
Molecular imaging utilizing peptide probes provides a noninvasive means for interrogating cells in vivo for a specific intracellular biochemical event [1,2]. Through an intramolecular optical quenching strategy, activatable probes with a fluorescent signal moiety remain optically silent until activation by the target enzyme This probe design confers a high degree of specificity and maximizes the signal-to-noise ratio for optimal detection of events that may occur at a relatively low frequency. This strategy is attractive for application to the eye, as optical imaging can be utilized to identify a fluorescent target using clinically available instruments. We demonstrated highly specific uptake by RGCs following intravitreal injection of fluorophore conjugated to our modified cell-penetrating peptide sequence [13], and subsequently utilized these probes in a rat model of NMDA-induced RGC degeneration to validate probe activity and localization ex vivo [18,19]
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