Sildenafil Treatment Inhibits Zebrafish Rod Photoreceptor Outer Segment Shedding

Abstract

 

Purpose

 

The rod photoreceptor outer segment (ROS) is a modified cilium composed of stacked, membranous, rhodopsin-filled discs. New discs are added at the base and old discs are shed from the tip of the ROS on a daily basis. The latter part of this renewal process is linked to the light cycle, such that shedding occurs following illumination. Given that phosphodiesterase (PDE) activity, a key component of phototransduction, is also stimulated by light, we sought to investigate whether PDE inhibition by sildenafil (commercially sold as Viagra) would mimic the dark state and inhibit shedding in zebrafish larval rods.

 

Methods

 

Transgenic zebrafish that expressed EGFP driven by the Xenopus rhodopsin promoter and a transmembrane-tagged mCherry protein under the control of a heat shock promoter were heat shocked at 5 days post fertilization (dpf) and treated with sildenafil until 8 dpf, 3 days post heat shock (dpHS). Retinal sections were imaged using confocal microscopy to obtain optical z-stacks of the photoreceptor layer. Images were analyzed by measuring the distance from the mCherry stripe to the tip of the ROS (D^S) of individual rod photoreceptors (Fig. 1). Measurements of rods from sildenafil-treated larvae were compared to rods from vehicle-treated control larvae.

 

Results

 

Sildenafil treatment inhibited ROS shedding. Drug-treated larvae had a significantly longer D^S measurement as compared to vehicle-treated control larvae and similar to that of dark-reared larvae. The growth distance (D^G) from the base of the ROS to the mCherry stripe was not affected by sildenafil treatment.

 

Conclusions

 

To our knowledge, this is the first demonstration of a drug that inhibits ROS shedding in an in vivo system. Further investigation of sildenafil treatment on models of human retinal degeneration disease may provide a basis for potential treatment of blinding disorders that are characterized by photoreceptor degeneration.

 

 

Fig. 1. Genetically encoded marker of ROS renewal. (A) Construct for generation of heat shock promoter-driven (hsp70), HA-tagged, transmembrane (TM)-bound, mCherry transgenic fish. SP, signal peptide. (B) Heat shock induction and displacement of mCherry over time. (C) Confocal z-projection of rods from an 8 dpf, 3 dpHS Tg(Xop:EGFP; hsp70:HA-mCherry^TM); alb^-/- larva. (D) Diagram of ROS growth (D^G), shedding (D^S), and total (D^T) distances.

 

Keywords: 648 photoreceptors • 689 retina: distal (photoreceptors, horizontal cells, bipolar cells) • 695 retinal degenerations: cell biology