Longitudinal fluorescent observation of retinal degeneration and regeneration in zebrafish using fundus lens imaging.

PURPOSE

Longitudinal observation of retinal degeneration and regeneration in animal models is time-consuming and expensive. To address this challenge, we used a custom fundus lens and zebrafish transgenic lines with cell-specific fluorescent reporters to document the state of individual retinal neurons in vivo.

METHODS

We empirically tested several versions of a custom fundus lens and assessed its capabilities under a stereomicroscope to image retinal neurons in transgenic zebrafish lines expressing fluorescent reporters. Vascular branch points provided spatial references enabling determination of whether changes induced by ablating photoreceptors were repaired over the course of several days.

RESULTS

Individual ultraviolet- and blue-sensitive cone photoreceptors were readily visualized in vivo, and green fluorescent protein-labeled blood vessels were used as landmarks to facilitate orientation. Sequential imaging of the same retinal areas over several weeks permitted documentation of photoreceptor reappearance in individual animals. Photoreceptor regeneration in these regions was evidenced by the reappearance of individual fluorescent cells.

CONCLUSIONS

This technique permits real-time in vivo serial examination of individual fish, permitting temporal analysis of changes to the retinal mosaic. The key benefits this technique offers include that the same retinal locations can be recovered and viewed at multiple time points, that in vivo observations are comparable to those made ex vivo, and that fewer animals need to be euthanized over the course of an experiment. Our results promise the ability to detect individual cells, including reappearing cone photoreceptors, and to monitor disease progression during screening of therapies in an adult animal model of late onset disease.