To treat healthy retinal pigmented epithelium (RPE) with the 3‐ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound‐healing response of these cells.

Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post‐irradiation and assessed for expression of proteins associated with wound‐healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post‐irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes.

Cultured RPE cells were ablated by 2RT laser in an energy‐dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle‐associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re‐expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy‐dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB‐crystallin.

The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound‐healing response leading to layer reformation.