In vivo longitudinal tracking of retinal pigment epithelial cells following laser treatment using Best1-cre-ZsGreen1 mice

Abstract

Purpose : To evaluate the retinal pigment epithelium (RPE) mobility following the laser treatment using transgenic mice expressing RPE-specific fluorescent proteins.

Methods : Transgenic mice expressing RPE-specific fluorescent proteins were generated by breeding the RPE-specific, cre-expressing mouse line (Best1-cre) with a floxed mouse strain for green fluorescent protein (ZsGreen1). Four laser burns were placed near the optic disc on each eye. Serial fundus photography was taken every week and images with different angles and magnifications were aligned using the 'Control Point Selection' tool, with the 'fitgeotrans' and 'imwarp' functions in MATLAB. The RPE mobility was evaluated using the ImageJ plugin ‘TrackMate’. On Day 28, mice were sacrificed and the RPE-flat-mount was made and correlated with the fundus photographic findings.

Results : RPE cells moved toward the laser burn to fill in the lesion. RPE cells migrated the most during the first week (Day 7 24.8 ± 23.9 μm; Day 14 10.4 ± 7.7 μm; Day21 10.6 ± 9.0 μm; Day28 10.1 ± 10.2 μm). The closer the RPE cell is to the laser burn, the greater the movement of the RPE cell (Day7 movement vs. distance from the laser burn; r=-0.487, p<0.001, Pearson correlation). The larger the size of the laser burn, the larger the movement of the RPE cell. We also discovered some interesting findings during RPE recovery following laser treatment. Large dysmorphic RPE cells migrated over the fluorescence of previously dead cells near the laser burn. Previously unobserved fluorescence, which could suggest new RPE cell proliferation, was observed near the laser burn. Cells migrating faster by overtaking neighboring cells were also observed.

Conclusions : After the laser treatment, RPE cells rapidly moved to fill in the defect within 1 week. Transgenic mice expressing RPE-specific fluorescence can be used to noninvasively observe the movement of RPE in vivo, helping to study the recovery mechanism after damage to RPE.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Serial fundus photos and a corresponding RPE flatmount image following laser treatment are presented. RPE cells undergo movement and elongation toward the laser burn. Intracellular fluorescence fades away as cells die. Large dysmorphic RPE cells over the fluorescence of previously dead cells are observed in the flatmount image.