Truncated photoreceptor outer segments recover when zebrafish are transferred back to normal light:dark conditions. Zebrafish were subjected to the CLL lesion protocol for 10 days, then returned to normal light:dark housing conditions and allowed to recover for 14 days. After the 14-day recovery period, retinas were harvested at 24 days post initial light (dpl). (A–C) Retinal sections were immunolabelled with anti-Green Opsin (green) labelling green cone PRs. Significant truncation of cone outer segments (OS) was observed at 10 dpl, and following the 14-day recovery, cone OS length was partially restored. (D) Green cone OS length was quantified using ImageJ (n = 5–6). At 10 dpl of CLL exposure, OS length was reduced by >50% (p < 0.0001). Partial recovery of OS length was achieved to ∼75% of baseline levels (p < 0.002). (E–G) Rod PRs were immunolabelled with zpr-3 marking rhodopsin (green). (F) At 10dpl, we observed significant truncation of the ROS and internalization of the zpr-3 signal. (G) Following the 14-day recovery under normal light conditions, ROS presented morphological recovery and OS length was partially restored. (H) ONL nuclei were quantified by hand count over a 300 µm linear distance. Following significant loss of ONL nuclei at 10dpl (p < 0.0001), after retinas recovered for 14 days, ONL nuclei counts were restored up to ∼75% of the 0 h baseline (p < 0.0001). (I–K) Microglia in retinal sections were labeled with a 4c4+ antibody (green). 4c4+ cells clearly reside at the outer margin of the ROS at every stage of the protocol. (L) Hand count of 4c4+ cells present in the outer retina PR-layer over a 300 µm linear distance reveals an increase in microglia after the onset of the light exposure, despite removal of the light stimulus (p < 0.01 at 28 dpl). (CC = green cone cell outer segments, ONL = outer nuclear layer, INL = inner nuclear layer, ROS = rod outer segments, RIS = rod inner segments, GCL = ganglion cell layer).
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