PMC

Optical coherence tomography and cSLO images taken at various time points post laser from a single animal. Optical coherence tomography and cSLO images of laser lesions 15 wpl shows regeneration and restoration of outer retinal layers. Arrowheads point to lesions on OCT. After 3 wpl, lesions are subtle on both OCT and cSLO.

Setup for OCT-guided laser photocoagulation of zebrafish retina. (A) Optical schematic of OCT-guided laser system. (B) Side view of zebrafish placed on rubber holder with contact lens in place just prior to application of laser. (C) Top down view with the arrow pointing to the contact lens. Ap, adjustable aperture; DM, dichroic mirror; f_c, collection; f_coll, collimating; f_oph, ophthalmic; f_r, relay; f_s, scanning lenses; G_x and G_y, two axis scanning mirrors.

Hematoxylin and eosin–stained histosections of zebrafish retina following laser injury. Cryosections of 10 μm are shown in each panel from various time points after laser. Scale bar: 100 μm. Black arrowheads point to the location of laser lesions. A dome-shaped area of disorganization is seen between 1 dpl and 7 dpl. Relamination of the ONL occurs between 2 and 3 wpl, but with an alteration of the contour (white arrow). At 15 wpl, only a subtle change in pigmentation remains. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium.

Optical coherence tomography and cSLO images of laser lesions up to 1 wpl. (A) Optical coherence tomography and cSLO images taken at various time points post laser lesion from a single animal. Arrowheads point to the central lesion on OCT. The representative B-scan images are centered on the central lesion, but all three lesions can sometimes be seen on a single B scan depending on the eye orientation (1, 2, and 3 dpl). Three discrete laser lesions can be seen on cSLO imaging. The optic nerve is visible at the bottom of each cSLO image. (B) Histogram of normalized lesion size measured by IRDF up to 15 wpl. Asterisk denotes time points without significant differences in lesion size. Total n for each time point: 0 dpl = 122, 1 dpl = 95, 2 dpl = 33, 3 dpl = 24, 1 wpl = 46, 3 wpl = 47, 6 wpl = 40, 9 wpl = 34, 12 wpl = 31, 15 wpl = 22.

TUNEL and DAPI staining of zebrafish retina following laser injury. (A) Confocal microscopy of 10-μm cryosections are shown in each panel from various time points after laser. Scale bar: 100 μm. TUNEL is shown in red and DAPI in blue. The control section did not have any laser injury. White arrowheads point to clusters of nuclei in the IPL overlying the laser lesions. The asterisk shows the location of the laser lesion in the 3-wpl panel. Disorganization and loss of nuclei can be seen in the ONL between 1 and 7 dpl. The ONL is relaminated between 2 and 3 wpl. Background TUNEL labeling is seen at the outermost (scleral) surface of the retina and also at the innermost surface (control, 2 and 3 wpl). (B) Histograms showing distribution of DAPI- and TUNEL-positive cells following laser injury (mean ± SEM, n = 3 all time points). See Methods. Time points not connected by the same letter are significantly different from each other (P < 0.05). Top left: DAPI-positive cells were counted between the RPE and ONL. Bottom left: DAPI-positive cells within the IPL. Top right: TUNEL-positive cells between the RPE and ONL. Bottom right: TUNEL-positive cells within the INLs and ONLs.