A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

The heretofore described light treatment protocol was compared to each individual method of LIRD. In dark-treated adult albino animals (Figures 3-5), the individual light treatments resulted in significant loss of rod (Figure 3) and cone (Figure 4) photoreceptors. However, both individual treatments primarily damaged photoreceptors in the dorsal half of the retina, leaving the ventral retina relatively protected from the light treatments (Figures 3 and 4). In addition, compared with the halogen light treatment, the UV light treatment damaged more cone photoreceptors in the dorsal half of the retina (compare Figures 4B with C, respectively). Combining the UV and halogen light treatments resulted in significantly greater loss to both rod and cone photoreceptors throughout the retina, largely eliminating the neuroprotection of the ventral half of the retina (Figures 3 and 4). Compared with the individual LIRD methods, a corresponding increase in proliferation was also observed in the ventral retina when the combined light treatment protocol was used (Figure 5).

Pigmented animals are more resistant to LIRD due to retinal pigmented epithelium, which absorbs light and protects the photoreceptors from phototoxicity. As expected, dark-adapted pigmented animals were nearly completely resistant to the halogen light treatment alone (Figures 6B, J, and N). We found that 48 hr after light onset, rod photoreceptors were reduced in the dorsal retina, but not ventral retina (Figures 6E, F, and Q). Cone photoreceptor nuclei were present in normal numbers (Figure 6R). Compared with untreated retinas, halogen light treatment alone also did not increase Müller glial cell proliferation 48 hr after light onset (Figures 7B and F). In albino animals, UV light treatment alone resulted in slightly greater damage to both rod and cone photoreceptors in the dorsal retina (Figures 3I and 4I). However, in pigmented animals, UV light treatment alone did not significantly reduce rod or cone cell number (Figures 6C, G, K, O, Q, and R). In addition, UV treatment alone only elicited a weak regenerative response in the dorsal half of the retina in pigmented animals (Figure 7C). In contrast to the individual LIRD results, combining the UV and halogen light treatments resulted in significantly greater photoreceptor damage and regenerative response in pigmented animals. Importantly, significant loss of both rods and cones was observed in both the dorsal and ventral retinas (Figures 6D, H, L, P, Q, and R). Compared with untreated and individual LIRD methods, a corresponding increase in proliferation was also observed in both dorsal and ventral halves of the retina when the combined light treatment protocol was used (Figure 5).

Figure 1. Photograph depicting the halogen light treatment setup. Two sets of four 250 W halogen lamps were spaced 29 cm on either side of two clear 1.8 L acrylic tanks. The aerator and tubing were placed so as not to obstruct the light. The lids of the tanks were cracked 2 cm to allow airflow from the fan. A thermometer was placed in one of the tanks for the temperature to be monitored. Click here to view larger image.

Figure 2. Photograph depicting the UV light treatment setup. The UV light source from a fluorescent stereomicroscope was securely fixed to a glass Petri dish ~2 cm off the bench top. A 250 ml glass beaker, partially wrapped in foil, was filled will 100 ml of system water. The fish were placed inside the 250 ml beaker. The 250 ml beaker was centered inside a 4 L beaker, which was filled with system water to the level of the water in the 250 ml beaker. The 4 L beaker was placed adjacent to the light filament centered at the 250 ml beaker. A large opaque shield was placed around the entire setup. Click here to view larger image.

Figure 3. Rod photoreceptor loss in albino zebrafish following various light damage paradigms. Retinal sections from adult Tg(nrd:egfp)/alb immunolabeled with GFP to show rod photoreceptor density (green) in the dorsal (A-D) and ventral (E-H) halves of the retina. A, E) Prior to light onset (0 hr), EGFP-positive rod photoreceptor nuclei (ONL) and outer segments (ROS) can be visualized. B, F) At 48 hr following halogen light onset (hLt), truncated ROS and significantly fewer rod nuclei (I) were present in the dorsal, but not ventral retina. C-G) At 48 hr after 30 min of UV exposure (hpUV), significantly fewer rod nuclei were present in the dorsal and ventral retinas compared to 0 hr (C, G), but not 48 hLt treatments (G, I). D-H) 48 hr following onset of combined light treatment (30 min UV exposure followed by halogen treatment; UV+48 hLt), almost no ROS were present in dorsal or ventral retinas, in addition to a significant loss of rod photoreceptor nuclei (I). I) Quantification of rod photoreceptor nuclei across 300 µm of the central dorsal or ventral retina. Significant differences (p ≤ 0.05) between groups are depicted as: "a" for different from 0 hr, "b" for different from 48 hLt and "c" for different from 48 hpUV. Scale bar in panel A represents 50 µm and applies to panels A-H. Click here to view larger image.

Figure 4. Double cone loss in albino zebrafish following various light damage paradigms. Retinal sections from adult albino zebrafish immunolabeled with Zpr-1 to show double cones (gold) in the dorsal (A-D) and ventral (E-H) halves of the retina following each of the light damage paradigms (48 hLt, 48 hpUV, and UV+48 hLt). A, E) At 0 hr, double cone nuclei were present in dorsal and ventral retinas. B, F) At 48 hLt, significantly fewer double cones were present in the ventral retina only (F, I). C-G) At 48 hpUV, significantly fewer double cones (I) and a large amount of debris was present in the dorsal retina only (C), while no changes were observed in the ventral retina (G). D-H) At UV+48 hLt, significantly fewer double cones (I) and very little debris was present in dorsal and ventral retinas. I) Quantification of double cones across 300 µm of the central dorsal or ventral retina. Significant differences (p ≤ 0.05) between groups are depicted as, "a" for different from 0 hr, "b" for different from 48 hLt and "c" for different from 48 hpUV. Scale bar in panel A represents 100 µm and applies to panels A-H. Click here to view larger image.

Figure 5. Changes is proliferation response following various light damage paradigms. Retinal sections from adult albino zebrafish immunolabeled with PCNA to show cell proliferation (red) in the dorsal (A-D) and ventral (E-H) halves of the retina following each of the light damage paradigms (48 hLt, 48 hpUV, and UV+48 hLt). A-E) At 0 hr, proliferation was absent from the inner nuclear layer (INL). B, F) At 48 hLt, single progenitor cells were present in the INL across the dorsal retina and in a small portion of the ventral retina. C-G) At 48 hpUV, columns of progenitor cells were present in the dorsal retina, while only single progenitor cells were present in a small portion of the ventral retina. D, H) At UV+48 hLt, large columns of progenitor cells were present across the dorsal and ventral retina. Scale bar in panel A represents 100 µm and applies to panels A-H. Click here to view larger image.

Figure 6. Rod and cone photoreceptor loss in pigmented zebrafish following various light damage paradigms. Retinal sections from adult wild-type (AB) zebrafish stained with TO-PRO-3 to show all nuclei (A-P; blue) and immunolabeled with Zpr-3 to show rod outer segments (A-H; magenta) or Zpr-1 to double cones (I-P; gold) in the dorsal (A-D, I-J) and ventral (E-H, M-P) halves of the retina following each of the light damage paradigms (48 hLt, 48 hpUV, and UV+48 hLt). A-H) Significant decreases in rod photoreceptor nuclei were present in the dorsal retina at 48 hLt (B, Q) and in the dorsal and ventral retina at UV+48 hLt (D, H, Q). I-P) Although cone nuclei were hypertrophied in the dorsal retina at 48 hpUV (K), no significant decreases in double cone nuclei were present at 48 hLt or 48 hpUV (J-K, N-O, R). Significant decreases in cone nuclei were present in the dorsal and ventral retina at UV+48 hLt (L, P, R). Q-R) Quantification of rod photoreceptor and double cone nuclei across 300 µm of the central dorsal or ventral retina. Significant differences (p ≤ 0.05) between groups are depicted as, "a" for different from 0 hr, "b" for different from 48 hLt and "c" for different from 48 hpUV. Scale bar in panel A represents 200 µm and applies to panels A-H. Click here to view larger image.

Figure 7. Proliferation response in pigmented zebrafish following various light damage paradigms. Retinal sections from adult wild-type (AB) zebrafish immunolabeled with PCNA to show cell proliferation (red) in the dorsal (A-D) and ventral (E-H) halves of the retina following each of the light damage paradigms (48 hLt, 48 hpUV, and UV+48 hLt). A-D) In the dorsal retina, only a few single progenitor cells were present in the INL at 48 hLt and 48 hpUV (B, C), while columns of progenitor cells were present at UV+48 hLt (D). E-H) In the ventral retina, no changes in proliferation were observed except at UV+48 hLt (H), at which time columns of progenitor cells were present. Scale bar in panel A represents 100 µm and applies to panels A-H. Click here to view larger image.