Author Contributions
Conceptualization, A.P., S.L. and P.L.; Data curation, W.A.I., L.-A.C., H.B. and P.L.; Formal analysis, A.P. and S.L.; Funding acquisition, P.L.; Investigation, A.P., S.L., W.A.I., L.-A.C., H.B. and P.L.; Methodology, A.P., S.L., L.-A.C. and P.L.; Resources, P.L.; Supervision, P.L.; Validation, P.L.; Writing—original draft, A.P. and S.L.; Writing—review & editing, A.P., S.L. and P.L.
Figure 1.
Representative scotopic and photopic ERGs recorded from light-exposed (10,000 lux) and control (unexposed) animals (n = 3–4 animals per group). For the exposed animals, all the ERGs were recorded immediately after the cessation of light exposure and compared to age-matched controls. For example, pups that were exposed for 1 day (P14–P15) were tested at P15 and compared to P15 controls. Horizontal calibration: 40 ms; vertical calibration: 400 µV (scotopic) and 100 µV (photopic). A 20 ms prestimulus baseline is included in all tracings. Vertical arrows indicate the flash onset. Abbreviations: a-wave (a) and b-wave (b). Light intensity used: 0.9 log cd.sec.m−2, flash duration 20 µs, inter stimulus interval 10ms (scotopic condition) and 1ms (photopic condition). A background light of 30cd/m2 was also used in photopic conditions.
Figure 1.
Representative scotopic and photopic ERGs recorded from light-exposed (10,000 lux) and control (unexposed) animals (n = 3–4 animals per group). For the exposed animals, all the ERGs were recorded immediately after the cessation of light exposure and compared to age-matched controls. For example, pups that were exposed for 1 day (P14–P15) were tested at P15 and compared to P15 controls. Horizontal calibration: 40 ms; vertical calibration: 400 µV (scotopic) and 100 µV (photopic). A 20 ms prestimulus baseline is included in all tracings. Vertical arrows indicate the flash onset. Abbreviations: a-wave (a) and b-wave (b). Light intensity used: 0.9 log cd.sec.m−2, flash duration 20 µs, inter stimulus interval 10ms (scotopic condition) and 1ms (photopic condition). A background light of 30cd/m2 was also used in photopic conditions.
Figure 2.
Graphic representation of the global retinal function [(A) scotopic a-wave; (B) scotopic b-wave and (C) photopic b-wave] recorded from light-exposed (10,000 lux; gray bars) animals following different exposure regimens and from age-matched control (unexposed; black bars) groups (n = 3–4 animals per group). Asterisks represent statistically significant differences (* p < 0.05) compared to age-matched control groups. For each exposed group, the percentage of control is shown below each graph. Amplitudes are reported as mean ± 1SD.
Figure 2.
Graphic representation of the global retinal function [(A) scotopic a-wave; (B) scotopic b-wave and (C) photopic b-wave] recorded from light-exposed (10,000 lux; gray bars) animals following different exposure regimens and from age-matched control (unexposed; black bars) groups (n = 3–4 animals per group). Asterisks represent statistically significant differences (* p < 0.05) compared to age-matched control groups. For each exposed group, the percentage of control is shown below each graph. Amplitudes are reported as mean ± 1SD.
Figure 3.
(A): Representative scotopic oscillatory potentials (OPs) recorded from light-exposed (10,000 lux; upper tracings) animals following different light exposure regimens and from age-matched control (unexposed; lower tracings) groups (n = 3-4 animals per group). Light intensity used: 0.9 log cd.sec.m−2. (B): Representative scotopic oscillatory potentials (OPs) tracings recorded to stimuli ranging between 0.9 and −4.5 log cd.sec.m−2 (as indicated on top of each tracing) from a representative P14–20 LIR animal (upper tracings) and an age-matched control animal (lower tracings). Numbers 1 to 9 indicate the total number of OPs at each intensity in both groups. Horizontal calibration: 20 ms; vertical calibration: 15 µV (exposed) or 40 µV (control). A 20 ms prestimulus baseline is included in all tracings. Vertical arrow indicates the flash onset. Abbreviations: control (Ctrl) and light-induced retinopathy (LIR) animals. OPs recording parameters: bandwidth 100-1000Hz, flash duration 20 µs, inter stimulus interval 10 ms. (C): Graphic representation of the number of OPs evoked to different light intensities ranging between 0.9 and −4.5 log cd.sec.m−2 (as indicated on top of each bar graph) in control animals (black bars) and following different light exposure regimens (grey bars). Asterisks represent statistically significant differences (* p < 0.05) compared to age-matched control groups. Values are reported as mean ± 1SD.
Figure 3.
(A): Representative scotopic oscillatory potentials (OPs) recorded from light-exposed (10,000 lux; upper tracings) animals following different light exposure regimens and from age-matched control (unexposed; lower tracings) groups (n = 3-4 animals per group). Light intensity used: 0.9 log cd.sec.m−2. (B): Representative scotopic oscillatory potentials (OPs) tracings recorded to stimuli ranging between 0.9 and −4.5 log cd.sec.m−2 (as indicated on top of each tracing) from a representative P14–20 LIR animal (upper tracings) and an age-matched control animal (lower tracings). Numbers 1 to 9 indicate the total number of OPs at each intensity in both groups. Horizontal calibration: 20 ms; vertical calibration: 15 µV (exposed) or 40 µV (control). A 20 ms prestimulus baseline is included in all tracings. Vertical arrow indicates the flash onset. Abbreviations: control (Ctrl) and light-induced retinopathy (LIR) animals. OPs recording parameters: bandwidth 100-1000Hz, flash duration 20 µs, inter stimulus interval 10 ms. (C): Graphic representation of the number of OPs evoked to different light intensities ranging between 0.9 and −4.5 log cd.sec.m−2 (as indicated on top of each bar graph) in control animals (black bars) and following different light exposure regimens (grey bars). Asterisks represent statistically significant differences (* p < 0.05) compared to age-matched control groups. Values are reported as mean ± 1SD.
Figure 4.
Representative reconstructions of the ONL of the inferior (left) and superior (right) retina (composed of 12–13 consecutive histological segments of 75 µm in width, each sectioned at every 340 µm from the ONH to the ora serrate for each hemiretina) obtained from control animals at P15 and P28. Abbreviations: ONL: outer nuclear layer; ONH: optic nerve head.
Figure 4.
Representative reconstructions of the ONL of the inferior (left) and superior (right) retina (composed of 12–13 consecutive histological segments of 75 µm in width, each sectioned at every 340 µm from the ONH to the ora serrate for each hemiretina) obtained from control animals at P15 and P28. Abbreviations: ONL: outer nuclear layer; ONH: optic nerve head.
Figure 5.
Spidergraph representation of the measurements of ONL thinning (immediately following bright light exposure) across the infero-superior axis in light exposed rats following different light exposure regimens (red) and in age-matched control (black) rats (n = 3–4 animals per group). ONL values were taken at every 340 µm from the optic nerve head to the ora serrata in both inferior (left) and superior (right) hemiretinas. Abbreviations: optic nerve head (ONH); control (C). The total ONL loss is indicated in percentage of control for each group above each spidergraph. Group data comparing the ONL thinning (total ONL, superior ONL and inferior ONL loss) between control (black) and exposed animals (red) after different light exposure regimens. Values are reported in mean ± 1SD.
Figure 5.
Spidergraph representation of the measurements of ONL thinning (immediately following bright light exposure) across the infero-superior axis in light exposed rats following different light exposure regimens (red) and in age-matched control (black) rats (n = 3–4 animals per group). ONL values were taken at every 340 µm from the optic nerve head to the ora serrata in both inferior (left) and superior (right) hemiretinas. Abbreviations: optic nerve head (ONH); control (C). The total ONL loss is indicated in percentage of control for each group above each spidergraph. Group data comparing the ONL thinning (total ONL, superior ONL and inferior ONL loss) between control (black) and exposed animals (red) after different light exposure regimens. Values are reported in mean ± 1SD.
Figure 6.
Representative reconstructions of the ONL of the inferior (left) and superior (right) retina (composed of 12–13 consecutive histological segments of 75 µm in width, each sectioned at every 340 µm from the ONH to the ora serrate for each hemiretina) obtained from juvenile exposed animals following different light exposure regimens. Abbreviations: ONL: outer nuclear layer; ONH: optic nerve head. The size of the photoreceptor hole in the superior retina is highlighted with red boxes.
Figure 6.
Representative reconstructions of the ONL of the inferior (left) and superior (right) retina (composed of 12–13 consecutive histological segments of 75 µm in width, each sectioned at every 340 µm from the ONH to the ora serrate for each hemiretina) obtained from juvenile exposed animals following different light exposure regimens. Abbreviations: ONL: outer nuclear layer; ONH: optic nerve head. The size of the photoreceptor hole in the superior retina is highlighted with red boxes.
Figure 7.
Estimation of photoreceptor loss per day in juvenile (black;
n = 3–4 animals per group) and adult (green;
n = 3–9 animals per group) LIR rats compared to control (blue;
n = 3–4 animals per group) rats. Adult data was taken from Joly et al., 2006 [
4] for comparison purposes.
Figure 7.
Estimation of photoreceptor loss per day in juvenile (black;
n = 3–4 animals per group) and adult (green;
n = 3–9 animals per group) LIR rats compared to control (blue;
n = 3–4 animals per group) rats. Adult data was taken from Joly et al., 2006 [
4] for comparison purposes.
Figure 8.
Representative IBA1 staining of microglial cells at each time point of light exposure in the retina of juvenile and adult LIR rats. Green: IBA1 stating. Blue: DAPI. A: control juvenile rat at P28; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 8.
Representative IBA1 staining of microglial cells at each time point of light exposure in the retina of juvenile and adult LIR rats. Green: IBA1 stating. Blue: DAPI. A: control juvenile rat at P28; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 9.
Representative regionalized IBA1 staining of microglial cells (Green) following light exposure in retina of juvenile LIR rats at P20 and P28. Retinal samples were collected at the region close to the optic nerve (A,D,G,J), at the photoreceptor damage are (B,E,H,K) and at the periphery (C,F,I,L) in the superior (A,B,C,G,H,I) and inferior (D,E,F,J,K,L) retinas at P20 (A to F) and P28 (G to L). Red frame: area of subretinal region and ONL showing migration of microglial cells. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 9.
Representative regionalized IBA1 staining of microglial cells (Green) following light exposure in retina of juvenile LIR rats at P20 and P28. Retinal samples were collected at the region close to the optic nerve (A,D,G,J), at the photoreceptor damage are (B,E,H,K) and at the periphery (C,F,I,L) in the superior (A,B,C,G,H,I) and inferior (D,E,F,J,K,L) retinas at P20 (A to F) and P28 (G to L). Red frame: area of subretinal region and ONL showing migration of microglial cells. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 10.
Representative GFAP staining of active Muller cell at each time point of light exposure in retina of juvenile and adult LIR rats. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 10.
Representative GFAP staining of active Muller cell at each time point of light exposure in retina of juvenile and adult LIR rats. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 11.
Representative CNTF and FGF2 staining at selected time point in juvenile control retinas. CNTF (A,B,C,D) and FGF2 (E,F,G,H) staining (in red) are restricted to the INL. A and E: P15; B and F: P17; C and G: P20. D and H: P28. Blue: DAPI. The red spots or lines found in the OPL are non-specific staining bringing by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 11.
Representative CNTF and FGF2 staining at selected time point in juvenile control retinas. CNTF (A,B,C,D) and FGF2 (E,F,G,H) staining (in red) are restricted to the INL. A and E: P15; B and F: P17; C and G: P20. D and H: P28. Blue: DAPI. The red spots or lines found in the OPL are non-specific staining bringing by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 12.
Representative FGF2 and GFAP double staining at each time point of light exposure in retina of juvenile and adult LIR rats. Red: FGF2 staining. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. The red spots or lines found in the OPL are non-specific staining bringing by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 12.
Representative FGF2 and GFAP double staining at each time point of light exposure in retina of juvenile and adult LIR rats. Red: FGF2 staining. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. The red spots or lines found in the OPL are non-specific staining bringing by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 13.
Representative CNTF and GFAP double staining at each time point of light exposure in retina of juvenile and adult LIR rats. Red: CNTF staining. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. The red spots or lines found in the OPL are non-specific staining generated by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Figure 13.
Representative CNTF and GFAP double staining at each time point of light exposure in retina of juvenile and adult LIR rats. Red: CNTF staining. Green: GFAP stating. Blue: DAPI. A: control juvenile rat at P15; B: juvenile rat at P15 after 1 day of light exposure; C: juvenile rat at P17 after 3 day of light exposure; D: juvenile rat at P20 after 6 day of light exposure; E: juvenile rat at P28 after 14 day of light exposure; F: control adult rat at P65; G: adult rat at P61 after 1 day of light exposure; H: adult rat at P63 after 3 day of light exposure; I: adult rat at P66 after 6 day of light exposure; J: adult rat at P74 after 14 day of light exposure. The red spots or lines found in the OPL are non-specific staining generated by secondary antibody which binds to blood vessels. Calibration bar: 50 µm. Immunohistological observations were based on 3-4 animals per group.
Table 1.
Immunohistochemistry reagents.
Table 1.
Immunohistochemistry reagents.
| Molecular Markers | Source | RRID | Dilution | Antibody |
---|
Primary | IBA1 | Wako Chemicals 019-19741 | AB_839504 | 1:500 | Rabbit Polyclonal |
| GFAP | Millipore sigma G3893 | AB_477010 | 1:100 | Mouse Monoclonal |
| FGF2 | Millipore sigma 05-118 | AB_309633 | 1:100 | Mouse Monoclonal |
| CNTF | Millipore sigma MAB338 | AB_2083064 | 1:100 | Mouse Monoclonal |
Secondary | Alexa Fluor® 488 | Abcam ab150077 | AB_2630356 | 1:1000 | Goat anti rabbit IgG |
| Alexa Fluor® 594 | Abcam ab150116 | AB_2650601 | 1:1000 | Goat anti mouse IgG |
Mount medium | ProLong®Gold Antifade Mountant with DAPI | Thermo fisher scientific P36935 | N/A | N/A | N/A |