4 RESULTS AND DISCUSSION
4.1 White LED at domestic lighting level to induce retinal injury
The representative ERG response curves of rats are shown in Figure 29A.
The normal retina showed a high b-wave peak, but the injured retina curved a low b-wave peak as a result of cell function loss. As shown in Figure 29B. Two LED groups and the white CFL group all demonstrated a significant decrease of b-wave amplitude at day 9 and day 28 after light exposure (ANOVA followed by Tukey post hoc test p < 0.001). The b-wave amplitude of the yellow CFL group did not decrease significantly at day 9; however, it had 21% of decrease at day 28 after light exposure. The data from each of the four exposure groups was not statistically different at 28+14 d as compared to 28 d of exposure, and this trend was also applied to the H&E staining results (data not shown). No significant development was found after 3 d of light exposure, and therefore data were not shown as well.
Figure 29 ERG responses after light exposure
Both LED groups demonstrated a significant decrease of b-wave amplitude at day 9 and day 28 after light exposure. The fluorescent lamp groups developed severe loss of b-wave amplitude until 28 d of light exposure. n = 3 for controls, n = 3 for 3 days of exposure groups, and n = 8 for each exposure group at each time of exposure (Curve scale: amplitude = 250 µV and stimulation = 50 msec). (**,
*** p < 0.01, 0.001, respectively, compared to the “normal” group by ANOVA with the Tukey post hoc test).
4.1.2 Retinal histology–H&E staining showing layer damages
As shown in Figure 30a-b. White LED light exposure can lead to morphologic alterations in the rat retina. The group that was exposed to 750 lux white LED light for 28 d exhibited the adverse effect of light exposure including the pyknotic photoreceptor nuclei (arrow), swelling of the inner segment (arrow head), and a disorganized outer segment (asterisk). As shown in Figure 30c-f.
The ONL thickness of white and blue LED groups decreased significantly at day 9 and day 28 (data not shown) after light exposure (ANOVA followed by Tukey post hoc test p < 0.01), whereas, the ONL thickness of the white and yellow CFL groups did not decrease significantly at day 9 after light exposure.
Figure 30 Retinal light injury after 9 d or 28 d of exposure analyzed by H&E staining
GCL: ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. IS: inner segment. OS:
outer segment. *RPE: the retinal pigment epithelium (usually next to the OS layer) is detached and cannot be found within this scope.
(A) (a) Normal retina layers, and (b) light exposure-induced retinal injury, including the absence of photoreceptors and INL degeneration. (B) The ONL thickness of the LED groups decreased significantly at day 9 and day 28 after light exposure, whereas the ONL thickness of white and yellow CFL groups did not decrease significantly at day 9 after light exposure. Both blue (c) and white LED (d) light exposure caused the disappearance of photoreceptors; the white CFL group (e) exhibited distortion of the OS and ONL; and the yellow CFL group (f) exhibited less movement in each layer. n = 3 for controls and n = 8 for each group after 9 or 28 days of exposure (** indicates
4.1.3 Apoptosis Detection - TUNEL staining detects nuclear apoptosis
The retinal TUNEL stains are shown in Figure 31. Light exposure induced significant retinal cell apoptosis in all groups. However, more apoptotic cells were shown in the retina of the LED groups than in the retina of the CFL lamp groups after 9 d of exposure (ANOVA followed by Tukey post hoc test p <
0.001 for LED groups; p < 0.01 for CFL groups).
Figure 31 Light-induced retinal cell apoptosis tested by TUNEL labeling
GCL: ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. RPE: the retinal pigment epithelium.
The damaged retina cells correspond to the positive labeling. (A) The result shows that more apoptotic cells (arrows) appear in the retina of the LED groups than that of the CFL groups after 9 days of light exposure. (B) The LED groups exhibit higher fluorescence intensity. n = 3 for controls and n = 8 for each exposure group (**, *** p < 0.01, 0.001, respectively, compared to the “normal”
group by ANOVA with the Tukey post hoc test; scale bar = 50 µm).
4.1.4 TEM demonstrations on the cellular injury
As shown in Figure 32 (samples were taken after 9 d of white LED light exposure). Nucleolus damage of photoreceptors occurred after exposure including early stage of nucleolus condensation (32b), karyolysis (32c), pyknosis (32d-e), and karyorrhexis (32f). Another crucial observation of photoreceptor injury included disruption of the inner and outer segments, which is shown in Figure 32g-l.
Figure 32 Retinal cellular injury studied by TEM
The photoreceptor nucleolus damage after LED light exposure result in (A) ONL nuclear deformations (arrows) shown as (a) normal ONL nucleus; (b) nucleolus condensation; (c) karyolysis;
(d and e) pyknosis; (f) karyorrhexis. (B) Photoreceptor deformations and (g) normal photoreceptor, IS and OS; (h and i) showing minor disruption; (j, k, and l) and IS disappearance followed by OS shrinkage and the formation of several small round shapes (scale bar = 2 µm for g, h, and k; scale bar = 1 µm for the rest of others). n = 3 for controls and n = 5 for white LED group after 9 days of
4.1.5 Immunohistochemistry (IHC) staining results indicating retinal light injury
Oxidative injury results in adducts on macromolecules that can be detected by immunostaining. The antibodies that specifically recognize these adducts provide evidence of the oxidative injury. Three antibodies were used to detect cell conditions in these experiments after 9 d of light exposure, including acrolein for lipid recognition (Figure 33A), 8-OHdG for DNA detection (Figure 33B), and nitrotyrosine for protein identification (Figure 33C). The results show that LED groups exhibit higher fluorescence intensity with 8-OHdG, acrolein and nitrotyrosine in ONL (ANOVA followed by Tukey post hoc test p < 0.001 for LED groups) and that the fluorescent lamps induced lower fluorescence intensity of 8-OHdG, acrolein and nitrotyrosine in ONL.
Figure 33 Retinal light injury labeling after 9 d of exposure by IHC
(A) Acrolein was used to detect the lipid adducts on macromolecules; (B) 8-OHdG was used to detect the DNA adducts; and (C) Nitrotyrosine was used for protein adduct recognition. The result shows LED groups exhibit higher fluorescence intensity on ONL, and the fluorescent lamp groups
B
4.1.6 Oxidative Stress -- superoxide anion O2-. shows the injury
As shown in Figure 34A. Lucigenin-stimulated superoxide anion (O2-)and total oxidative products were computed for all groups. After 3 d of blue LED light exposure, the retina O2- exceeded 60000 in 8 min, the white LED group exhibited a high total count close to 40000, and the fluorescent groups accumulated smaller total counts from 20000 to 30000. However, the plot exhibited an opposite trend when the exposure duration was increased to 9 d (Figure 34B). This result suggests that retinal oxidative stress may be induced by light exposure in the early stage.
Figure 34 A reactive oxygen species assay after 3 d and 9 d of light exposure
CL: chemiluminescence
(A) After 3 d of blue LED light exposure, the lucigenin-stimulated superoxide anion (O2-) exceeded 60000 in total count; the white LED group had a high total count close to 40000; and the fluorescent groups accumulated less total counts from 20000 to 30000, whereas normal rats exhibited only a count of approximately 1000. n = 3 for controls and n = 3 for each exposure group. (B) After 9 d of exposure, the O2- total count for the blue LED light group decreased to 8000; the white LED light group decreased to 18000; and both fluorescent light groups remained at the same level at 20000 to 30000. n = 3 for controls and n = 8 for each exposure group (**, *** p < 0.01, 0.001, respectively, compared to the “normal” group by ANOVA with the Tukey post hoc test).
ROS - Lucigenin
Reactive oxygen species -- superoxide anion O2-.
CL Intensity (in thousands)
Reactive oxygen species -- superoxide anion O2-.
CL Intensity (in thousands)
4.2 Mechanism of LED induced retinal injury and its wavelength dependency