Light-emitting-diode induced retinal damage and its wavelength dependency in vivo

Abstract

Aim: To examine light-emitting-diode (LED)-induced retinal neuronal cell damage and its wavelength-driven pathogenic mechanisms.

Methods: Sprague-Dawley rats were exposed to blue LEDs (460 nm), green LEDs (530 nm), and red LEDs (620 nm). Electroretinography (ERG), Hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunohistochemical (IHC) staining, Western blotting (WB) and the detection of superoxide anion (O₂^-·), hydrogen peroxide (H₂O₂), total iron, and ferric (Fe³⁺) levels were applied.

Results: ERG results showed the blue LED group induced more functional damage than that of green or red LED groups. H&E staining, TUNEL, IHC, and TEM revealed apoptosis and necrosis of photoreceptors and RPE, which indicated blue LED also induced more photochemical injury. Free radical production and iron-related molecular marker expressions demonstrated that oxidative stress and iron-overload were associated with retinal injury. WB assays correspondingly showed that defense gene expression was up-regulated after the LED light exposure with a wavelength dependency.

Conclusion: The study results indicate that LED blue-light exposure poses a great risk of retinal injury in awake, task-oriented rod-dominant animals. The wavelength-dependent effect should be considered carefully when switching to LED lighting applications.

Keywords: LED light injury; blue light injury; iron; light injury mechanisms; oxidative stress; retinal light injury.

Figure 1. LED light sourcespectral power distribution (SPD) curves

A: Yellow phosphor-converted white LED to exhibit correlated color temperature (CCT) at 6500 K; the first peak appeared at 460 nm with 0.028 W/nm showing the blue content and the second peak with a bell shape presenting a large portion of yellow content; B: Blue light factor B(λ) distribution; C: Single-wavelength blue light LED peaked at 460 nm with 102.3 µW/cm² in radiometric units. Green light LED peaked at 530 nm with 102.8 µW/cm². Red light LED peaked at 620 nm with 102.7 µW/cm².

Figure 2. Electroretinography responses

All three LED groups demonstrated a significant decrease in the b-wave amplitude after light exposure. The blue LED group showed the highest function loss; n=40 for the control group and n=54 for each exposure group. Curve scale: amplitude=250 µV and stimulation=50ms. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group.

Figure 3. Histological analysis

A: Normal retinal layers in the control group compared to different LED light exposure-induced retinal injuries, including the absence of photoreceptors and INL degeneration; B: The ONL thickness of the exposure groups decreased significantly after 28d of light exposure. The blue LED group exhibited the strongest loss; n=6 for the control group and n=8 for each exposure group. GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; PIS: Photoreceptor inner segment; POS: Photoreceptor outer segment. The retinal pigment epithelium (usually next to the POS layer) is detachedand cannot be found within this scope in A. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group; scale bar=50 µm.

Figure 4. Retinal cellular injury studied by TEM

A: Normal ONL nucleolus on the left; nucleolus condensation in phase 1 remodeling and pyknosis in phase 2; B: Normal POS on the left; POS deformations in phase 1 remodeling and round POS debris in phase 2; C: Normal RPE nucleus on the left; shrinking RPE nucleus in phase 1 and condensation in phase 2. There are 36 sections analyzed and 244 of pyknotic RPE nuclei observed after the blue light exposure; n=4 for the control group and n=6 for the exposure group. Scale bar=100 nm for the control and phase 2 in B; scale bar=0.5 µm for the rest of other images.

Figure 5. Molecular apoptotic marker detection

A: The damaged retinal cells correspond to the positive labeling. The results showed that more apoptotic cells presented in the retina of the exposure groups; B: The blue LED group exhibited the highest fluorescence intensity; C: The WB results showed apoptotic marker, PARP-1, had higher activation after 3, 9d of blue or green light exposure; D: The PARP-1 expression showed much significant activation after 3, 9d of exposure; n=4 for the control group and n=8 for each exposure group. GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; PIS: Photoreceptor inner segment; POS: Photoreceptor outer segment. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group.

Figure 6. Retinal light injury molecular labeling by IHC

A, B: 8-OHdG was used to detect the DNA adducts; C, D: Acrolein was used to detect the lipid adducts on macromolecules; E, F: Nitrotyrosine was used for protein adduct recognition. The result shows all exposure groups exhibited higher fluorescence intensity in the ONL. The blue LED group exhibited the highest response, whereas the green and red groups' response was lower; n=6 for the control group and n=8 for each exposure group. GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; PIS: Photoreceptor inner segment; POS: Photoreceptor outer segment. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group; scale bar=50 µm.

Figure 7. WB assay of anti-oxidant enzymes

A, B: After 3d of light exposure, the antioxidant genes HO-1 and cytosolic GPx1 were up-regulated after blue LED light exposure as an antioxidant response to all LED light insults, but the blue LED showed the strongest expression. The apoptotic marker PARP-1 showed greater densities after blue and green light exposure. C, D: Clear wavelength-dependent expressions were found after 9d of light exposure. E: SOD2 was down-regulated, corresponding to the wavelengths after 3 or 9d of light exposure, but the blue light activated a higher expression after 28d of exposure due to the delayed/adaptive antioxidant response; n=6 for the control group and n=8 for each exposure group. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group.

Figure 8. WB assay of iron metabolism markers

A strong association between iron metabolism and light injury resulting from 3d of blue LED light exposure (A); CP (B), Ft (C); and Fpn (D) were up-regulated, but Tf (E) and the TrfR (F) were down-regulated as the wavelength decreased; n=8 for the control group and each exposure group. ^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group.

Figure 9. Iron metabolism and superoxide products

A: Lucigenin-stimulated superoxide O₂⁻· had reached 60 000 of CL, and the green and red LED light exposure groups accumulated 10 000 to 20 000 of CL in 8min after 3d of blue LED light exposure; B: Blue light exposure increased H₂O₂ concentrations and reached a high concentration of 0.13 nmol/retina, whereas the green and red groups contained 0.089 to 0.083 nmol/retina, respectively; C: Blue LED light exposure significantly increased the total iron concentration to 2 nmol/retina, whereas the normal retina only contains 0.23 nmol/retina of total iron. The longer wavelength exposures also increased the total iron concentration. The green LED group increased to 1 nmol/retina and the red LED group increased to 0.89 nmol/retina; D: Blue LED light exposure significantly increased the ferric concentration to 1.6 nmol/retina, whereas the normal retina only contains 0.15 nmol/retina. The green LED exposure increased to 0.56 nmol/retina and the red LED exposure increased to 0.33 nmol/retina. n=6 for controls and n=8 for each exposure group (^aP<0.05, ^bP<0.01, ^cP<0.001 compared with the control group). E: Diagram of the oxidative pathway in the outer retina and RPE. As the retina absorbed the light under a high O₂ condition, O₂⁻· is initially generated and converted to H₂O_2, then ultimately to H₂O. The O₂⁻· could easily convert to a toxic hydroperoxyl radical (HO₂·) during the process. The abnormal accumulation of H₂O₂ under the high iron condition leads to a Fe²⁺ being converted to the more injurious Fe³⁺. Although the Fe²⁺-melanin complex is readily oxidized by H₂O₂ and O₂, few highly noxious hydroxyl radicals (OH·) escape the melanin polymer via a Fenton reaction and may develop injurious reactions. (Concept modified from Rozanowska, Malgorzata Barbara, 2009. http://www.photobiology.info/Rozanowska.html accessed 18 March 2015).