Analytical Methods

3.4.1 Electroretinography (ERG)

As shown in Figure 25 and Figure 26, ERG was performed as described

all rats before and after light exposure using ERG (Acrivet, Hennigsdorf, Germany). After 18 h of dark adaptation, rats were anesthetized using an intramuscular injection of 100 mg/kg ketamine and 5 mg/kg xylazine (WDT eG, Garbsen, Germany). One drop of tropicamide (0.5%) (Mydriaticum Stulln, Pharma Stulln, Germany) was applied for pupil dilation before ERG measurement. One drop of Alcaine (0.5%) (proxymetacaine hydrochloride;

Alcon Pharmaceuticals Ltd, Puurs, Belgium) was applied for local anesthesia before placing the active electrode onto the cornea (see Figure 25). Two subcutaneous needle electrodes (Ambu Neuroline Twisted Pair Subdermal, Bad Nauheim, Germany) served as the reference and ground electrodes. The reference needle was subcutaneously inserted between the eyes, and the ground needle was subcutaneously inserted between the rear legs to obtain the proper impedance levels, which were less than 10 kΩ at 25 Hz. LED flashes were stimulated without background illumination, and the flash interval was 1 s with a flash duration of 3 ms. The weighted average of 10 stimulations was computed by the program to produce the final detection values.

Figure 26 Diagram of retina response components to ERG stimulation ¹⁵

3.4.2 Hematoxylin and eosin (H&E staining)

Retinal histology was performed as described previously with modifications

sectioning was performed (4% paraformaldehyde in 0.1 M phosphate buffer [pH 7.4] for 1 h at 48°C), and the eyeballs were dehydrated in EtOH, infiltrated in xylene, and embedded in paraffin. Radial 5 μm sections were stored at 48°C.

The histologic analysis included quantification of the outer nuclear layer (ONL) and retina morphology alteration using a light microscope. The midsuperior aspect of the retina was examined for all histological analyses in this study.

3.4.3 Transmission electron microscopy (TEM) analysis

TEM was performed as described previously ⁶⁴ after 9 d of light exposure.

The processes were performed at the Electron Microscopy Facility at the Department of Pathology at National Taiwan University Hospital (Taipei, Taiwan). Retina slices of 1 mm (Figure 27A) were prefixed in 2.5%

glutaraldehyde in PBS, postfixed with 2% osmium tetroxide, and dehydrated for 10 min each in sequential baths of 30%, 50%, 70%, 90%, and 100% ethanol.

The specimens were placed into propylene oxide for 30 min, followed by a mixture of propylene oxide and epoxy resin for an additional 1 h; the samples were subsequently embedded into a gelatin capsule with epoxy resin at 60°C for one day. Subsequently, 80 to 90 nm ultrathin sections were obtained using an ultramicrotome. The sections were stained with 2% tannic acid in distilled water (DW) for 5 min, followed by 2% uranyl acetate in DW for 15 min and a lead-staining solution for 5 min. In the final step, the sections were coated with a thin copper grid-film and placed in a vacuum chamber for scanning (Figure 27B). As shown in Figure 28A, the specimens were examined using TEM with a high-resolution instrument at 80 kV (JEOL JEM-1400, Peabody, MA, USA), and

Figure 27 Specimen of a retina slice

(A) (B) Figure 28 TEM observation instrument (JEOL JEM-1400)

(A) (B)

3.4.4 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)

The TUNEL assay was performed using a FragEL^TM DNA fragmentation detection kit (Calbiochem, Darmstadt, Germany) following the standard protocol with a minor modification to detect apoptotic cells after 9 d of light exposure.

Tissue sections were deparaffinized, rehydrated, and blocked using endogenous peroxidase with H2O2 for 30 min. Antigen retrieval was achieved by pressure-cooking in a 0.1 M citrate buffer at pH 6 for 10 min followed by cooling at room temperature before incubation with the enzyme. The TUNEL enzyme (1 h at 37°C) and peroxidase converter (30 min at 37°C) were applied to the 10 μm sections after incubation for 5 min in a permeabilizing solution of 0.1% Triton-X in 0.1% sodium citrate. The tissues were counter-stained with DAPI, and the DNA strand breaks were labeled with fluorescein FITC-Avidin D. The fluorescent signals were obtained by adding FITC-Avidin, which bound to the biotinylated-dU of the damaged DNA. After staining, image analysis was used to quantify the relative fluorescence intensity of the TUNEL-positive cells, with the number of TUNEL-stained nuclei quantified in 4 random slides per sample.

Sections were visualized on a fluorescent microscope over the entire retina excluding the RPE layer (Nikon Instruments Inc., NY, USA). The number of TUNEL-positive cells for each section was counted by Image-Pro Plus software (v.6.0).

3.4.5 Immunohistochemistry (IHC)

Immunohistochemistry was performed as described previously ^{67, 68} after 9 d of light exposure. In brief, cryosections of the retina samples were incubated

deoxyguanosine (8-OHdG) (1:50, JAICA, Tokyo, Japan)], lipids [Acrolein, (1:200, Advanced Targeting Systems, San Diego, CA USA)] and proteins [nitrotyrosine, (1:200, Abcam, Millipore Billerica, MA, USA)]. The same quantification method used for the TUNEL analysis was applied in the IHC analyses. The relative fluorescence intensity corresponding to the number of IHC-positive cells for each section was measured and quantified by Image-Pro Plus software (v.6.0).

3.4.6 Free radical assay (reactive oxidative species, ROS)

Measurement of reactive oxygen species in the retina was performed as described previously ⁶⁸ after 3 or 9 d of light exposure. In brief, 0.2 mL of homogenized extraction was loaded with 0.1 mL of 0.9% saline onto a 3-cm dish with a stir bar placed at the center. The dish was placed into the chemiluminescence analyzer chamber (Tohoku CLA-FS1, Miyagi, Japan). The ROS were quantified after adding the enhancer Lucigenin to the chemiluminescence analyzer. After 60 s of background detection, 1 mL of a Lucigenin (bis-N-methylacridinium nitrate) solvent (2.5 mg of Lucigenin dissolved in 50 mL 0.9% saline) was added for stimulation. The stimulated O2-.

and total oxidative products were captured every 10 s and computed for 7 min after 1 min of baseline detection.

3.4.7 Western blotting (WB)

Total protein was extracted from the retina by lysing the sample in radioimmunoprecipitation assay (RIPA) buffer [0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA] and protease

boiled for 5 min. A 100-mg sample was separated on 10% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Immobilon-P;

Millipore Corp., Billerica, MA, USA). The membranes were incubated with hemeoxygenase-1 [(HO-1); Abcam, Cambridge, MA, USA], ceruloplasmin [(CP); Santa Cruz Biotechnology, Dallas, Texas, USA], anti-cytosolic glutathione peroxidase [(GPx1); Abcam, Millipore Billerica, MA, USA], anti-poly (ADP-ribose) polymerase-1 [(PARP-1); Cell Signaling Technology Inc., Danvers, MA, USA], anti-superoxide dismutase [(SOD2);

Santa Cruz Biotechnology Inc., Dallas, Texas, USA], and anti-β-actin (Abcam, Millipore Billerica, MA, USA) antibodies. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody and visualized by chemiluminescence (GE Healthcare). The density of the blots was determined using image analysis software after scanning the image (Photoshop, ver.7.0;

Adobe Systems, San Jose, CA, USA). The optical densities of each band were evaluated by comparison with the density of the β-actin bands.

3.4.8 Hydrogen peroxide (H2O2) assay

The assay was performed using a hydrogen peroxide colorimetric/fluorometric assay kit (BioVision, Milpitas, CA, USA) following the standard protocol to detect H2O2 concentrations after 3 days of light exposure. In brief, total protein was extracted from the retina and centrifuged for 15 min immediately after the extraction. Each well was loaded with 20 μl samples and brought to a volume of 50 μl with assay buffer. Reagents and H2O2

standards were mixed and then incubated for 10 min. A background detection

3.4.9 Total iron and ferric (Fe³⁺) assay

The assay was performed using a QuantiChrom Iron Assay (DIFE-250) Kit (BioAssay Systems, Hayward, CA, USA) following the standard protocol to detect total iron concentrations after 3 days of light exposure. In brief, total protein was extracted from the retina, and 50 μl of the extraction was loaded into a 96-well plate. Then, 200 μl of working reagent was added and incubated for 40 min at room temperature, followed by an optical density reading at 510–630 nm.

Sample readings were compared with the standard curve to calculate the concentrations.