Antibodies and other reagents
Antibodies used in this study are listed in Table 1 in the Additional file 1. Tropicamide and 0.5% proparacaine were obtained from Xingqi Pharmacectical (Shengyang, China) and Alcon Laboratories (Fort Worth, Texas), respectively. The primary antibody dilution buffer, the avidin-biotin complex (ABC) kit for 3-Amino-9-ethylcarbazole (AEC) staining was bought from Boster (Wuhan, China), and 4,6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame, CA). Rodamine-conjugated phalloidin was obtained from Invitrogen (Carlsbad, CA). Total protein assay kit was from Bio-Rad (Hercules, CA). The enhanced chemiluminescence (ECL) detection system and the horse radish peroxidase-conjugated secondary antibody were purchased from Cell Signaling (Danvers, MA). Hematoxylin and eosin (H&E), and all other chemical reagents used in the experiments, unless indicated, were purchased from Sigma-Aldrich (St. Louis, MO).
Animal and treatment
A breeding pair of the bigenic mouse line that harbors a human amyloid precursor protein with Swedish mutations (K595N/M596L, APPswe) and a mutant human presenilin 1 (PS1-dE9, PS1) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) . As described, this mouse line was originally derived from a hybrid of C57BL/6 J and C3H/HeJ, which carries rd1 mutation (Pde6brd1) that confounds spontaneous retinal degeneration. To exclude possible disturbance of rd1 gene, all rd1-positive mice were identified by PCR genotyping and excluded in breeding (see Additional file 1). Mice were normally maintained in the institutional transgenic mouse facility with 12/12 h light-dark cycle with food and water ad libitum. APPswe/PS1 bigenic mice and none-transgenic littermates (non-Tg) at the age of 6 months were grouped (N = 12) for treatments. When the groups of control mice were kept in normal conditions, CILE for the treated groups of mice was replaced by a source of 10,000-Lux cool full spectrum light (wavelength ranges from 380 nm - 780 nm) for 3 months (light-3 m) or 6 months (light-6 m). The light intensity exposed to the animals was confirmed by a light meter (Thermo Fisher Scientific, Pittsburgh, PA). Temperature in animal cages during the light exposure was maintained between 22-24°C. To ensure the efficacy of light exposure, both eyes of each mouse were also topically given 1% atropine long-lasting emulsion once a week during the entire period of CILE. All animal procedures were conducted in accordance with the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and an approval of the Institutional Animal Care and Use Committee (IACUC) of the Zhongshan Ophthalmic Center of Sun Yet-sen University.
Fundus photography was performed as described with minor modification . Briefly, following papillary dilation with 1% tropicamide and 2.5% phenylephrine in HCl solution and sedation with 4.3% chloral hydrate in PBS (intraperitoneal injection, i.p.), mice were placed under the microscope operation system platform (OMS-800, Topcon, Japan) and fundus images were directly captured using a Canon G9 digital camera attached to the scope over the eye that was covered by one drop of 1% hydroxypropyl methylcellulose over the cornea to compromise refractor errors.
Scotopic electroretinogram (ERG) was recorded as reported previously . Each group of animals was assayed with a RETIPORT ERG recording system (RETI Technologies, Gaithersburg, Germany). Before measuring, all animals were kept in dark overnight for at least 8 hours for dark-adaptation. Following papillary dilation with 1% tropicamide and 2.5% phenylephrine in HCl solution, sedation with 4.3% chloral hydrate in PBS (i.p.) and corneal anesthesia with 0.5% proparacaine, the gold loop electrodes were placed on the cornea, the reference electrode was plug into the mouth underneath of the tongue, and a ground electrode was subcutaneously inserted into the midway of the tail. The rod-only responses were recorded after stimulation by white light flashes with intensity of 0.008 cd/m2s (-25 dB), whereas the maximal response from the mixed rod/cone- responses (max response) was recorded with light flashes of 2.5 cd/m2s (0 dB). Three independent stimuli within 3 sec intervals were recorded as a single ERG recording. At least 3 recordings were obtained from each eye. The amplitudes and the latency of a and b waves were measured.
Mice were euthanized with 4.3% chloral hydrate (0.1 ml/g) followed by transcardiac perfusion with ice-cold 0.1 M phosphate buffered saline (PBS, pH 7.4). Eyes were then immediately enucleated and fixed in 4% paraformaldehyde (PFA) in PBS (pH7.4) overnight at room temperature (RT) or dissected to extract retina for preparation of tissue lysates for Western blots. Fixed eyes were then preserved in PBS containing 20% sucrose and 0.05% sodium azide at 4°C, gradually dehydrated in 70% - 100% isopropanol, and embedded in paraffin blocks. Retinal cross sections (4 μm thickness) through the center of pupil-optic nerve head were prepared for histopathological analysis. To be prepared for transmission electron microscopy (EM), about 2 mm × 2 mm size of retina with attached RPE/choroid/sclera was dissected from the proximity of the optic nerve head (within 5 mm range) from some enucleated eye cups following PBS perfusion. Dissected retina-chorid-sclera tissues were immediately fixed in 2.5% glutaraldehyde and 1% PFA in 0.1 M sodium cacodylate-HCl (pH 7.4) (24 h at 4°C) followed by further post-fixation in 1% osmium tetroxide in 0.1 M cacodylate buffer-HCl (pH 7.4) for 4 h, gradually dehydrated in 50%-100% alcohol followed by immersion in propylene oxide, and embedded in epoxy resin for preparation of ultrathin sections. RPE/choroid flatmounts were prepared as described previously . In general, fixed eyes were split equatorially and retinas were carefully removed from the eyecup under a stereoscope. After the extraocular muscles were removed, the posterior eye segment containing the RPE/choroid complex of each eye was spread into four quarters by four radial cuts.
Histopathology, immunohistochemistry and immunofluorescence microscopy
Hematoxylin and eosin (H&E) staining was performed for general histopathological assessment as previously described . For immunohistochemistry, paraffin-embedded retinal cross sections were deparaffinized, rehydrated, and autoclaved (121°C × 5 minutes in 10 mM citrate buffer, pH 6.0) for antigen retrieval. Following quenching of endogenous peroxidase activity with 3% H2O2 (20 min at RT), sections were blocked with 5% goat serum in PBS containing 0.1% Triton-X 100 and 20 mM L-lysine incubated with an appropriate primary antibody as listed in Table 1 (Additional file 1) at 37°C for 1 h. Specific immunoreactivity was then visualized by microscopy following incubation with a biotinylated secondary antibody, ABC kit and AEC staining. To detect Aß, outer retina flatmounts were treated by 70% formic acid for 5 min, washed with PBS, blocked with 4% bovine serum albumin in PBS containing 0.1% Triton X-100 (30 min, RT), and incubated with an appropriate primary antibody in primary antibody dilution buffer overnight at 4°C. Following appropriate washing with PBS, retinal flatmounts were incubated with an appropriate fluorophore-conjugated second antibody, counterstained with DAPI, coverslipped onto microslides, and visualized using a Zeiss LSM510 Meta Confocal Microscope. For quantification, four non-overlapped areas were randomly selected along the equatorial zone which is about 300 μm away from the optic disc, under the magnification of 400X. Images were captured using an Axioplan 2, Zeiss camera and further quantified with Image Pro-Plus1.42q (National Institutes of Health, USA). Total Aß-positive area, number of IBA-1-immunoractive cells, and total length of IBA-1-immunoractive dendrites of microglia within the four selected regions were quantified. Disorganization of RPE alignment was scored as percentage of phalloidin-negative area over the entire RPE flat mounts.
Western blotting was conducted as previously described [46, 47]. Briefly, dissected retinas were homogenized in lysis buffer (50 mM Tris-HCl/pH7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, protease inhibitor cocktail mixture) and pelleted. The concentrations of total protein in the supernatants were assayed using Bio-Rad Protein Assay kit. About 35 μg total protein of lysate from each samples was resolved in 10% SDS-polyacrylamide gel, blotted on PVDF membrane followed by incubation with Blotto (0.1% Tween-20 and 5% milk in PBS, × 1 h), a specific primary antibody in 5% milk in PBST (overnight at 4°C) and with a horse radish peroxidase-conjugated secondary antibody in RT with appropriate washing. The immunoreactivity was visualized with ECL.
Transmission electron microscopy
The ultrathin (70 nm) sections were prepared using a Leica UltraCut S Microtome, counterstained with uranyl acetate and lead citrate, and examined under a JEOL 100CX II electron microscope (JEOL, Tokyo, Japan) at 80 kV. Microscopic images were acquired using X-ray films.
In all of the graphs, the data points represent the means ± S.E.M. from all individuals in each group of animals (N = 6-12). Applicable comparisons were performed by one-way analysis of variance followed by Student's t-test for multiple groups or independent samples or T-test for two groups by SPSS13.0 software. The difference between groups was considered as statistically significant when the value of p was ≤ 0.05.