Blood levels of oxLDL increase with age [⁴⁰] and upon ingestion of high cholesterol diet [²⁰], as corroborated (Figure 1A). Since CD36 clears circulating oxLDL [²⁹], we tested if CD36 deficiency would lead to accumulation of oxLDL in the sub-retinal region. CD36-deficient mice presented abundant sub-retinal oxLDL accumulation (Figure 1B,C), despite regular diet; oxLDL was minimally detected in comparably raised wild type (WT, CD36+/+) congeners. CD36-dependent cellular uptake of oxLDL was confirmed upon systemic administration of fluorescent tagged oxLDL; specific uptake of DiI-oxLDL was seen in the RPE of CD36+/+ mice (Figure 1D), whereas no fluorescence was detected in RPE of CD36−/− mice in vivo (Figure 1E). Likewise, exposure of RPE cells from CD36+/+ mice to DiI-oxLDL revealed internalization of the oxLDL, while no internalization was seen in RPE of CD36−/− mice (Figure 1F,G).

Lipids are the main component in basal laminar deposits [⁸,¹³,⁴¹]. We evaluated if sub-RPE accumulation of oxLDL in CD36−/− mice (Figure 1C) is associated with BM thickening. Electron microscopy of the sub-retinal region revealed age-dependent debris detected in older (12 months-old) but not younger (4 months-old) CD36−/− mice (Figure 2A-F).

Mice deficient in the LDL receptor ligand, ApoE (ApoE−/−) involved in uptake of LDL, fed a HFHC diet, exhibit an atherosclerosis phenotype with high blood oxLDL levels [²¹]; along the lines of the concept presented herein, these animals also exhibit laminar deposits in BM, as seen in AMD [²⁴]. To further address the role of CD36 on oxLDL accumulation and BM thickness, oxLDL plasma levels and electron microscopy of the sub-retinal region were performed in ApoE−/− mice and in ApoE/CD36 double knockout mice (ApoE−/−/CD36−/−). oxLDL levels were highest in the 4 months-old HFHC-fed ApoE/CD36 double knockout mice (Figure 3A). In addition, BM thickening was further augmented in these relatively young ApoE−/−/CD36−/− mice (Figure 3B-D). Collectively, data indicate that CD36 deficiency is associated with augmented sub-retinal deposits.

Additional experiments were conducted to determine if CD36 stimulation could prevent thickening of BM in HFHC-fed ApoE−/− mice. For this purpose animals were treated daily either with NaCl or the CD36 agonist EP80317 [³⁶] from 8 to 18 weeks of age. HFHC diet significantly increased the thickness of BM in saline-treated ApoE−/− mice (Figure 4B,D), as previously reported [²⁴]. Whereas, treatment of ApoE−/− mice with EP80317, which reduces plasma cholesterol levels [³⁶], prevented the accumulation of sub-RPE deposits and thickening of BM (Figure 4C,D).

Along with BM thickening, ApoE−/− mice also present photoreceptor dysfunction [²⁴,²⁵], as evidenced with the significantly attenuated rod-cone a-wave amplitude of saline-treated ApoE−/− compared to WT mice (Figure 5A, B). Similarly, the significantly attenuated rod and rod-cone b-waves (−2.7 log cd.sec.m-2 and 0.6 log cd.sec.m-2, respectively) are also suggestive of inner retinal dysfunction in saline-treated ApoE−/− compared to WT mice (Figure 5A, C); treatment of ApoE−/− mice with EP80317 significantly attenuated the loss of the scotopic rod and rod-cone responses (Figure 5A-C).

The ApoE−/− and ApoE/CD36 double deficient (ApoE−/−CD36−/−) mice, obtained as described previously [³⁵], were housed at local animal facilities under 12 hours light-12 hours dark cycles and fed ad libitum with a normal (ND) or a high fat-high cholesterol diet (HFHC) (D12108, cholate, AIN-76A semipurified diet, Research Diets Inc., NewBrunswick, NJ). CD36−/− mice and their controls wild-type littermates (CD36+/+) were reproduced separately under ND unless otherwise indicated. Eight-week-old ApoE−/− mice were treated daily with EP803017 (300μg/Kg) [³⁶] or vehicule (0.9% NaCl) by subcutaneous injections for a period of 10 weeks prior to sacrifice. All mice were sacrificed by carbon dioxide inhalation or by intraperitoneal injection of pentobarbital sodium overdose, prior to ocular enucleation. All experimental procedures were done in accordance with the Institional Animal Ethics Committee and the Canadian Council on Animal Care guidelines for use of experimental animals.

Eyes were fixed for 1 h in 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After 1 h, the eyes were dissected, fixed for another 3 h, postfixed in 1% osmium tetroxide in cacodylate buffer, and dehydrated in graduated ethanol solutions. The samples were included in epoxy resin and oriented. Ultra-thin sections (80 nm) were contrasted by uranyl acetate and lead citrate and were observed with an electron microscope JEOL 100 CX II (JEOL) with 80 kV, and measurements of Bruch's membrane thickness were made on three representative animals.

Eight at 12-day-old CD36+/+ and CD36−/− mice were sacrificed, enucleated and eyes were maintained at room temperature overnight in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) and then incubated 30 min with 2 mg/ml trypsin/collagenase I at 37 °C. After trypsin inhibition with DMEM containing 10% fetal calf serum, the RPE layer was harvested. The RPE was plated in 8-wells labtek (Nunc) at a rate of RPE from one eye per well in DMEM containing 10% FCS, 1% penicillin/streptomy- cin. Cells were maintained for 7 days before the phagocytosis assay.

Confluent RPE monolayers were challenged with DiI-oxLDL (Biomedical Technology Inc, MA, USA). When RPE reached 80% of confluence, cells were incubated with 30μg/ml DiI-oxLDL for 5 hours in DMEM containing 5% lipoprotein-deficient serum (Biomedical Technology Inc) at 37°C. After incubation, no-ingested DiI-oxLDL were removed by washing three times with PBS and cells were fixed for 10 min in paraformaldehyde 4%. The cells were then permeabilized for 30 min in 1% triton X-100, blocked with 1% bovine serum albumin in PBS for 30 min and incubated with 4', 6-Diamidino-2-phenyl-indole (DAPI) (1:4000, Sigma-Aldrich) for 5 min. The slides were mounted and observed with a Nikon Eclipse E800.

CD36+/+ and CD36−/− mice (2-month-old) were anesthetized and intravenous injection of 100 μl of tag DiI coupled or not to oxLDL (Biomedical Technology Inc) were given in the tail vein. The mice were euthanatized 24 hours after injection and eyes were collected and fixed in 4% paraformaldehyde in PBS for 2 hours. The tissue was then mounted in OCT (Tissue Tek), cut in thin sections (10μm). Nuclei were labeled with DAPI (1:4000) and sections were mounted with Gelmount (Biomeda). Fluorescence was observed with a Nikon Eclipse E800.

Twelve-month-old CD36+/+ and CD36−/− mice eyes were fixed in paraformaldehyde 4% in PBS for 1 hour at room temperature and rinsed in PBS before embedded in OCT. Frozen transverse sections 10 μm thick were cut and permeabilized for 10 min in 1% Triton X-100. Postfixation was performed with methanol or ethanol, depending on the antibody used. Immunolabeling with primary antibodies (1:100) rabbit polyclonal oxLDL (RayBiotech Inc.) was performed overnight at room temperature. After washing in PBS, secondary antibodies coupled with Alexa Fluor 488 (1:100, Molecular Probes) were applied for 1 hour at room temperature. Nuclei were labeled with DAPI (1:4000) and sections were mounted with Gelmount. Fluorescence was observed with an Olympus BX51 microscope. All immunostaining were repeated at least three times, and staining without primary antibody served as negative controls.

Ten- and 24-month-old C57Bl6/J mice (Jackson Laboratory) under ND and 12-week-old ApoE−/− mice under ND or HFHC diet and 12-week-old ApoE−/−CD36−/− under HFHC diet were anesthetized. Blood was drawn from the inferior vena cava mice using EDTA as an anticoagulant and butylated hydroxytoluene (20 μM final concentration) as antioxidant. Plasma was collected by centrifugation for 15 min at 1,000 g. Mouse plasma oxLDL level was determined by use of an ELISA kit (Accurate Chemical and Scientific Corp, USA).

Dark adapted scotopic full-field electroretinograms (ERG) (intensities:-6.3 log cd.sec.m⁻²through 0.6 log cd.sec.m⁻²) were obtained from 18-week-old ApoE−/− mice on HFHC diet treated or not with EP803017 and WT control-age matched mice as we described in detail [^37-39]. Amplitudes of ERG a-wave and b-wave components were measured according to a method previously described [³⁷,³⁸]. Briefly, the amplitude of the a-wave was measured from baseline to trough, and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave.

Data between groups (other than for ERG) were compared using non-parametric Mann Whitney U-test. For ERG data, 2-way repeated measures ANOVA (P < 0.05) with Bonferroni post-tests were used to compare WT mice to ApoE−/− mice, and to determine the effect of EP80317 treatment on the different parameters of the ERG as the repeated factor and treatment group as the independent factor. All analysis and graphic representations were performed with Prism software (version 4.0c; GraphPad Software); values are represented as mean ± standard error of the mean (SEM). P values were calculated for a confidence interval of 95%; hence P values of less than 0.05 were considered significant.