Aβ(1-42) peptide and its inactive reverse control peptide Aβ(42-1) were prepared as described in our previous study (19). Briefly, 0.5 mg of lyophilized Aβ peptide (Sigma-Aldrich, Shanghai, China) was dissolved in 140 μl hexafluoroisopropanol (HFIP) and incubated for 20 min, followed by the addition of 900 μl distilled H₂O and a 20-min incubation in the HFIP/water mixture. Subsequently, the solvent was evaporated from the resulting supernatant under constant stirring at room temperature for 5 days. Oligomerization was verified by dot blot assay as previously described (19).

To elucidate the in vivo effects of Aβ peptide on the retina, C57BL/6 mice received a single unilateral subretinal injection of Aβ peptide (Fig. 1). Briefly, 5-month-old C57BL/6 mice were anesthetized by an intraperitoneal injection of 300 mg/kg chloral hydrate (Sangon Biotech, Shanghai, China). The pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical, Osaka, Japan); 0.5% proparacaine hydrochloride (Alcon, Shanghai, China) was applied for topical anesthesia. Following complete pupillary dilation, the anesthetized animals were placed under an Nikon SMZ-1 stereoscopic microscope, and the corneas were carefully punctured through the pars plana with a 30-gauge needle to produce a hole sufficiently large enought to insert a 33-gauge blunt needle (Hamilton Co., Reno, NV, USA). The blunt needle tip was inserted through the corneal puncture and advanced into the anterior chamber. A slight resistance to the movement of the needle indicated the penetration of the retina and entrance into the subretinal matrix. The mice were divided into 3 groups as follows: i) mice injected with oligomeric Aβ(1-42) [OAβ(1-42) (1 nmol/2 μl, n=24); ii) mice injected with OAβ(42-1) (1 nmol/2 μl, n=8), and iii) mice injected with 2 μl of phosphate-buffered saline (PBS) (calcium- and magnesium-free, n=8).

All animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All surgical interventions and animal care procedures were approved by the Ethics Committee of Tongji University, Shanghai, China.

Flash ERG (FERG) was recorded using an ERG recording system (APS-2000AER; Kanghua Ruiming Technology, Chongqing, China). The animals were allowed to adapt to the dark overnight and prepared for electroretinography and the scotopic electroretinogram was recorded. The animals were anesthetized with an intraperitoneal injection pf 300 mg/kg chloral hydrate (Sangon Biotech). Following pupillary dilation and the topical anesthesia of the cornea, gold loop electrodes were placed on the cornea with a drop of 2.5% hydroxypropyl methylcellulose. Subsequently, the reference electrode was placed into the mouth of the animal underneath the tongue, and a ground electrode was subcutaneously inserted into the midway of the tail. The ERG waveforms were then recorded in response to a flash at 1.125 cd^*s/m². The amplitude of the a-wave was measured from the baseline to the trough of the a-wave. The amplitude of the b-wave was measured from the trough of the a-wave to the peak of the b-wave. The amplitude of the c-wave was measured from the baseline to the peak of the c-wave.

On day 7 following surgery, the mice were terminally anesthetized and subjected to whole-body perfusion with 4% paraformaldehyde (PFA) in PBS and the whole eyes were then enucleated and fixed in 4% PFA overnight. Only sections including the optic nerve were used for histological and immunohistochemical analyses. Sections (5-μm-thick) of paraffin-embedded specimens were stained with hematoxylin and eosin (H&E). The slides were dehydrated and placed on a coverslip.

For immunohistochemical analysis, antibody staining was performed on sections of paraffin-embedded eyes. Antibodies to RPE65 (ab67042) and p16^INK4a (ab54210) were obtained from Abcam (Hong Kong, China) and used at a dilution of 1:100. The paraffin-embedded sections were heated to 60°C for 30 min, and then deparaffinized and rehydrated in graded alcohol series. The paraffin-embedded sections were incubated for 1 h in PBS containing 5% FCS to reduce non-specific binding, and then overnight at 4°C with the p16^INK4a and RPE65 primary antibodies. After washing, the sections were incubated in a solution of 1:200 of goat anti-mouse 594-conjugated secondary antibody or goat anti-rabbit 488-conjugated secondary antibody (Abcam). The sections were then washed, stained for 5 min with DAPI, and washed again in PBS. The sections were then mounted and examined under a fluorescence microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany).

For ultrastructural analysis, the enucleated eyes were dissected at the equator immediately and the posterior eye cups were fixed in 2.5% glutaraldehyde in phosphate buffer at 4°C overnight. The central 2×2 mm tissue temporal to the optic nerve was post-fixed with 2% osmium tetroxide and alcohol dehydrated and embedded in epoxy resin. The ultra-thin sections were stained with lead citrate and uranyl acetate, and examined under an electron microscope (CM120; Philips, Eindhoven, The Netherlands).

For the funduscopic examination of the mice, confocal scanning laser ophthalmoscopy (cSLO) was performed using a device available for human fundus imaging (Spectralis HRA; Heidelberg Engineering, Heidelberg, Germany). Fundus images were captured using a 790-nm diode laser for infrared (IR) and 488 nm for autofluorescence (AF).

RPE-choroid preparations for western blot analysis and RT-PCR were dissected from the freshly harvested eyes and preserved at −80°C until further processing. The RPE-choroid complex was sonicated in radio-immunoprecipitation assay (RIPA) buffer containing proteinase inhibitors for 15 min. Protein content was determined by a bicinchoninic acid assay (Pierce, Rockford, IL, USA). Aliquots (50 μg of total protein) of each sample from the RPE-choroid were loaded per lane onto 10% SDS-PAGE gels for electrophoresis and then transferred onto PVDF membranes. The membranes were blocked and incubated with the primary antibody, rabbit polyclonal antibodies against p16^INK4a (1:500; Abcam) and rabbit polyclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase [GAPDH (Cat. no. ab37168), 1:1,000] overnight. The membranes were then washed and incubated with horseradish peroxidase-coupled secondary antibodies for 2 h. The blots were washed and developed with chemiluminescence reagent. The membranes were exposed to ImageQuant LAS 4000 imaging, and densitometric analysis was performed using Photoshop CS4.0 software.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was then reverse transcribed into cDNA with RT Master Mix (Takara Biotechnology, Dalian, China). The expression levels of IL-8 and IL-6 were measured using quantitative PCR mix (Takara Biotechnology). The primers used for PC were as follows: IL-6 sense, 5′-TTCCATCCAGTTGCCTTCTT-3′ and antisense, 5′-CATTTCCACGATTTCCCAGA-3′; IL-8 sense, 5′-CCTT GGTCTTCCTGCTTGA-3′ and antisense, 5′-ATCGTTG TTCCCATCCACAT-3′. Real-time PCR was performed in a ABI7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH (sense, 5′-AGCAGTCCCGTACA CTGGCAAAC-3′ and antisense), 5′-TCTGTGGTGATGT AAATGTCCTCT-3′ was used for normalization and the relative gene expression was expressed as the relative ‘fold change’ calculated using the ΔΔCt method.

To elucidate the in vivo effects of Aβ peptide on visual function, C57BL/6 mice received a subretinal injection of Aβ peptide, and visual function was evaluated by ERG on day 7 post-injection. Photoreceptors are the source of the negative-going a-wave (21,22). Rod bipolar cells are the source of the b-wave in the dark-adapted retina (23–25). RPE cells are the source of the positive-going c-wave (26). Representative waveforms of the maximal ERG scotopic response evoked by a single flash at 1.125 cd^*s/m² are illustrated in Fig. 2A. No significant differences were observed between the Aβ(42-1)-injected mice and the PBS control mice. However, a moderate but significant decrease in a-, b- and c-wave amplitude was detected in the Aβ(1-42)-injected mice (Fig. 2A and B). These results indicated that the subretinal injection of Aβ(1-42) induced an impairment of visual function in the animals.

To demonstrate the deleterious effects of Aβ(1-42) on the retina, we compared its effects on retinal histology and RPE structure with those of PBS and the inactive reverse peptide Aβ(42-1). The H&E-stained sections of RPE/neural retina were examined under a microscope (Fig. 3A). The mice injected with PBS or the reverse peptide showed a normal appearance of retinal sections (Fig. 3A, left and middle panels). By contrast, degenerative alterations, including extensive vacuolation (V symbol), hyperpigmentation (arrow), hypopigmentation (asterisk) and thickness of the BrM, were observed in the outer retina of the Aβ(1-42)-injected mice (Fig. 3A, right panel).

Subsequently, the RPE structures were examined by TEM. The PBS- and Aβ(42-1)-injected mice showed normal structures of the RPE, well-developed RPE infoldings and a consistent thickness of the BrM (Fig. 3B, left and middle panels). In the Aβ(1-42)-injected mice, the RPE cells were highly vacuolated with membranous debris, the normal basal infoldings were replaced by amorphous material deposits, the number of pigment granules decreased and the BrM was thickened (Fig. 3B, right panel). Taken together, the results from histopathological analysis and TEM demonstrated that the Aβ(1-42)-injected mice developed some degenerative alterations on the neural retina layer, the RPE and BrM, which are similar to some of the morphological characteristics of patients with AMD (27).

We then investigated whether the subretinal injection of Aβ(1-42) induces RPE cell senescence. cSLO, a clinical imaging method, is used to examine the fundus autofluorescence (FAF) pattern as a means of assessing the health of the RPE in AMD. Therefore, funduscopic examination was used to assess RPE alterations/senescence in vivo. IR-cSLO revealed that the Aβ(1-42)-injected mice developed an increase in the number of drusen-like lesions characterized by irregularly shaped bright regions (Fig. 4A, right panel), and the increased granular autofluorescent spots were observed by AF-cSLO in almost the same areas (Fig. 4B, right panel). By contrast, no significant distribution of fluorophores was observed in the ocular fundus of the PBS- or Aβ(42-1)-injected mice (Fig. 4A and B). The analysis of cell senescence on RPE/neural retinal sections by p16^INK4a staining confirmed that Aβ(1-42) induced RPE cell senescence in the mice on day 7 post-injection (Fig. 4C, right panel). However, in the retinal sections of the PBS- or Aβ(42-1)-injected mice, no p16^INK4a-positive RPE cells were detected (Fig. 4C, left and middle panels). Subsequently, the expression level of p16^INK4a in RPE cells was determined by western blot analysis; the injection of Aβ(1-42) significantly increased p16^INK4a expression by (3.12±0.46)-fold compared with the injection of the reverse peptide or PBS (Fig. 4D). Taken together, these data strongly suggest that Aβ(1-42) significantly induces RPE cell senescence in the retinas of mice.

Hallmarks of inflammation, including elevated IL-6 and IL-8 expression, are significantly associated with the severity of AMD. Recent evidence indicates that cellular senescence is accompanied by a marked increase in the secretion of 40–80 factors, which has been termed the SASP (42). The key SASP factors, such as IL-6 and IL-8, are known to stimulate chronic inflammation in age-related diseases (42). Thus, to determine whether the subretinal injection of Aβ alters SASP gene expression in RPE cells, the RPE-choroid layer was isolated and IL-6 and IL-8 mRNA expression was examined by RT-PCR. Aβ(1-42) injection significantly increased the mRNA expression of IL-6 (6.8±0.8-fold, P<0.05) and IL-8 (3.7±1.2-fold, P<0.05), compared with PBS-injected group (Fig. 5), whereas the injection of Aβ(42-1) did not induce any significant changes in the mRNA expression of IL-6 and IL-8, compared with the PBS-injected group (Fig. 5).