A2E and lipofuscin distributions in macaque retinal pigment epithelium are similar to human

Abstract

The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research.

Figure 1. Color AF images and associated spectra of macaque RPE

AF images of whole RPE eyecups from a 7 year-old (a) and an 18 year-old (b) macaque (λ_exc = 430-480 nm; λ_em > 490 nm). Bright green autofluorescent spots are dissection artifacts. Microscopic imaging (10x len, λ_exc = 450-490 nm; λ_em > 510 nm) of an 18 year-old RPE and lipofuscin in the center (c), mid-periphery (d), and far-periphery (e). Approximate regions are indicated in the schematics of the whole RPE (f). Comparison of overall average (± SD) fluorescence intensities from old (n=4) and young (n=3) RPE samples from central, mid-peripheral, and far-peripheral regions across the RPE (f). Spectra from 7 year-old (g) and 18 year-old (h) whole RPE tissues indicating changes in the spectral composition of lipofuscin at the regions across the eyecup denoted in the insets. The location of the optic nerve head is denoted by an asterisk on each sample. Scale Bars: a, b = 5 mm; c, d, e = 60 μm.

Figure 2. Lipofuscin AF and MALDI images of A2E

a) The distribution of A2E across the RPE in a 7 year-old macaque. b) MALDI image of singly oxidized A2E (m/z 608) across the macaque RPE in the same tissue. c) Lipofuscin AF image collected utilizing an IVIS 200 bioluminescence imaging system (λ_exc = 450 – 490 nm, λ_em = 575 – 650 nm) shown on the indicated false color scale. d) Overlay of lipofuscin AF (green) and the molecular image of A2E (red) in the same tissue. All Scale bars = 5 mm. The optic nerve head is denoted by an asterisk.

Figure 3. Mass spectral data in macaque RPE

a) Average mass spectrum (MALDI profile) of the 7 year-old macaque across the entire RPE eyecup in the m/z 550-1000 range. The peaks for A2E and single oxidized A2E (A2E+ox.) are indicated. All peaks were normalized to the A2E peak. Mass spectra from central (b) and peripheral (c) regions indicated on the tissue outline in the m/z range of 550-1000. Spectra are shown on the same scale.

Figure 4. The ratio of oxidized A2E to A2E

The values represent data from average mass spectra of young (< 10 years, n=3) and mature (> 10 years, n=4) macaque tissues.

Figure 5. The fragmentation of the A2E molecule

Structure of A2E with expected sites of fragmentation. The sites of the fragments recovered in the tissue analysis are highlighted with masses listed.