Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Dec 2;31(12):2426-2436.
doi: 10.1021/jasms.0c00119. Epub 2020 Jul 24.

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High-Resolution Imaging Mass Spectrometry

David M G Anderson  1 , Jeffrey D Messinger  2 , Nathan H Patterson  1 , Emilio S Rivera  1 , Ankita Kotnala  1   2 , Jeffrey M Spraggins  1 , Richard M Caprioli  1 , Christine A Curcio  2 , Kevin L Schey  1
Affiliations

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High-Resolution Imaging Mass Spectrometry

David M G Anderson et al. J Am Soc Mass Spectrom. .

Abstract

The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases. The retina's exquisite architecture comprises numerous cell types that are aligned horizontally, yielding structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. MALDI imaging mass spectrometry (IMS) is now capable of uniting low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach fortified with accurate multi-image registration was used to localize lipids in human retina tissue at laminar, cellular, and subcellular levels. Multimodal imaging results indicate differences in distributions and abundances of lipid species across and within single cell types. Of note are distinct localizations of signals within specific layers of the macula. For example, phosphatidylethanolamine and phosphatidylinositol lipids were localized to central RPE cells, whereas specific plasmalogen lipids were localized to cells of the perifoveal RPE and Henle fiber layer. Subcellular compartments of photoreceptors were distinguished by PE(20:0_22:5) in the outer nuclear layer, PE(18:0_22:6) in outer and inner segments, and cardiolipin CL(70:5) in the mitochondria-rich inner segments. Several lipids, differing by a single double bond, have markedly different distributions between the central fovea and the ganglion cell and inner nuclear layers. A lipid atlas, initiated in this study, can serve as a reference database for future examination of diseased tissues.

Keywords: age-related macular degeneration; lipid atlas; macula; photoreceptors; retinal pigment epithelium.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Workflow of sample preparation and multimodal imaging. (1) The cornea is removed before fixation with 4% paraformaldehyde for 48 h at 4 °C. The iris and lens are removed before imaging. (2) The eye lacking cornea, iris, and lens mounted in jig to obtain OCT image. (3) The eye is placed in a dissection guide as described in the text to capture a belt with the optic nerve, macula, and temporal periphery. The belt is embedded in 2.6% CMC in a cryomold. The caps are discarded. (4) Cryosections at 12–14 μm throughout the entire eye are thaw-mounted on either glass or ITO slides. (5) ITO slides are imaged for autofluorescence (AF) before being coated with matrix via sublimation for acquisition of IMS data. (6) Highly accurate data registration is performed from IMS, postacquisition AF, pre AF, and H&E stained tissue.
Figure 2.
Figure 2.
Six MALDI IMS signals with distinct laminar localizations in human macula from an 83-year-old female donor. (A) Schematic diagram of retina, Bruch’s membrane, and choriocapillaris, indicating cell types in their characteristic layers. The outer nuclear layer (ONL) contains cell bodies of cones (C) and rods (R) and their inner fibers, all interleaved with Müller glia (M). Photoreceptor inner segments (IS) contain many mitochondria. Photoreceptor outer segments (OS) contain phototransduction-related proteins in membrane disks. The RPE apical processes are specialized for metabolic exchange with photoreceptors and retinoid processing. Layers: ILM, inner limiting membrane; NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, HFL Henle fiber layer, ELM, external limiting membrane; RPE retinal pigment epithelium, ChC, choriocapillaris; BrM, Bruch’s membrane; cell types, A, astrocyte; Am, amacrine; B, bipolar; E, endothelium; G, ganglion cell; H, horizontal cell; P, pericyte. (B) OCT B-scan of a donor eye macula prior to sample preparation, R and C indicating retina and choroid, respectively. The retina is at the top, the choroid (vasculature) is on the bottom, and the hyperreflective interface is the retinal pigment epithelium (RPE) layer. (C) Zoomed image of tissue section stained with H&E to indicate cell layers at the level shown in (A). NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, HFL Henle fiber layer, ONL outer nuclear layer, IS-OS photoreceptor inner and outer segments, RPE retinal pigment epithelium, Ch choroid. (D) Panoramic H&E stained tissue post-IMS data acquisition showing macula and extended stretch of temporal peripheral retina. (E) Overlay of MALDI IMS images of selected lipid signals localizing to distinct retinal layers and RPE as indicated. Colors defined in panel F. (F) Individual ion signals overlaid on top of H&E stained tissue image.
Figure 3.
Figure 3.
MALDI IMS signals consistent with localization to photoreceptor and RPE compartments. (A) Schematic diagram of outer retina and Bruch’s membrane, excerpted from Figure 2A. Blue, pink, yellow, and green bands indicate layers formed by highly compartmentalized and vertically aligned photoreceptors and RPE cells in panels B and C. See Figure 2 for explanation of cellular and subcellular content of each layer. Layers: OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; RPE, retinal pigment epithelium; BrM, Bruch’s membrane; R, Rod; C, cone photoreceptors. (B–F) Images and H&E stained tissue images overlaid in peripheral retina displaying signals from multiple lipid classes that localize to subcellular compartments of the photoreceptor cells. (B) Overlay showing four separate signals defined in panels C–F. (C) Localized to ONL. (D) Localized to photoreceptor inner and outer segments. (E) Localized to mitochondria-rich photoreceptor inner segments. (F) Localized to RPE apical processes.
Figure 4.
Figure 4.
MALDI IMS shows complementarity of signals in retina and RPE. (A) Signals localized to the foveal center and extending into the macular NFL, IPL, HFL also localize to the peripheral RPE. (B) Signals localized to RPE underlying the macula, not confined to the fovea.
Figure 5.
Figure 5.
Lipid species varying in only one double bond can exhibit different MALDI IMS distributions. (A) H&E stained tissue image of a normal macula, with the foveal pit in the center. (B) Zoomed view of panel A showing retinal layers. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; HFL, Henle fiber layer; ONL, outer nuclear layer; PR/IS, photoreceptor inner segments; PR/OS, photoreceptor outer segments; RPE, retinal pigment epithelium; ChC, choriocapillaris. (C) m/z 885.5499, PI(18:0_20:4) distributes in the foveal center, GCL, INL, and HFL. (D) m/z 887.558, PI(18:0_20:3), with one double bond less than the species in panel C, localizes to the HFL of the foveal center and the inner INL. (E) m/z 913.587, PI(18:0_22:4) localizes to the foveal center, GCL, INL, and HFL, like the species in panel A, but with a lesser lateral extent of INL. (F) m/z 915.601, PI(18:0_22:3), with one double bond less than the species in panel E, localizes to the HFL of the foveal center.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Masland RH The Neuronal Organization of the Retina. Neuron 2012, 76 (2), 266–280. - PMC - PubMed
    1. Rossi EA; Roorda A The relationship between visual resolution and cone spacing in the human fovea. Nat. Neurosci. 2010, 13 (2), 156–7. - PMC - PubMed
    1. Liu A; Chang J; Lin Y; Shen Z; Bernstein PS Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration. J. Lipid Res. 2010, 51 (11), 3217–3229. - PMC - PubMed
    1. Agbaga M-P; Merriman DK; Brush RS; Lydic TA; Conley SM; Naash MI; Jackson S; Woods AS; Reid GE; Busik JV; Anderson RE Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors. J. Lipid Res. 2018, 59 (9), 1586–1596. - PMC - PubMed
    1. Harkewicz R; Du H; Tong Z; Alkuraya H; Bedell M; Sun W; Wang X; Hsu YH; Esteve-Rudd J; Hughes G; Su Z; Zhang M; Lopes VS; Molday RS; Williams DS; Dennis EA; Zhang K Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function. J. Biol. Chem. 2012, 287 (14), 11469–80. - PMC - PubMed

MeSH terms