Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry

Abstract

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.

Keywords: ceramide; docosahexaenoic acid; lipid characterization; lipid localization; very long chain polyunsaturated fatty acids.

Fig. 1.

Organization of the human retina. A: Diagram illustrating the distribution of rod and cone photoreceptors. The origin of the optic nerve in the retina, the optic disk, lacks photoreceptors, thus creating a blind spot within the visual field. Cone photoreceptors become concentrated around the optic axis in a central region, the macula, which measures 4.5–6.0 mm in diameter. The center-most area of the macula, the fovea, contains the highest concentration of cones, which produce high acuity bright light color vision. As cones diminish peripherally, rod photoreceptors accumulate and extend to the retina margin. The peripheral regions of the retina function in dim light and produce monochromatic vision. Features along the retinal surface are measured in millimeters (retinal eccentricity) from the fovea. The vertical axis denotes photoreceptor density. [Redrawn and modified with permission from (35)]. B: H&E-stained retinal section (82-year-old Caucasian female). The orientation of the section is nasal to temporal to reveal both the optic nerve and the cone-rich macular region, surrounded by the rod-rich periphery. Guide lines relate relevant features on the histological section to corresponding areas on the diagram above. The fovea is indicated at the center of the macula and several retinal layers are indicated [ganglion cell/nerve fiber layer (GC/NFL), INL, ONL, choriocapillaris (CC), sclera, and optic nerve].

Fig. 2.

Positive ion MALDI IMS of PC and SM lipids in human ocular tissue. A: H&E stain of ocular tissue section immediately adjacent to the section used for MALDI imaging. Total positive ion MALDI mass spectra of the accessory tissue (B), optic nerve (C), and retina (D) were obtained directly from the ocular section. Extracted positive ion MALDI images of the [M+Na]⁺ of TAG(52:2) (m/z 881.8) (E), SM(d18:1/18:0) (m/z 753.6) (F), PC(34:1) (m/z 782.6) (G), PC(36:1) (m/z 810.6) (H), PC(40:6) (m/z 856.6) (I), and the [M+H-H₂O]⁺ of Cer(d18:1/18:0) (m/z 548.5) (J). K: Merged positive ion MALDI image of Cer(d18:1/18:0) (blue), PC(40:6) (red), and PC(36:1) (green). GCL, ganglion cell layer.

Fig. 3.

Negative ion MALDI IMS of PI, PS, and PE lipids in human ocular tissue. A: H&E stain of ocular tissue section immediately adjacent to the section used for MALDI imaging. Total negative ion MALDI mass spectra of the accessory tissue (B), optic nerve (C), and retina (D) were obtained directly from the ocular section. Extracted negative ion MALDI images of the [M-H]⁻ of SM(d16:0/18:0)+DHAP (m/z 853.8) (E), PE(P-18:1/18:1) (m/z 726.5) (F), PI(18:0/20:4) (m/z 885.6) (G), PS(18:0/22:6) (m/z 834.5) (H), PE(P-18:0/22:6) (m/z 774.5) (I), and PE(18:0/22:6) (m/z 790.5) (J). K: Merged positive ion MALDI image of PE(P-18:0/22:6) (green), PE(18:0/22:6) (red), and PS(18:0/22:6) (blue). GCL, ganglion cell layer.

Fig. 4.

Positive ion MALDI IMS of VLC-PUFA-containing PC lipids in human ocular tissue. A: H&E stain of ocular tissue section immediately adjacent to the section used for MALDI imaging. B: Inset enlarged from (A) showing a region where the photoreceptor layer is detached from the inner retina (arrow). C: Total positive ion MALDI mass spectra of the retina directly from the ocular section. The solid black diamonds indicate background ions from the embedding agent. Extracted positive ion MALDI images of the [M+Na]⁺ of PC(40:6) (m/z 856.6) (D), PC(44:12) (m/z 900.6) (E), and PC(56:11) (m/z 1,070.7) (F). G: Merged positive ion MALDI image of PC(56:11) (green), PC(44:12) (red), and PC(40:6) (blue). Arrows denote the layer of photoreceptors that have been detached from the inner retina. GCL, ganglion cell layer;

Fig. 5.

Merged positive ion MALDI image of A2E (m/z 592.5) (RPE, green), PC(40:6)+Na (m/z 856.6) (photoreceptors, red), and PC(36:1)+Na (m/z 810.6) (inner retina, blue), illustrating stratification of lipid species into discrete identifiable retinal layers.