Abstract
In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Müller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Müller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Müller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Müller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Müller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value ≤ 0.05) between the areas containing Müller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Müller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Müller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.
Highlights
Neurons in the retina and it is their axons that form the optic nerve that carries the electrical signals from the retina to the visual centers in the brain[1,2]
In order to identify a lipid signature for Retinal Ganglion Cells (RGCs) and Müller cells, a strategy was developed that integrates in situ and in vitro experiments within a lipidomics workflow (Fig. 1)
Prior to performing the lipidomic analysis, the purity and quality of the cultures established was verified by immunocytochemistry using antibodies that recognized RGCs and Müller glia
Summary
Neurons in the retina and it is their axons that form the optic nerve that carries the electrical signals from the retina to the visual centers in the brain[1,2]. The spatial distribution and identity of lipid and retinoid metabolites are characteristic of specific retinal cell layers[31,33,35,36], as seen in Abca4–/– mice (a model of Stargardt disease) when analyzed using high spatial resolution MALDI-IMS These studies mainly focused on the lipids in the photoreceptor cell layers (the Outer segment and ONL) and the retinal pigment epithelium (RPE)[36]. Despite their importance as causative and diagnostic molecules for retinal disease, there is little known about the lipid profiles in the normal retina and the differences in the lipid composition of the cells that make up the retina To address this issue, we set out here to identify the lipid signature of Müller glia and RGCs in sections of the porcine retina, and in primary cell cultures, using MALDI-IMS. Our model of glaucoma in pig displays a same pattern of neuron degeneration as that seen in h umans[41]
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