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David T Stark, David M.G. Anderson, N. Heath Patterson, Jacky Man Kwong Kwong, Kevin Schey, Richard M. Caprioli, Joseph Caprioli; Discovery-based identification of lipid signaling molecules in evolving glial scars after optic nerve crush. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5624.
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© ARVO (1962-2015); The Authors (2016-present)
Adult central nervous system axons fail to regenerate after injury. Contributing factors include limited neuronal intrinsic growth capacity and an unfavorable glial environment. Lipid mediators have been identified as novel myelin-associated inhibitors of axon growth (MAIs) in adults and guide axons during development, but they have attracted less attention than inhibitory signaling proteins of the glial milieu. We developed a discovery-based approach to characterize the spatial distribution of lipid species in evolving glial scars in a rat optic nerve crush (ONC) model.
ONC was performed and eyes with attached optic nerves were collected at various post-crush intervals. Sections of peripapillary retina and nerve were analyzed with matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Images of histologically discriminant ions were overlaid with H&E and tentative molecular assignments made via the LIPIDMAPS database. Some assignments were confirmed with tandem mass spectrometry. Retinal ganglion cell (RGC) death was quantified with the specific marker Rbpms.
At post-crush day 7, 20% of RGCs survived (95% CI [11.9%, 28.4%]). Lipid sulfatide MAIs were cleared from the center of the crush site, but they remained abundant distally and proximally. In contrast, an ion consistent with the growth cone guidance cue arachidonoyl lysophosphatidyl inositol [LPI(20:4)] accumulated in the crush site. While cholesterol was cleared from the crush site, lysosome-specific bis(monoacylglycero)phosphates (BMPs) and cholesteryl esters accumulated markedly.
LPI(20:4) may promote RGC axon growth via the lysophosphatidyl inositol (LPI) receptor LPI1 (orphan GPR55). Accumulation of LPI(20:4) suggests that cellular elements within an evolving glial scar release attractive guidance cues while clearing MAIs. Free cholesterol is the most abundant lipid constituent of myelin on a molar basis. Accumulation of BMPs at the crush site likely reflects elevated endocytosis of debris and lysosomal biogenesis in activated microglia. The appearance of cholesteryl esters at the crush site suggests severe impairment of cholesterol homeostasis. This approach may be useful for identification of candidate pro-regeneration lipid signaling molecules.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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