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J. Du; Cell-Type Specificity and Temporal Expression of an Eph/ephrin Signaling System at the Optic Nerve Head (ONH) of DBA/2J Glaucomatous Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4355.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the spatial-temporal pattern and cell-type specificity of Eph/ephrin expression at the ONH of DBA/2J mice. To additionally document retinal ganglion cell (RGC) axon responses to Eph proteins following local application onto axons in vitro.
DBA/2J mice that spontaneously develop pigmentary glaucoma were examined from 3-11 mos. of age. Expression of Eph receptor tyrosine kinases and their ligands, the ephrins, was studied by in situ hybridization on optic nerve tissue sections. Cell of origin was identified by double labeling using Eph and ephrin specific fluorescence in situ hybridization together with fluorescence immunostaining using cell specific markers. To determine whether adult RGC axons can respond to Eph molecules, axons from C57/Bl6 mouse retinal explants were exposed to Fc tagged Eph protein delivered by micropipettes or bath applied. Changes in intra-axonal calcium were visualized using Fluo-4AM imaging. Axons were exposed to Fc protein alone in control experiments.
Specific upregulation of EphB2 and ephrinB2 mRNA was detected at the DBA/2J ONH at 9-11 mos., coinciding with onset of axon loss. EphB2 and ephrinB2 expression was not detected in younger animals, animals without axon loss, or control 10 mos. old C57/Bl6 mice. EphA4 was found at the ONH throughout life and no mRNA upregulation was detected corresponding to disease. EphB2 was expressed by specialized F4/80, MOMA2-positive macrophage/microglia that do not express the typical microglia marker Iba-1. EphrinB2 was expressed by Iba-1 positive microglia or GFAP positive astrocytes. EphA4 was detected in GFAP positive astrocytes. No T cells were found at the ONH of animals with axon loss. Adult RGC axons exhibited binding of Eph and ephrins and axons exposed to EphB2-Fc protein (1-15 uM) showed dose-dependent elevations in intra-axonal Ca2+. Not all axons responded to EphB2 and the onset of responses was heterogeneous from 30 secs. to 2 mins.
EphB and ephrinB expression at the DBA/2J ONH is correlated with axon loss. The ability of RGC axons to respond to EphB2 suggests that an EphB and ephrinB signaling system involving different macrophage/microglia subtypes and astrocytes might be at work at the glaucomatous ONH to locally affect RGC axon physiology. The importance of the ONH as the putative site of pathogenesis, together with the known roles of Eph/ephrins in axonal injury, suggests that the potential role of these molecules in glaucoma deserves further study.
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