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Caitlin Mac Nair, Cassandra Schlamp, Valery I Shestopalov, Robert W Nickells; Pannexin 1 expression in macroglia modulates their activation after optic nerve crush. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4948.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve crush (ONC) causes RGC death associated with activation of the retinal macroglia and microglia. Bax-deficient RGCs are resistant to crush and display an attenuated glial activation response, indicating a relationship between RGC death and glial activation. Previously we showed that the P2X7R agonist BzATP triggers macroglial activation, while the P2X7R antagonist oxATP suppresses it after ONC, suggesting that ATP may act as a signal from injured RGCs to trigger glial activation. One mechanism of ATP release after crush may be through ATP-permeable PANX1 hemichannels, which are enriched in RGCs.
The Panx1 gene was ablated from either RGCs, macroglia, or all cells in Panx1-loxP mice using cell-specific expression of CRE. These mice were then subjected to ONC, and macroglial activation (as a function of Gfap upregulation) was monitored by QPCR and immunofluorescence. Additionally, mouse retinas were analyzed for RGC survival after crush.
All genotypes exhibited a similar baseline expression of Gfap in naïve eyes. The comparison of relative Gfap levels between crushed and contralateral retinas showed an increase of Gfap in the crushed eye of WT mice and mice with PANX1-deficienct RGCs. Mice with Panx1-/- macroglia and total knockouts exhibited minimal change between experimental and contralateral eyes. Immunofluorescence data revealed Müller cell upregulation of GFAP in only the crushed eye across all genotypes. When absolute levels of Gfap were compared to naïve eyes, wild type contralateral control eyes exhibited a 2.5-fold increase compared to a 12-fold increase in the crushed eye. Conversely, the mice with macroglia deficient for PANX1 showed a 15-fold increase in both eyes, even though Müller cell activation only presented in experimental eyes. Crush induced similar rates of cell loss in WT and PANX1-deficient macroglial mice by 14 days, but by 21 days the latter displayed protection of the RGCs.
Our results show that ONC injury induces an increase in Gfap levels in the retina of both injured and contralateral eye. The contralateral effect is attributable to only astrocytes and is markedly increased in mice with PANX1-deficient macroglia.
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