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Miduturu Srinivas, Abduqodir Toychiev, Stewart A Bloomfield, NEFELI SLAVI; Astrocytic Cx43 in Retinal Hypoxia: Gap junctions Vs Hemichannels. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3453.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal astrocytes influence the abnormal growth of blood vessels in ischemic retinopathies. In the oxygen induced retinopathy (OIR) mouse model, astrocytes degenerate shortly after exposure to hypoxia (~48 hours), an event associated with pathologic angiogenesis. A prominent feature of astrocytes is the abundant expression of connexin 43 (Cx43). In a companion abstract (Slavi et al., ARVO 2017) we show that deletion of Cx43 increases astrocyte density and promotes reparative angiogenesis in OIR. Cx43 forms gap junction channels (GJs) and hemichannels (HCs). Both channel configurations have been implicated in the propagation of secondary cell death in pathologic conditions. Here, we dissect the contribution of GJs and HCs in hypoxia-induced astrocyte degeneration.
Mouse litters were exposed to 75% oxygen from postnatal day (p) 7 to p12 and then returned to room air. The strength of GJ coupling and activation of HCs was measured at different points during relative hypoxia in both the central, avascular retina and the peripheral, vascularized retina. For GJ channel function, a single astrocyte was loaded with neurobiotin (5 mg/ml) and the extent of tracer spread was measured. For HC activity, uptake of ethidium bromide (EtBr, 4 µM) into astrocytes was measured in the presence or absence of Gap19 peptide, which is known to block HCs but not GJs.
In the central retina, the number of tracer coupled astrocytes was low at 0 hours (76.6±23.3 cells; n=5 mice) but markedly increased after 6 hours in relative hypoxia (193±20 cells; n=8). Coupling remained high after 24 hours in hypoxia (137±30 cells; n=6 mice). The observed elevation of coupling was abolished after inhibition of casein kinase 1δ (CK1 δ) (n=3), which phosphorylates Cx43 and promotes assembly of GJs into plaques. No difference in the number of coupled cells in the peripheral retina was observed between these time points (p>0.05; n=3 mice per group). Uptake of EtBr into astrocytes was found in both the central and peripheral retina at 0, 6 and 24 hours of hypoxia (80 cells/mm2; n=3, p>0.05) and was not blocked by the Gap 19 peptide.
GJ coupling between astrocytes is elevated early in the hypoxia phase of OIR due to phosphorylation of Cx43 by CK1δ. Increases in GJ channel function and not HC activity appear to underlie the harmful effects of Cx43 in OIR.
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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