Abstract
Purpose :
Retinal ischemia is a common cause of visual impairment and blindness. Ischemia leads to morphological and functional alterations in retinal astrocytes, which in turn is known to have profound effects on retinal ganglion cell (RGC) viability. Connexin43 (Cx43), a protein that forms gap junction (GJ) channels, is abundantly expressed in astrocytes. Here, we examined how changes in coupling between astrocytes impact RGC survival in acute ischemic injury.
Methods :
Conditional knockout (KO) of Cx43 from astrocytes was established using the Cre-loxP recombination system by means of a GFAP promoter to drive Cre expression. Cx43 channels were also pharmacologically inhibited by injecting intravitreally SBO15 (20 μM). Ischemia-Reperfusion (I/R) injury in the retina was induced in 1-2 month old wild-type (WT) and Cx43 KO mice by raising the intraocular pressure (IOP) of the left eye to 75-80 mmHg for 60 min followed by reperfusion. The untreated contralateral eye was used as a control. Eyes were processed after 1, 3 or 7 days post-injury (dpi). Immunohistochemistry was performed to determine RGC survival, using the RGC-specific marker Brn3a and to examine connexin expression. To assess GJ-mediated coupling, individual astrocytes were loaded with the GJ-permeant dye Neurobiotin and dye spread to adjacent cells was counted using ImageJ software. Student’s t-test was used for statistical analyses.
Results :
I/R at 7 dpi resulted in a 75% loss of RGCs and a significant increase in astrocyte reactivity. Genetic ablation of astrocytic Cx43 significantly (p < 0.05) increased the numbers of Brn3a-positive cells (11.2 ± 3.3 cells in WT I/R; n=11 versus 23.6 ± 3.5 cells in KO I/R per 0.1 mm2 of retinal area; n=7). A similar protective effect on RGCs was observed after pharmacological inhibition of Cx43 channels with SBO15. Expression of Cx43 in astrocytes of WT I/R retina increased by 2-fold compared to control. Similarly, astrocytic GJ coupling at 1 dpi (108 ± 26.5 coupled cells; n=3) was 2-fold higher compared to controls (49.6 ± 8.87 coupled cells; n=3), and it remained high even after 7 dpi.
Conclusions :
Our results indicate that Cx43-mediated astrocytic coupling is harmful to RGCs during acute ischemia. Inhibition of astrocytic Cx43 channels may represent a potentially useful strategy to promote ganglion cell survival during ischemic conditions.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.