Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration
Figure 2
The effect of EDN2 loss on mutant PR survival in vivo and in retinal explants.
(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained Edn2+/+ and Edn2−/− retinas. (B,C) The loss of EDN2 in Tg(RHO P347S) retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in Pde6brd1/rd1 retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, Pde6brd1/rd1 and Tg(RHO P347S) retinas in mice expressing or lacking EDN2 (**p<0.005). (E) qRT−PCR assays of the Edn2 mRNA, normalized to Gapdh mRNA, in in vivo WT, WT explants and Pde6brd1/rd1 explants (n = 3;*p<0.05). Values were compared to the mean Edn2 mRNA levels in WT in vivo samples (arbitrarily given a value of 1). Edn2 transcripts were significantly increased in WT as well as Pde6brd1/rd1 explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured ex vivo from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in Pde6brd1/rd1; Edn2−/− retinal explants did not increase PR survival. Both Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.