Figure 4
Necroptosis blockade attenuated retinal inflammation and rescued degeneration in rd1 mice. Nec-1 was injected intravitreally at P9 in rd1 mice and retina were collected and analyzed at P14. (a) Western blotting revealed elevated levels of necroptotic mediators (RIP1 and RIP3) in the rd1 retinae comparing with C57 ones. Reduced expression of RIP1 and RIP3 was observed in the rd1 after Nec-1 treatment. (b-d) Nec-1 significantly downregulated expression of TNF-α and CCL2 at both RNA and protein levels. (e) Retinal neural function was measured by ERG at P14. ERG was performed using single-flash recordings at two light intensities of 3.0 and 10.0 scotopic cd s/m2, respectively. C57 mice were used as normal control. No response was detected in PBS-treated rd1 mice at P14. After Nec-1 injection, significant improvements of both a-wave and b-wave amplitudes were observed. n=8 eyes per group. (f) Histologic alterations of the retinae were evaluated by H&E staining on retinal paraffin sections. Nec-1-treated rd1 mice exhibited an increase in the thickness of the inner and outer nuclear layers in comparison with the PBS-treated ones. IPL, inner plexiform layer; OPL, outer plexiform layer; ONL:, outer nuclear layer. n=6 eyes and six microscope fields per eye (g) Confocal microscopy showed that the number of TUNEL+ and cleaved-caspase-3+ cells in the outer nuclear layer was decreased after Nec-1 administration compared with PBS treatment, indicating Nec-1 prevented the photoreceptors from apoptosis. For each panel, images below showed higher magnification of the selected areas of the above images respectively. n=6 eyes and six microscope fields per eye for statistical analysis. Scale bar: 25 μm. *P<0.05; **P<0.01; ***P<0.001
