Fig. 7: Treatment with N-Acetyl-L-cysteine prevents elevated intraocular pressure-induced retinal injury by inhibiting oxidative stress in Pitx2^KI mutant retinas.

A N-Acetyl-l-cysteine in 40% PBS was injected intraperitoneally (25 mg/kg/day) into Pitx2^WT and Pitx2^KI mutant mice for 5 weeks (from 5-week to 10-weeks of age) as the treated groups. Equivalent volumes of 40% PBS were delivered intraperitoneally to the Pitx2^WT and Pitx2^KI mice as the vehicle-treated control groups. All animals were sacrificed at 10 weeks of age to conduct analyses. B IOP analysis in Pitx2^WT and Pitx2^KI mice subjected to vehicle and N-Acetyl-l-cysteine treatment. Individual IOP values are shown by dots in the histograms (n = 10 in each group). C ROS activity was measured by DHE staining of retinal sections from Pitx2^WT and Pitx2^KI mutant mice subjected to vehicle or N-acetyl-l-cysteine treatment. The red staining is positive for ROS content. Nuclei were counter-stained with DAPI (blue). Scale bars: 50 μm. D Quantification of the fluorescence intensity of DHE staining showed an obvious decrease in ROS formation in retinas from treated-Pitx2^KI mutant mice (n = 10 in each group). E Representative images of H&E staining of retinal section from Pitx2^WT and Pitx2^KI mice subjected to vehicle or N-Acetyl-l-cysteine treatment. Scale bars: 25 μm. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. F Quantification of the nuclei number in GCL shows that the Pitx2^KI mutants have significantly decreased number of nuclei in the GCL (n = 6 in each group). G Immunofluorescent enface view of retinal flatmounts from vehicle- or N-Acetyl-l-cysteine-treated Pitx2^WT and Pitx2^KI mice following labeling with anti-Brn3a to detect RGC. Scale bars: 25 μm. H Quantification of the number of RGC showed overall decreased number of RGL Brn3a positve cells. However, comparison of Pitx2^KI vehicle group, Pitx2^KI treated group showed significantly more RGC remaining in N-Acetyl-l-cysteine-treated Pitx2^KI mice (n = 12 in each group). I Quantitation indicates that an obvious increase in ∆ RGC number in Pitx2^KI retinas (n = 10 in each group). ∆ RGC number = RGC number (Treated group)–RGC number (vehicle group). A positive number indicates an increase in RGC number after N-Acetyl-l-cysteine treatment. *P < 0.05; **P < 0.01; ***P < 0.001. #, not significant. The data are presented as mean ± SEM.