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. Author manuscript; available in PMC: 2023 Aug 17.
Published in final edited form as: Neuron. 2022 Jun 28;110(16):2607–2624.e8. doi: 10.1016/j.neuron.2022.06.003

Figure 7. CRISPR ablation of the identified TFs protects RGCs in a glaucoma model.

Figure 7.

(A) Schematic illustration of the viscobeads occlusion experimental glaucoma model. Concentrated viscobeads were injected into the mouse anterior chamber to block aqueous drainage.

(B) A representative photograph of viscobeads accumulated at mouse iridocorneal angle 5 min after injection. Arrows show the white viscobeads were restricted at the iridocorneal angle.

(C) Viscobeads distribution in the mouse anterior chamber following intracameral injection. Left: A representative transmission electron microscopy (TEM) image of an intact mouse iridocorneal angle. Right: A representative fluorescence image showing rhodamine B labelled viscobeads accumulation at mouse iridocorneal angle. Scale bar, 10 μm (left) and 100 μm (right).

(D) Intraocular pressure (IOP) in the mice before and after the injection of viscobeads (n=10) or saline (n=5) and the naïve group (n=5). Data are shown as mean ± SD. (E, F) Representative wholemount retina confocal images from an intact mouse (E) and a mouse after 8 weeks from viscobeads injection (F). RBMPS antibody was used for the immunohistochemical staining of RGCs. Scale bar for the left panel for (F, G), 2 mm. Scale bar for the left panel for (F, G), 100 μm.

(G) Quantification of RGC survival 8 weeks after the viscobeads (n=31) or saline (n=5) injection and the naïve group (n=5). Data are shown as mean ± SD.

(H-K) Knockout of individual TFs protects RGCs in the glaucoma mice. (H) Schematic illustration. AAV2 vectors encoding sgRNAs were injected intravitreally at 2 weeks before beads injection. (I) IOP elevation in different group of mice. (J) Representative images of retinal sections showing RGC survival with sgRNA injections targeting indicated TFs. Scale bar, 20 μm. (K) Quantification of RGC survival after 8 weeks of viscobeads injection, including intact condition (n=7), control (non-targeting) sgRNA injection (n=10), ATF3 sgRNA injection (n=13), ATF4 sgRNA injection (n=9), C/EBPγ sgRNA injection (n=9), CHOP sgRNA injection (n=10), ATF3+ATF4 sgRNA injection (n=10), ATF3+C/EBPγ sgRNA injection (n=12). Data are shown as mean ± s.e.m.. **p<0.01, ***p<0.001, calculated by one-way ANOVA.