Chronic mild and acute severe glaucomatous neurodegeneration derived from silicone oil-induced ocular hypertension

Abstract

Recently, we established silicone oil-induced ocular hypertension (SOHU) mouse model with significant glaucomatous neurodegeneration. Here we characterize two additional variations of this model that simulate two distinct glaucoma types. The first is a chronic model produced by high frequency (HF) pupillary dilation after SO-induced pupillary block, which shows sustained moderate IOP elevation and corresponding slow, mild glaucomatous neurodegeneration. We also demonstrate that although SO removal quickly returns IOP to normal, the glaucomatous neurodegeneration continues to advance to a similar degree as in the HF group without SO removal. The second, an acute model created by no pupillary dilation (ND), shows a greatly elevated IOP and severe inner retina degeneration at an early time point. Therefore, by a straightforward dilation scheme, we extend our original SOHU model to recapitulate phenotypes of two major glaucoma forms, which will be invaluable for selecting neuroprotectants and elucidating their molecular mechanisms.

Figure 1

Low to high IOP elevations achieved by high to low frequencies of pupillary dilation after SO-induced ocular hypertension. (A) Schedule of dilation to relieve pupillary blocking after SO intracameral injection. HF: high frequency, twice a week; LF: low frequency, four times in eight weeks; ND: no dilation. wpi: weeks post SO injection. (B) Longitudinal IOP measurements of experimental eyes and contralateral control (CL) eyes at different time points after SO injection. ND groups at 3, 5 and 8wpi are independent groups that are sacrificed immediately after IOP measurement. (C,D,E) Comparisons of average IOP of the three groups at 3, 5 and 8wpi. Data are presented as means ± s.e.m, 3wpi: HF: n = 26, LF: n = 28, ND: n = 24; 5wpi: HF: n = 26, LF: n = 27, ND: n = 24; 8wpi: HF: n = 21, LF: n = 30, ND = 30. **: p < 0.01, ****: p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test.

Figure 2

Mild to severe glaucomatous RGC soma and axon degeneration in HF, LF and ND eyes at 5wpi. (A) Representative OCT images of mouse retinal area surrounding the ON head at 5wpi. GCC indicated by double end arrows. (B) Quantification of GCC thickness, represented as percentage of GCC thickness in the SO eyes, compared to the CL eyes. Control (CON): n = 16; HF-5w: n = 25; LF-5w: n = 27; ND-5w: n = 22; ND-3w: n = 24, * P < 0.05, ***: p < 0.001, ****: p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (C) Upper panel, confocal images of wholemount retinas showing surviving RBPMS-positive (red) RGCs of naïve, HF, LF and ND eyes at 5wpi. Scale bar, 100 µm. Middle panel, confocal images of a portion of the peripheral retina showing surviving RBPMS-positive (red) RGCs in the corresponding groups. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD in the corresponding groups. Scale bar, 10 µm. (D,E) Quantification of surviving RGCs in the peripheral retina and surviving axons in ON of the corresponding groups at 5wpi, represented as percentage of SO eyes compared to CL eyes. (F) Quantification of P1-N2 amplitude, represented as percentage of P1-N2 amplitude in the SO eyes, compared to CL eyes. Data are presented as means ± s.e.m, Control: n = 12; HF: n = 22; LF: n = 16; ND-5w: n = 23; * P < 0.05, ** P < 0.01, ***: P < 0.001, ****: P < 0.0001; one-way ANOVA with Tukey’s multiple comparison test.

Figure 3

Mild to severe glaucomatous RGC soma and axon degeneration in HF, LF and ND eyes at 8wpi. (A) Representative OCT images of mouse retinal area surrounding the ON head. GCC: ganglion cell complex, including RNFL, GCL and IPL layers; indicated by double end arrows. (B) Quantification of GCC thickness, represented as percentage of GCC thickness in the SO eyes, compared to the CL eyes. Control (CON): n = 17; HF: n = 30; LF: n = 30; ND: n = 14. **: p < 0.01, ****: p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (C) Upper panel, confocal images of wholemount retinas showing surviving RBPMS-positive (red) RGCs of naïve, HF, LF and ND eyes at 8wpi. Scale bar, 100 µm. Middle panel, confocal images of a portion of the peripheral retina showing surviving RBPMS-positive (red) RGCs in the corresponding groups. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD in the corresponding groups. Scale bar, 10 µm. (D,E) Quantification of surviving RGCs in the peripheral retina and surviving axons in ON of the corresponding groups at 8wpi, represented as percentage of SO eyes compared to CL eyes. (F) Quantification of P1-N2 amplitude, represented as percentage of P1-N2 amplitude in the SO eyes, compared to CL eyes. Data are presented as means ± s.e.m. Control (CON): n = 12; HF: n = 26; LF: n = 30; ND: n = 27, * p < 0.05, ** p < 0.01, ***: p < 0.001, ****: p < 0.0001; one-way ANOVA with Tukey’s multiple comparison test.

Figure 4

Degeneration of the inner retina: severe in ND, but much milder in HF and LF groups. (A,D) Representative light microscope images of semi-thin transverse sections of retina stained with toluidine blue in the corresponding groups at 5 and 8wpi. Scale bar, 50 µm. (B,E) Quantification of inner retina thickness at 5 and 8wpi, represented as percentage of SO eyes compared to CL eyes. (C,F) Quantification of outer retina thickness at 5 and 8wpi, represented as percentage of SO eyes compared to CL eyes. Data are presented as means ± s.e.m. 5wpi: Control (CON): n = 8; HF: n = 10; LF: n = 10; ND: n = 10; 8wpi: Control (CON): n = 8; HF: n = 10; LF: n = 10; ND: n = 6, * p < 0.05, ** p < 0.01; one-way ANOVA with Tukey’s multiple comparison test.

Figure 5

Decreased blood supply in the inner retina: severe in ND, much milder in HF and LF groups. Time series of retinal fundus fluorescein angiography (FFA) images at different time points after fluorescein injection show significantly delayed retinal vessel filling and marked capillary non-perfusion in the ND group, whereas the HF group is similar to the control (CON) group, at 5wpi. Three animals of each group were imaged.

Figure 6

SO removal reverses IOP elevation but not glaucomatous neurodegeneration in HF group. (A) Longitudinal IOP measurements of HF SOHU eyes before and after SO removal and CL eyes at different time points after SO injection. (B) Comparisons of average IOP of the SOHU eyes with or without SO-removal at 5wpi. Data are presented as means ± s.e.m, n = 12. ****: p < 0.0001, Student’s t test. (C) Representative OCT images of mouse retina area surrounding ON head at 5wpi. GCC indicated by double end arrows. (D) Quantification of GCC thickness, represented as percentage of GCC thickness in the SO eyes, compared to the CL eyes. Control (CON): n = 13; HF: n = 19; SO-removal: n = 19, ****: p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (E) Upper panel, confocal images of wholemount retinas showing surviving RBPMS-positive (red) RGCs of naïve, HF, and SO removal eyes at 5wpi. Scale bar, 100 µm. Middle panel, confocal images of a portion of the peripheral retina showing surviving RBPMS-positive (red) RGCs in the corresponding groups. Scale bar, 20 µm. Lower panel, light microscope images of semi-thin transverse sections of ON stained with PPD from the corresponding groups. Scale bar, 10 µm. (F,G) Quantification of surviving RGCs in the peripheral retina and surviving axons in ON of the corresponding groups at 5wpi, represented as percentage of SO eyes compared to CL eyes. Data are presented as means ± s.e.m, Control (CON): n = 12; HF: n = 22; SO-Removal: n = 22, ***: p < 0.001, ****: p < 0.0001; one-way ANOVA with Tukey’s multiple comparison test.