Sustained ocular hypertension induces dendritic degeneration of mouse retinal ganglion cells that depends on cell type and location

Abstract

Purpose: Glaucoma is characterized by retinal ganglion cell (RGC) death and frequently associated with elevated IOP. How RGCs degenerate before death is little understood, so we sought to investigate RGC degeneration in a mouse model of ocular hypertension.

Methods: A laser-induced mouse model of chronic ocular hypertension mimicked human high-tension glaucoma. Immunohistochemistry was used to characterize overall RGC loss and an optomotor behavioral test to measure corresponding changes in visual capacity. Changes in RGC functional properties were characterized by a large-scale multielectrode array (MEA). The transgenic Thy-1-YFP mouse line, in which a small number of RGCs are labeled with yellow fluorescent protein (YFP), permitted investigation of whether subtypes of RGCs or RGCs from particular retinal areas were differentially vulnerable to elevated IOP.

Results: Sustained IOP elevation in mice was achieved by laser photocoagulation. We confirmed RGC loss and decreased visual acuity in ocular hypertensive mice. Furthermore, these mice had fewer visually responsive cells with smaller receptive field sizes compared to controls. We demonstrated that RGC dendritic shrinkage started from the vertical axis of hypertensive eyes and that mono-laminated ON cells were more susceptible to IOP elevation than bi-laminated ON-OFF cells. Moreover, a subgroup of ON RGCs labeled by the SMI-32 antibody exhibited significant dendritic atrophy in the superior quadrant of the hypertensive eyes.

Conclusions: RGC degeneration depends on subtype and location in hypertensive eyes. This study introduces a valuable model to investigate how the structural and functional degeneration of RGCs leads to visual impairments.

Figure 1.

Laser photocoagulation of the aqueous humor outflow in mouse eyes. (A) Schematic diagram of the laser treatment. (B) Sections of a control eye with open angle and a laser-treated eye with angle-closure. Stars indicate the areas that were expanded below. Scale bars: 200 μm. (C) IOP increases after laser treatment. IOP was measured at 1, 3, and 7 days, and then weekly for 26 weeks after laser treatment. 0 to 4 weeks: n = 77 mice; 5 to 8 weeks: n = 51; 9 to 12 weeks: n = 29; 13 to 16 weeks: n = 14; 17 to 26 weeks: n = 11. Error bar: SEM, same throughout.

Figure 2.

Ganglion cell density is decreased following laser treatment. (A) Whole-mounted retinas immunostained by Brn-3a antibody. Scale bar: 500 μm. (B) Cell density of Brn-3a (left) and Brn-3b (right) from laser-treated (colored bars) and untreated eyes (black bars) at 1, 2, and 6 months after the laser treatment. **P < 0.01; ***P < 0.005 in the Student's t-test. (C) Axon loss after laser treatment. Left: Sections to show stained axons in untreated and treated optic nerves. Scale bar: 10 μm. Right: Percentage axon loss at 1 to 12 weeks after laser treatment. n = 5 to 6 mice in each group. (D) Retinal sections from laser-treated and untreated eyes immunostained by PKCα and calretinin antibodies show that the overall morphology of the inner retina was unaffected by IOP elevation. Scale bar: 50 μm.

Figure 3.

Decrease of visual acuity and contrast sensitivity with IOP elevation. (A) Photos of laser-treated eye and control eye. (B) Visual acuities were measured before and at 2 months and 5 to 6 months after laser treatment. (C) Contrast sensitivity at 2 months after laser treatment. **P < 0.01; and ***P < 0.001 in Student's t-test.

Figure 4.

Functional degeneration of RGCs after laser treatment. (A) Schematic checkerboard stimulation. Each checker projects onto a 100 × 100-μm area of the retina and its luminance value is randomly chosen from a Gaussian distribution centered around the mean luminance level. The stimulus was updated at 30 frames per second. (B) The spatiotemporal receptive field of each cell was estimated through STA analysis. The legend bar indicates the contrast of each pixel in the STA in units of SD from the mean luminance. (C) The representation of the receptive field as a 1-dimensional Gaussian along the x (blue) and y (red) axes in (B). Cells in which the peak STA contrast exceeds 4 SDs (red dashed line) are labeled as visually responsive. (D) Percentages of visually responsive cells from untreated and treated eyes. n = 7, and P = 0.05 in Student's t-test. (E) RGCs from hypertensive eyes had smaller receptive field (RF) center sizes compared to controls (P = 0.0037 in Student's t-test, and P = 0.0038 in K-S Test).

Figure 5.

At 2 months after laser treatment, the average dendritic field and soma areas of RGCs may become smaller. (A) A projected image of confocal Z-stacks of a Thy-1-YFP RGC shows the dendritic coverage area circled by a green circumferential line and the soma area by the orange line. Scale bar: 20 μm. (B) At 2 months after laser treatment, the dendritic field sizes of all RGCs from treated (red) and untreated (black) eyes were plotted against their soma sizes. (C) The relative cumulative distributions of the dendritic field sizes from treated (black) and untreated (gray) eyes. Separation of points: 10,000 μm². (D) The relative cumulative distributions of the soma size from treated and untreated eyes. Separation of points: 20 μm². P values were calculated by the K-S test (same in Figs. 6, 7).

Figure 6.

The degeneration of RGC dendrites starts from the vertical axis and the shrinkage of RGC somas starts from the superior quadrant. After immunostaining, retinas were flat-mounted like clover leaves with four quadrants: superior, inferior, temporal, and nasal (labeled as the dark area in the circle). Thy-1-YFP–positive cells from different regions were measured and compared between treated (gray) and untreated (black) eyes. Scale bar: 500 μm.

Figure 7.

ON RGCs are more susceptible to IOP elevation than ON-OFF RGCs. (A, D) Representative images of an ON-RGC (A) and an ON-OFF-RGC (D). The orthogonal views at the bottom show that ON cell dendrites were laminated in the ON sublamina and the ON-OFF dendritic tree in both ON and OFF sublaminae. Scale bar: 20 μm. (B, E) The relative cumulative distributions of the dendritic field sizes of ON cells (B) and ON-OFF cells (E) from superior and inferior quadrants. (C, F) The relative cumulative distributions of the soma sizes of ON cells (C) and ON-OFF cells (F) from superior quadrant. Untreated: black; and Treated: gray.

Figure 8.

SMI-32–positive ON cells from the superior quadrant exhibited a smaller mean dendritic field size. (A) Immunostaining of SMI-32 antibody and double-staining of SMI-32 and YFP/GFP antibodies on retinal sections. Scale bar: 100 μm (left); scale grid: 20 μm (right). (B) The relative cumulative distributions of the dendritic field and soma sizes of all SMI-32 positive cells. P values were calculated by the K-S test. (C) The relative cumulative distributions of the dendritic field and soma sizes of SMI-32–labeled ON cells in the superior quadrant. (D) Dendritic tracing of an SMI-32– and Thy-1-YFP–positive RGC. Scale grid: 20 μm. (E) The total dendritic length of SMI-32– and Thy-1-YFP–positive RGCs from the superior quadrant of treated and untreated retinas. P = 0.05 in Student's t-test.