Transgenic mice expressing mutated Tyr437His human myocilin develop progressive loss of retinal ganglion cell electrical responsiveness and axonopathy with normal iop

Abstract

Purpose: To characterize age-related changes of retinal ganglion cell (RGC) function, IOP, and anatomical markers of axon/glia integrity in a transgenic mouse expressing Tyr437His mutant of human myocilin protein.

Methods: Retinal ganglion cell electrical responsiveness was tested with pattern electroretinogram (PERG) in 11 transgenic mice expressing mutated myocilin at different ages over 18 months under ketamine/xylazine anesthesia. Twelve age-matched C57BL/6J mice also were tested as controls. Intraocular pressure was measured with a Tonolab tonometer. Immunohistochemistry for GFAP and neurofilament was performed on dissected optic nerve heads.

Results: In transgenic mice expressing mutated myocilin, the PERG amplitude progressively decreased with increasing age by approximately 50%, whereas the PERG peak latency increased by approximately 40 ms (ANOVA, P < 0.05). In contrast, PERGs of young and old control mice had similar amplitudes and peak latencies. In transgenic mice, GFAP staining was more intense and extended than in control mice, and increased with increasing age; neurofilament staining showed swollen and partially degenerated axons in old transgenic mice. The IOP of young transgenic mice was similar to that of control mice and did not significantly change with increasing age.

Conclusions: Transgenic mice expressing mutated human myocilin display progressive age-related changes in RGC electrical responsiveness that are not associated with IOP elevation but are associated with marked astrogliosis and axonopathy. Our results support the view that MYOC expression in the optic nerve may impact structural, metabolic, or neurotrophic support to RGC axons, thereby influencing their susceptibility to glaucomatous damage independently of IOP.

Keywords: intraocular pressure; mouse; myocylin; optic nerve; pattern electroretinogram; retinal ganglion cell.

Figure 1

Age-related PERG changes in Tg mice expressing Tyr423His human myocilin (top) compared with control C57BL/6J (B6) mice (bottom). (A, B, F, G) Pattern electroretinogram waveforms recorded in individual eyes of young and old mice (black: 2 months old; gray: 18 months old). (C, H) The corresponding grand-average PERG waveforms. Pattern electroretinogram amplitudes (D, E) and peak latencies (E, J) as a function of age in Tg mice (top) and B6 mice (bottom). Open symbols represent PERG amplitudes and latencies measured in individual mice (average of the two eyes). Filled symbols adjacent to open symbols represent the corresponding means ± SEM. Note in (D, E, I, J) that in Tg mice the PERG amplitude progressively decreases with increasing age while the PERG peak latency increases. In control mice, the PERG amplitude and peak latency are invariant with age.

Figure 2

Age-related IOP measurements in Tg mice expressing Tyr423His human myocilin (A) compared with control C57BL/6J (B6) mice (B). Each symbol represents the average of the two eyes. (C) Mean ± SEM IOP for each age group of Tg and B6 mice.

Figure 3

Immunostaining for neurofilament (green) and GFAP (red) in longitudinal sections through the optic nerve head and optic nerve of four different Tg mice expressing Tyr423His human myocilin (top, 2-month-olds; bottom, 18-month-olds). In older Tg mice (E, G), RGC axons appear swollen and partially degenerated compared with young Tg mice (A, C). In both young and old Tg mice, GFAP staining is more intense and widespread than that of representative wt mice (I). In older Tg mice (F, H), GFAP staining is more extended than in young Tg mice (B, D). In (I), GFAP (red), neurofilament (green), and DAPI staining are shown superimposed.

Figure 4

Pattern visually evoked potentials in young (A) and old (B) Tg mice expressing Tyr423His human myocilin (n = 3, 6 eyes). (C) Grand-average VEP waveforms for young (black line) and old (gray line) Tg mice. (D, E) Pattern visually evoked potential amplitudes (D) and peak latencies (E) measured in individual mice (average of the two eyes). Filled symbols adjacent to open symbols represent the corresponding means ± SEM. Pattern visually evoked potentials were recorded from a screw electrode implanted in the monocular cortex contralateral to the eye stimulated with a contrast-reversing grating of 0.05 cycles per degree and high contrast.