Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation

Abstract

Mutations in PITX2 cause Axenfeld-Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.

Keywords: Pitx2; glaucoma; mouse model.

Figure 1

Anterior segment morphology in egl1 mice. (A–I) Representative slit lamp image from C57BL/6J (A) and egl1 mice (B–I). (A) Representative slit lamp image showing clear and avascular cornea and centrally located pupil in uniformly shaped iris. (B) Normal cornea from an egl1 mouse. (C–I) Anterior segment lesions in egl1 mice. The egl1 mice developed a variety of anterior segment lesions (indicated by triangles) such as localized faint opacity in paracentral zone of the cornea (C), dense corneal opacity with neovascularization (D,E), iris thinning (D,E) and pupil deviation (E), diffuse opacity covering the entire cornea (F), and iris synechiae (the iris adhesion to the cornea) (G–I). (H) Enlarged view of area indicated by arrow in (G). (J–L) Representative H&E staining images of cross-section of the ciliary body and iridocorneal angle from C57BL/6J (J), egl1 mouse with normal IOP (K) and after 4 weeks of IOP elevation (L). The iridocorneal angle remained open and ciliary body appeared normal triangular in C57BL/6J and the egl1 mice with normal IOP. Anterior synechiae (the iris attached in the iridocorneal angle) and ciliary body atrophy were seen in eyes with elevated IOP. Scale bar = 50 µm.

Figure 2

IOP elevation in egl1 mice. (A) Conscious IOP measurement setup. (B) Plastic cone and mouse restrainer for IOP measurement. (C) Close view of conscious IOP measurement. (D) The IOP was monitored in egl1 mice longitudinally. Significant IOP elevation was detected at 4 weeks of age and remained at all ages examined. Data are presented as means ± SEM (n = 44). **: p < 0.01, ****: p < 0.001. (E) The IOP values from each time point were plotted by three ranges, <20 mmHg, 20–25 mmHg, and >25 mmHg. The IOP values demonstrated a wide range in mice of the same age.

Figure 3

Total aqueous humor outflow facility in young and aged egl1 animals. (A) The aged animals demonstrated significantly lower outflow facility compared to the young animals. Data are presented as means ± SEM (n = 5 and 7 for 2- and 12-months old animals, respectively). ****: p < 0.001. (B) Individual outflow facility was plotted against IOP measurement. The outflow facility and IOP measurement show a trend of negative correlation, but this did not achieve statistical significance.

Figure 4

RGC degeneration in egl1 mice. (A) Quantification of RGCs was performed before and at 2 and 8 weeks after IOP elevation. Significant RGC loss was observed after 8 weeks of IOP elevation. Data are presented as means ± SEM (n = 11–17). **: p < 0.01. (B) Representative images show RBPMS (red) immunolabeled retinal whole mounts from mice before (left) and 8 weeks after (right) IOP elevation. The density of RGCs, demonstrated by RBPMS positive cells, decreased after 8 weeks of IOP elevation. Scale bar = 50 µm. (C) Individual RGC count was plotted against IOP exposure described as AUC. There was a moderate negative correlation between RGC counts and IOP exposure. R square: 0.43, p < 0.001.

Figure 5

PERG reduction in egl1 mice. (A) Representative PERG waveforms recorded from egl1 mice before (black) and 2 weeks after (red) IOP elevation. IOP elevation decreased and delayed the PERG response. (B,C) Bar graphs show decreased PERG responses (B) and increased PERG latencies (C) after 2 weeks of IOP elevation. Data are presented as means ± SEM (n = 5–6) *: p < 0.05, **: p < 0.01.

Figure 6

Glaucomatous ONH changes in egl1 mice. Representative immunofluorescence staining images show increased expression of GFAP and Iba-1 and disorganized NF in the egl1 mice. Images were taken in the rectangular boxed area in (A). (B–D) Columns show GFAP (red), Iba-1 (green), and NF (green) staining in C57BL/6J wildtype and egl1 mice with various IOP levels. Nuclei were labeled with DAPI (blue). Scale bars = 50 μm.