Aberrant Collagen Composition of the Trabecular Meshwork Results in Reduced Aqueous Humor Drainage and Elevated IOP in MMP-9 Null Mice

Abstract

Purpose: Homeostatic turnover of the trabecular meshwork extracellular matrix (ECM) is essential to regulate aqueous humor outflow and to maintain intraocular pressure homeostasis. In this study, we evaluated aqueous humor turnover, intraocular pressure, and trabecular meshwork organization in MMP-9 null mice.

Methods: Intraocular pressure and aqueous humor turnover were measured in MMP-9 null versus wild-type mice. Morphology of the anterior segment of the eye, with special attention to the structural organization of the trabecular meshwork, was investigated by means of optical coherence tomography, light microscopy, and transmission electron microscopy. Furthermore, using quantitative real-time polymerase chain reaction and immunostainings, we evaluated the ECM composition of the trabecular meshwork. Finally, the integrity and function of the retina and optic nerve were assessed, via optical coherence tomography, histologic techniques, and optomotor testing.

Results: MMP-9 null mice displayed early-onset ocular hypertension and reduced aqueous humor turnover. While transmission electron microscopic analysis did not reveal any abnormalities in the cellular organization of the trabecular meshwork, detailed investigation of collagen expression indicated that there is an aberrant trabecular meshwork ECM composition in MMP-9 null mice. Notably, at the age of 13 months, no glaucomatous neurodegeneration was seen in MMP-9 null mice.

Conclusions: Our observations corroborate MMP-9 as an important remodeler of the collagenous composition of the trabecular meshwork and provide evidence for a causal link between MMP-9 deficiency, trabecular meshwork ultrastructure, and ocular hypertension.

Figure 1

MMP-9 null mice exhibit elevated IOP owing to reduced aqueous humor drainage. (a) Longitudinal follow-up of IOP in wild-type and MMP-9 null mice revealed that IOP elevation already manifests from the age of 2 months and persists at all ages measured thereafter, in MMP-9 null mice (n ≥ 8, 2-way ANOVA, P < 0.0001). (b) Ultrasound pachymetry revealed no difference in CCT between wild-type and MMP-9 null mice (n ≥ 8, 2-way ANOVA). (c) Fluorophotometric measurements revealed that aqueous humor turnover is delayed in MMP-9 null animals (age 13 months) (n = 5, 2-way repeated-measures ANOVA, P < 0.05). (d) Representative series of fluorescence images captured from wild-type and MMP-9 null mice at 10-minute intervals after application of fluorescein. Scale bar: 1 mm.

Figure 2

MMP-9 null mice exhibit normal overall anterior segment morphology. (a) Spectral- domain OCT imaging of the anterior segment of the eye showed an open iridocorneal angle (*) in all mice under study. (b) In addition, morphometric analysis of the anterior chamber depth (arrow) indicated that there is no difference between wild-type and MMP-9 null mice (age 13 months) (n ≥ 13, Student's t-test). Scale bar: 500 μm. (c) Postmortem histologic analysis of the anterior segment revealed no overt changes in the ciliary body (cb), trabecular meshwork (tm), and Schlemm's canal (sc) of MMP-9 null mice (n = 5) as compared to age-matched wild-type mice (n = 7). Scale bar: 50 μm.

Figure 3

MMP-9 null mice exhibit normal laminar organization of the trabecular meshwork. (a) Representative transmission electron microscopy images of the trabecular meshwork of wild-type and MMP-9 null mice, illustrating normal laminar organization of the trabecular meshwork and the presence of giant vacuoles (gv) in the inner wall endothelium of Schlemm's canal (sc). Scale bar: 2 μm. (b) Morphometric analysis did not detect any difference in the thickness of the trabecular beams (tb), the trabecular meshwork, or the intertrabecular space (n ≥ 3, Student's t-test).

Figure 4

Collagen synthesis is affected in MMP-9 null mice. (a) qPCR analysis of Col1a1 mRNA expression reveals a (nonsignificant) reduction of approximately 50% in collagen I synthesis in MMP-9 null mice in comparison to wild-type mice (n = 6, Student's t-test). (b) Overview picture of a collagen I immunostaining of the anterior chamber angle region, showing reduced collagen I expression in the trabecular meshwork and the sclera of MMP-9 null mice. Scale bar: 50 μm. (c) High-magnification image of collagen I staining in the trabecular meshwork, revealing nearly complete absence of collagen I staining in the trabecular beams of MMP-9 null mice (*). Scale bar: 10 μm. (d) qPCR analysis of Col4a3 mRNA expression reveals a (nonsignificant) reduction of approximately 50% in collagen IV synthesis in MMP-9 null mice in comparison to wild-type mice (n = 6, Student's t-test). (e) Overview picture of collagen IV immunostaining of the anterior chamber angle region reveals that immunostaining intensity is slightly reduced in the trabecular meshwork of MMP-9 null mice. Scale bar: 50 μm. (f) High-magnification image of collagen IV staining in the trabecular meshwork confirms reduced collagen IV immunolabeling. Scale bar: 10 μm.

Figure 5

Sirius Red staining on sections of the iridocorneal angle reveals a disturbed collagen turnover in MMP-9 null mice. (a, b) Sirius Red staining reveals immature collagen (i.e., non–cross-linked collagen monomers) in red, and mature collagen (i.e., cross-linked bundles of collagen fibers) in green. Scale bar: 50 μm. (c) MMP-9 null mice display an altered ratio of immature versus mature collagen, compared to wild-type animals: more mature collagen and less immature collagen is seen (n = 6; Student's t-test). (d) These observations could be explained by the hypothesis schematically depicted here. Owing to the lack of MMP-9 proteolytic activity in MMP-9 null mice, degradation of mature collagen is impeded. As a result of feedback signaling mechanisms, this results in an increased suppression and/or decreased activation of collagen gene transcription, which leads to a lowered synthesis of immature collagen monomers.

Figure 6

MMP-9 null mice exhibit no signs of retinal atrophy or optic neuropathy. (a, b) Morphometric analysis of the thickness of the total retina (TR, arrow) and the thickness of the GCC (arrow) on SD-OCT images of the retina revealed no significant differences between 13-month-old wild-type (n = 15) and MMP-9 null mice (n = 13) (Student's t-test). Scale bar: 50 μm. (c, d) Retinal ganglion cell density, quantified as the number of Brn3a⁺ cells per mm² retina and represented via pseudocolor density mapping here, was similar in wild-type and MMP-9 null mice and did not change between the age of 3 and 13 months (Student's t-test). Scale bar: 1 mm. (e) Postmortem histologic analysis of the retina revealed no overt changes in cytoarchitecture of 13-month-old wild-type mice (n = 7) versus age-matched MMP-9 null mice (n = 5). Scale bar: 20 μm. (f) Measurements of the optic nerve diameter revealed no difference between 13-month-old wild-type (n = 7) and MMP-9 null mice (n = 6) (Student's t-test). (g) Quantification of axonal density in the optic nerves of 13-month-old wild-type (n = 7) and MMP-9 null mice (n = 6) revealed no effect of MMP-9 deficiency on axonal integrity (Student's t-test). (h) Toluidine blue staining on optic nerve cross-sections of wild-type and MMP-9 null mice. Scale bar: 10 μm.