Ultrastructural abnormalities of the trabecular meshwork extracellular matrix in Cyp1b1-deficient mice

Abstract

Cytochrome P450 1B1 (CYP1B1) is highly expressed in human and murine ocular tissues during development. Mutations in this gene are implicated in the development of primary congenital glaucoma (PCG) in humans. Mice deficient in Cyp1b1 (Cyp1b1(-/-) ) present developmental abnormalities similar to human primary congenital glaucoma. The present work describes the ultrastructural morphology of the iridocorneal angle of 21 eyes from 1-week-old to 8-month-old Cyp1b1(-/-) mice. Morphometric and semiquantitative analysis of the data revealed that 3-week-old Cyp1b1(-/-) mice present a significantly (P < .005) decreased amount of trabecular meshwork (TM) collagen and higher TM endothelial cell and collagen lesion scores (P < .005) than age-matched controls. Collagen loss and lesion scores were progressively increased in older animals, with 8-month-old animals presenting severe atrophy of the TM. Our findings advance the understanding of the effects of CYP1B1 mutations in TM development and primary congenital glaucoma, as well as suggest a link between TM morphologic alterations and increased intraocular pressure.

Keywords: CYP1B1; collagen fibrils; congenital glaucoma; electron microscopy; eye; mouse; oxidative stress; trabecular meshwork.

Figure 1

Iridocorneal angle; 2 week-old wild-type mice. Normal morphology of the iridocorneal angle (ICA). C= cornea, Cb= ciliary body, aTM=anterior trabecular meshwork, pTM= posterior trabecular meshwork, *= Schlemm’s canal. Transmission electron microscopy, uranyl acetate. 710x.

Figure 2

Trabecular meshwork; 2 week-old wild-type mice. Normal trabecular meshwork arranged in multiple trabecular beams composed of a core of collagen fibers (*) surrounded by trabecular cells (arrows). # = Schlemm’s canal. Transmission electron microscopy, uranyl acetate. 2650x.

Figure 3

Trabecular meshwork; 2 week-old wild-type mice. Higher magnification presenting the normal relationship between the trabecular beam’s components. Collagen fibers (*), trabecular cells cytoplasmic projections (arrows) and elastic fiber aggregates (arrow heads). Transmission electron microscopy, uranyl acetate. 8800x.

Figures 4, 6 and 8

Trabecular meshwork; 3–week-, 6-week- and 3-month-old wild-type mice, respectively. Normal trabecular meshwork. Please note the relationship between the collagenous core (*) of the trabecular beams and trabecular cells (arrows). sc = Schlemm’s canal. Transmission electron microscopy, uranyl acetate. 2650x.

Figures 5, 7 and 9

Trabecular meshwork; 3 week, 6week and 3 month-old Cyp1b1^−/− mice, respectively. Irregular trabecular beams with accentuation of the inter-trabecular spaces (*), fragmentation of the collagen fibers (arrows) and irregular and reactive trabecular cells (open arrows). sc = Schlemm’s canal. Transmission electron microscopy, uranyl acetate. 2650x.

Figure 10

Iridocorneal angle; 8-month-old Cyp1b1^−/− mice. Complete collapsed of the trabecular meshwork (TM) with formation of one atrophic and irregular trabecular beam spanning the anterior and posterior TM. ICA= Iridocorneal angle, C= cornea, Cb= ciliary body, ir= iris, *= Schlemm’s canal. Transmission electron microscopy, uranyl acetate. 710x.

Figure 11

Trabecular meshwork; 8-month-old Cyp1b1^−/− mice. Higher magnification of the collapsed trabecular tissue presenting irregular trabecular cells (arrows) in close proximity with scant extracellular matrix (*). sc = Schlemm’s canal; Transmission electron microscopy, uranyl acetate. 2650x.

Figure 12

Trabecular meshwork; 8-month-old Cyp1b1^−/− mice. Higher magnification of the trabecular beam presenting marked degeneration of the collagen fibers (arrows) and irregular trabecular cell projections (arrow heads); Transmission electron microscopy, uranyl acetate. 8800x.

Figure 13

Morphometric quantification of the relative amounts of collagen in the trabecular meshwork of Cyp1b1^−/− mice. 3-week-old Cyp1b1^−/− mice presented significantly less collagen in the trabecular meshwork than wild-type, and younger control and mutant mice. TM collagen loss progressed with age. Means with different letters are significantly different (n=42 [3/group/age], Mean ± SD, Student’s t-test, p = 0.0004). w= week, m= month, WT = wild type.

Figure 14

Semiquantitative scoring analysis of the distribution of the collagen lesions in the trabecular meshwork of Cyp1b1^−/− mice. Cyp1b1^−/− mice presented significantly higher median scores than wild-type mice in all ages. There was a significant increase in median score between 2- and 3-week-old and 6-week and 3-month-old Cyp1b1^−/− mice. (n=42 [3/group/age], Wilcoxon–Mann–Whitney, Median ± range, *differences between Cyp1b1^−/− and wild-type, p=0.002; ** differences between Cyp1b1^−/− mice, p=0.005). w= week, m= month, WT = wild-type.

Figure 15

Semi-quantitative scoring analysis of the trabecular cell ultrastructural morphology of Cyp1b1^−/− mice. Cyp1b1^−/− mice presented significantly higher median scores than wild-type mice in all ages (*, p=0.002). There was a significant increase in median score between 2- and 3-week-old Cyp1b1^−/− animals (p=0.005). (n=42 [3/group/age], Wilcoxon–Mann–Whitney, Median ± range,). w= week, m= month, WT = wild-type.