Scleral structural alterations associated with chronic experimental intraocular pressure elevation in mice

Abstract

Purpose: To study changes in scleral structure induced by chronic experimental intraocular pressure elevation in mice.

Methods: We studied the effect of chronic bead-induced glaucoma on scleral thickness, collagen lamellar structure, and collagen fibril diameter distribution in C57BL/6 (B6) and CD1 mice, and in collagen 8α2 mutant mice (Aca23) and their wild-type littermates (Aca23-WT) using electron and confocal microscopy.

Results: In unfixed tissue, the control B6 peripapillary sclera was thicker than in CD1 mice (p<0.001). After 6 weeks of glaucoma, the unfixed CD1 and B6 sclera thinned by 9% and 12%, respectively (p<0.001). The fixed sclera, measured by electron microscopy, was significantly thicker in control Aca23 than in B6 or CD1 mice (p<0.05). The difference between fresh and fixed scleral thickness was nearly 68% in untreated control B6 and CD1 mice, but differed by only 10% or less in fresh/fixed glaucoma scleral comparisons. There were 39.3±9.6 lamellae (mean, standard deviation) in control sclera, categorized as 41% cross-section, 24% cellular, 20% oblique, and 15% longitudinal. After glaucoma, mean peripapillary thickness significantly increased in fixed specimens of all mouse strains by 10.3 ±4.8 µm (p=0.001) and the total number of lamellae increased by 18% (p=0.01). The number of cellular and cross-section lamellae increased in glaucoma eyes. After glaucoma, there were more small and fewer large collagen fibrils (p<0.0001). Second harmonic generation imaging showed that the normal circumferential pattern of collagen fibrils in the peripapillary sclera was altered in significantly damaged glaucomatous eyes.

Conclusions: Dynamic responses of the sclera to experimental mouse glaucoma may be more important than baseline anatomic features in explaining susceptibility to damage. These include decreases in nonfibrillar elements, alterations in lamellar orientation, an increased number of smaller collagen fibrils and fewer larger fibrils, and relative increase in the number of scleral fibroblast layers.

Figure 1

Mean intraocular pressure in control and glaucoma groups by strain. The mean intraocular pressure (IOP; mmHg) readings of the control eye and glaucoma treated eyes at five different time points post bead injection are shown in the panels as follows: A: B6, n=5; B: CD1, n=5; C: Aca23-WT, n=5; and D: Aca23, n=5.

Figure 2

Scleral tissue division. The method of identifying areas of the sclera for measurement of scleral thickness, lamellar orientation, and fibril diameter distribution is shown. A: For sclera thickness measurements in fresh tissue slices, the sclera was divided into six regions, labeled R1 (peripapillary) to R6 (limbus). B: The sclera was divided into the following pieces; (1) Four zones; superior, nasal, inferior, and temporal. (2) The optic nerve head and peripapillary sclera were removed with a 1.5 mm in diameter hole punch. (3) Razor cuts were made at the superior to assist with identification during plastic embedding and sectioning. (4–7) From each quadrant, two specimens (representing Regions 2+3 and Region 4 in A above) were selected for epoxy embedding, as was the peripapillary specimen.

Figure 3

Collagen lamella orientation. Using transmission electron microscopy (TEM), the lamellae of the sclera were quantified and their thickness measured by dividing them into the following four types based on their orientation of collagen fibrils: cross-section (fibrils anterior-posterior=CR), longitudinal (parallel to equator, nasal-temporal=L), oblique (O), and lamellae consisting of sclera fibroblasts (cellular=CL; scale bar=500 nm).

Figure 4

Collagen fibril diameter distribution by sclera location in CD1 control. The distribution for 100 collagen fibrils from a control CD1 mouse taken from the inner, mid-, and outer sclera shows that the mid-sclera has a broader range of fibril diameter and higher mean fibril diameter. The inner and outer sclera were different when compared to the larger fibrils found in the middle portion of the sclera (≤0.0001 and ≤0.0001 respectively, t test).The inner and outer scleral collagen diameter measurements, however, were not statistically different from one another.

Figure 5

Collagen fibril diameter distribution: glaucoma compared to control. A histogram of collagen fibril diameter density (fibers/um²) is plotted for the control mouse sclera (green line, n=20 mice; 5 CD1, 5 B6, 5 Aca23-WT, and 5 Aca23) and glaucomatous sclera (blue line, n=19 mice; 5 CD1, 5 B6, 5 Aca23-WT and 4 Aca23; indicating density on the right y-axis). The ratio of the glaucoma density to control density at each diameter is represented as a linear regression (red line: r²=0.62, indicates values of ratio glaucoma to control, left y-axis). The glaucoma group has a higher proportion of smaller collagen fibrils and a lower proportion of larger fibrils with the crossover from more, smaller to fewer, larger fibrils occurring near the mean fibril diameter for controls (vertical dotted line).

Figure 6

Regression lines and values for all four mouse strains. Histogram of collagen fibril diameter density (fibers/um²) is plotted for the control mouse sclera (green line) and the glaucoma sclera (blue line) for each mouse strain. The ratio of the glaucoma density to control density at each diameter grouping is represented as a linear regression (red line) for each of the four mouse strains as follows: A: B6 (n=5, R²=0.01); B: CD1 (n=5, R²=0.08); C: Aca23-WT (n=5, R²=0.36), and D: Aca23 (n=5, R²=0.82). The Aca23 and CD1 glaucoma groups had a higher proportion of smaller collagen fibrils and a lower proportion of larger fibrils compared to their controls (both p<0.0001).

Figure 7

Second harmonic generation images. Second harmonic generation (SHG) imaging shows collagen orientation in the sclera of an older CD1 mouse; the 1 mm² sclera piece includes the optic nerve canal (seen as the dark central area). The superior is located at the top of the images, while blood vessels (*) surround the optic nerve head on the nasal, inferior and temporal portion of the sclera. A: The peripapillary (posterior) sclera in control specimen had collagen fibrils oriented in a circumferential fashion around the optic nerve. B: In eyes exposed to elevated intraocular pressure (IOP), there was a retraction of this circumferential zone away from the optic canal, especially in the superior area. The white boxes in first two images (A, B) highlight the superior region. C: A 20× image of the posterior sclera shows no retraction in control tissue at the circumferential zone of the superior region. D: The retraction of collagen fibrils can be seen at the circumferential zone of the superior quadrant near the optic canal in 6 week glaucoma-treated eyes (20X). Moreover, 40× images of the mid-sclera show collagen assembled in a basket weave formation, where E: untreated tissue and F: glaucoma-treated tissue, which shows a slight change in fibril assembly. Scale bars are equal to 100µm for images A and B, and 10 µm for images C, D, E, and F.