Experimental scleral cross-linking increases glaucoma damage in a mouse model

Abstract

The purpose of this study was to assess the effect of a scleral cross-linking agent on susceptibility to glaucoma damage in a mouse model.CD1 mice underwent 3 subconjunctival injections of 0.5 M glyceraldehyde (GA) in 1 week, then had elevated intraocular pressure (IOP) induced by bead injection. Degree of cross-linking was measured by enzyme-linked immunosorbent assay (ELISA), scleral permeability was measured by fluorescence recovery after photobleaching (FRAP), and the mechanical effects of GA exposure were measured by inflation testing. Control mice had buffer injection or no injection in 2 separate glaucoma experiments. IOP was monitored by Tonolab and retinal ganglion cell (RGC) loss was measured by histological axon counting. To rule out undesirable effects of GA, we performed electroretinography and detailed histology of the retina. GA exposure had no detectable effects on RGC number, retinal structure or function either histologically or electrophysiologically. GA increased cross-linking of sclera by 37% in an ELISA assay, decreased scleral permeability (FRAP, p = 0.001), and produced a steeper pressure-strain behavior by in vitro inflation testing. In two experimental glaucoma experiments, GA-treated eyes had greater RGC axon loss from elevated IOP than either buffer-injected or control eyes, controlling for level of IOP exposure over time (p = 0.01, and 0.049, multivariable regression analyses). This is the first report that experimental alteration of the sclera, by cross-linking, increases susceptibility to RGC damage in mice.

Keywords: Collagen; Cross-linking; Extracellular matrix; Glaucoma; Glyceraldehyde; Mouse; Retinal ganglion cell; Sclera.

Figure 1

Analysis of scleral inflation data X and Y DIC coordinates (raw values) are shown from the nasal scleral edge for an eye at P₀ (red circles) and P=30 mm Hg (purple squares). The ellipse fitted points (using DIC values) at P₀ (filled blue circles and line) and deformed positions at P=30 mm Hg for the same eye (teal squares and line). The comparison of raw data points and the fitted ellipse show the modest degree of smoothing that occurred (see Section 2.4. Biomechanical Inflation Testing).

Figure 2

Comparison of IOP—strain for GA-treated and control sclera The mean IOP—strain values are plotted for 4 groups of mice with loading from reference IOP to 30 mm Hg. The groups are Control (purple asterisk) and 1x treatment with GA (red square) at the right and 2x treatment with GA (blue diamond) and 3x treatment with GA (orange circle) at the left. Standard error bars are shown for control and 3x GA groups. The fitted lines have also been added (for each group) in each respective color. Data from the nasal meridional analysis (temporal meridional and circumferential analyses gave similar results) show the significant stiffening effect after either 2 or 3 treatments with GA.

Figure 3

Electroretinogram data for Scotopic B-Wave The graph plots the mean amplitude for control (blue triangles, N=5) and glyceraldehyde treated eyes (green diamonds, N=5) at 10 different scotopic, B-wave, flash intensities (amplitudes in μV (microvolts)).

Figure 4

Mouse peripapillary scleral area The mouse peripapillary sclera, optic nerve head, and optic nerve in epoxy section. The sclera divides into two portions, one splitting off posteriorly to join the dura mater and one continuing directly to the ONH. We measured the scleral thicknesses in two areas, one just prior to the division, which is referred to as the outer peripapillary scleral thickness (solid line square) and one after the division, next to the optic nerve head, referred to as the inner peripapillary sclera (dotted line square; scale bar =100μm).

Figure 5

IOP Difference for Buffer+GL, GA+ GL and GA groups Mean paired IOP difference between left eyes (treated) and right eyes (control) of each animals. From glaucoma experiment 1, the buffer-injected (blue diamonds, N=41) and GA-injected (red squares, B=40) show no significant increase pre-glaucoma (GL) induction after their subconjunctival injection sequences (both p > 0.4). After glaucoma induction both GA and buffer groups have significant elevations through the first 2 weeks (p <0.0001), but no significant difference between the two experimental groups (all p > 0.27). Animals that were injected with GA alone, but without glaucoma induction had no change in IOP (at either 1, 4, 7, 14 or 42 days) in their injected compared to control eyes (green triangles, N=10). Mean paired difference +/− standard error.

Figure 6

Distribution of RGC axon loss of individual eyes in first glaucoma experiment This graph plots the distribution of RGC axon loss for each individual mouse by group; 3 times treated GA eyes are represented in red squares (N=1–41) and the buffer injected eyes are represented in blue diamonds (N=1–40). Each eye (numbered 1–41) is ranked in ascending order of percent axon loss, and is plotted to show the distribution of axon loss in each experimental group; 3x GA eyes showed greater amount of axon loss in the 10–60% range when compared to buffer-injected controls.