Figure 9.

Wld^s profoundly protects from glaucomatous damage. (A) The IOP distributions of mice of each genotype overlap extensively. The boxes show the upper and lower quartiles and the bars show the extremes including outliers. The centerline of each diamond is the mean and the upper and lower points of each diamond represent the 95% confidence intervals of the mean. Mice with a single copy of the Wld^s fusion gene are hemizygous (Hemi). At both 9 and 12 mo, there were no significant differences in IOP (ANOVA) between the neuroprotected, Wld^s mice (see below), and mice of any other genotype. At 10.5 mo, the wild-type (WT) mice had an unusually high skewing of their IOP compared with typical values at this age (see Anderson et al., 2005; Libby et al., 2005a), and their IOP was significantly higher than mice of all other genotypes (P < 0.04 for all comparisons). This skewing did not account for the decreased axon loss in Wld^s mice. This is clear because (1) there was extensive overlap between all genotypes, and (2) the IOP distribution of Wld^s mice was extremely similar (P = 0.7) to Bax ^+/− heterozygotes mice (Het), whose axons were not protected (see below). (B) Axon protection in Wld^s mice. Distributions of optic nerve damage in mice of the indicated genotypes are shown at two important time points (Materials and methods). The Wld^s allele significantly rescues axons from glaucomatous degeneration at 12 mo of age (P < 0.0001, chi square comparing Wld^s Hemi to their WT littermates). Importantly, Wld^s increased the number of eyes with no detectable glaucoma. Bax ^+/− heterozygosity conferred no protective effect when compared with wild-type mice (right). Though not statistically significant, there tended to be a greater degree of protection in D2.Wld^sBax ^+/− double mutants (right), compared with Wld^s single mutants (left). The number of eyes analyzed for each genotype at 10.5 mo was typically ≥40 but 20 for D2.Bax ^+/− heterozygotes. At 12 mo, typically ≥63 eyes were studied for each genotype but 21 for D2.Bax ^+/− heterozygotes. (C–F) Wld^s preserves RGC axons and optic nerve morphology as seen in semi-thin sections. Most wild-type (C) and D2.Bax ^+/− nerves (E) had severe glaucoma with very substantial axon loss (see Fig. 3) and extensive glial scarring. In contrast, the majority of D2.Wld^s (D) and D2.Wld^sBax ^+/− nerves (F) had no or early glaucoma, with <5% of axons damaged (Fig. 3) and no evidence of glial hypertrophy or scarring. (G) Axon counts for a randomly selected sample of DBA/2J and D2.Wld^s nerves with no or early glaucoma (n = 6 for each genotype). The averages are not statistically different (P = 0.235), and none of the D2.Wld^s nerves had decreased axon counts. Because Wld^s more than doubled the number of eyes with no or early glaucoma, half of these counted eyes were rescued. (H–J) Retinal flatmounts showing that in Wld^s protected eyes RGC somata survive (H, preglaucoma; I, severe glaucoma; J, no or early glaucoma). (K) This was confirmed by counting RGC layer cells in eyes with no or early glaucoma. Again, half of the D2-Wld^s eyes were rescued and none had cell numbers below the range of wild-type values (n = 10 each genotype, P > 0.1). As expected, in both wild-type and D2.Wld^s mice with severe optic nerve damage the majority of RGCs were lost. The retinal images shown are from a matched region of the superior, peripheral retina. (L) Despite the profound rescue, a mild degree of somal shrinkage (∼10%) was seen in D2.Wld^s eyes with no detectable glaucoma. The shrinkage appears to occur generally across cells of different sizes (see Fig. S4). (M) Wld^s strongly preserved PERG amplitude, a measure of RGC activity, in randomly selected mice (number of eyes = 16, 14, 18 for D2-Gpnmb⁺, DBA/2J, and D2.Wld^s, respectively). Bars, 100 μm.