Inhibition of histone deacetylases 1 and 3 protects injured retinal ganglion cells

Abstract

Purpose: Thy-1 is a marker of retinal ganglion cell (RGC) differentiation. Optic nerve injury triggers reduction of Thy-1 promoter activation followed by retinal ganglion cell (RGC) death. This study determined whether MS-275, an inhibitor of the histone deacetylases 1 and 3, can inhibit these changes.

Methods: Mice expressing cyan fluorescent protein (CFP) under control of the Thy-1 promoter received MS-275 (subcutaneous) or vehicle three times per week starting 1 week before optic nerve crush and continuing for 6 weeks. The same retinal area was imaged using the blue-light confocal scanning laser ophthalmoscope before and after optic nerve crush every week, and fluorescent spots were counted manually. The eyes were then processed for histopathologic analysis.

Results: The mean proportions of fluorescent retinal neurons remaining in the vehicle group following optic nerve crush were 36 ± 8, 18 ± 6, 13 ± 10, 12 ± 4, 13 ± 5, and 13 ± 5% at weeks 1 through 6, respectively (n = 6). In contrast, the mean proportions of fluorescent retinal neurons remaining in the group treated with MS-275 were 59 ± 19, 39 ± 11, 34 ± 12, 33 ± 15, 32 ± 13, and 27 ± 15% at weeks 1 through 6, respectively (n = 7, P < 0.05 at weeks 1 through 5). Rate analysis showed that MS-275 slowed the rate of loss during the first 2 weeks by 23% (P < 0.05) and subsequently was similar. Histopathologic analysis revealed 27 ± 13% greater ganglion cell layer (GCL) neurons in the eyes from mice that received MS-275 treatment (P < 0.02).

Conclusions: These results indicate that treatment with MS-275 protects against the loss of RGC differentiation and promotes RGC survival following optic nerve injury.

Figure 1.

Body weight of mice that received vehicle control and MS-275. Error bars indicate SD. There was no significant body weight change in any of the groups (P = 0.71, N = 6 for vehicle group and P = 0.89, N = 7 for the MS-275 group).

Figure 2.

Inverted images of fluorescent retinal neurons in same retinal area obtained prior to optic nerve crush (baseline) and at weeks 1, 2, 3, and 6 after optic nerve crush showing protective effect of MS-275. Note that prolonged treatment with MS-275 did not generally induce fluorescence in cells that were not fluorescent at the beginning of the study.

Figure 3.

Counts of retinal neurons that continue to express CFP after optic nerve crush expressed as a percentage of baseline count. N = 6 in the vehicle control group and 7 in the MS-275 group. Bars indicate SD. Asterisk indicates P < 0.05, t test.

Figure 4.

The mean rate of loss of fluorescent retinal neurons in the first two weeks, the second two weeks, and the third two weeks after optic nerve crush (N = 6 in the vehicle control group and 7 in the MS-275 group). Bonferroni t-test analysis of all pair-wise comparisons indicated that the mean rate of loss was significantly slowed by MS-275 treatment during the first two weeks (asterisk). It also indicated that the rate of loss during the first two weeks was significantly faster in each treatment group than the rate of loss in the second two weeks or third two weeks for both treatment groups (not indicated).

Figure 5.

Comparison of fluorescent retinal neurons in sham operated eyes of mice treated with vehicle control or MS-275 and imaged by bCSLO prior to initiating the study (baseline) and at the end of the study (week 6). Note that for both the control vehicle treated mice and MS-275-treated mice, the cells observed at the end of the study were the same as was observed at the beginning of the study.

Figure 6.

Conventional histological sections of the central retina (left column), mid-peripheral retina (middle column), and peripheral retina (right column), from mice that received vehicle (top two rows) or MS-275 (bottom two rows) illustrating MS-275 treatment protection of GCL neurons. Among the sham operated eyes, retinas from vehicle-treated animals (A–C) or MS-275-treated animals (G–I) had normal appearance. Among the eyes that received optic nerve crush, retinas from MS-275-treated animals (J–L) had greater numbers of GCL neurons than vehicle-treated animals (D–F). Note that the other retinal layers from each of these experimental conditions had normal appearance. Figure labels: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar = 20 μm.