These results indicate MS-275 protects against loss of retinal Thy-1 promoter activation that occurs following optic nerve crush, as well as protects against reduction in the number of GCL neurons. Specifically, the proportion of initially fluorescent retinal neurons in each group that remained fluorescent after optic nerve crush was up to 175% greater in the animals that received MS-275 than in corresponding control animals. The rate of decline in the number of fluorescent retinal neurons was significantly slowed by the MS-275 treatment during the first two weeks after optic nerve crush. Both groups reached a plateau by the third week with counts of the fluorescent retinal neurons remaining over 2-fold greater in the animals that received MS-275 than in the animals that received vehicle. In addition to RGC differentiation, histopathologic analysis of the eyes at the end of the study that showed the number of remaining GCL neurons in the mice that received MS-275 was significantly greater than in the mice that received vehicle. Together, these results indicate that MS-275 protects both RGC differentiation and RGC survival following optic nerve crush.
The current method for analysis of the bCSLO images is modification of the method of Leung et al.
18,19 that scored brightly fluorescent retinal neurons and excluded dimly fluorescent neurons. More than 96% of the brightly fluorescent retinal neurons in Thy1-CFP23Jrs mice were RGCs since they could be labeled by injection of a retrograde tracer into the superior colliculus.
19 A subsequent study by Raymond et al. showed that the dimly fluorescent retinal neurons in this strain included cholinergic amacrine cells.
22 Quantitative analysis showed 80% of all fluorescent neurons were RGCs and 20% were cholinergic amacrine cells. Because the fluorescent retinal neurons that respond to optic nerve crush first become dimmer before CFP expression ceases, it is likely that dimly fluorescent cholinergic amacrine cells are included in the fluorescent retinal neuron counts. As cholinergic amacrine cell death following optic nerve injury is minimal,
23 it is probable the observed decline in retinal neurons expressing CFP primarily reflects down regulation of Thy-1 gene expression in RGCs.
The possibility that the increased numbers of fluorescent retinal neurons in the Thy1CFP23Jrs mice might reflect induction of CFP expression in some of the originally nonfluorescent amacrine cells was investigated and found to be either minimal or nonexistent. Specifically, careful comparison of the images from the eyes that received optic nerve crush (
Fig. 2) and in the sham eyes (
Fig. 5) showed that MS-275 treatment did not induce fluorescence in cells that were not fluorescent prior to the beginning of the study. Thus, MS-275 preserved fluorescence in cells that were initially fluorescent without inducing initially nonfluorescent cells to become fluorescent.
The absence of any significant change in body weight during the course of the study suggests that MS-275 had minimal impact on the general metabolism of the study mice. Hence, the increased numbers of fluorescent retinal neurons imaged in vivo and the greater numbers of GCL neurons observe in the histopathological analysis in the mice that received MS-275 likely reflects a neuroprotective effect. In view of the potentially wide spread effects of HDAC activity alterations on global gene expression
6 ; however, further study will be needed to confirm this point.
Turning to the experimental results, the number of fluorescent retinal neurons observed in the crush-operated eyes of MS-275-treated mice was substantially greater than the mice that received vehicle. At 2 weeks after crush, when the number of fluorescent retinal neurons in vehicle-treated mice first declined to less than 20% of baseline counts, the remaining fluorescent retinal neurons in the crush-operated eyes of mice that received MS-275 was 40% of baseline counts. Thus, after subtracting the 20% proportion of amacrine cells present at baseline, there remained at least 20% of the original fluorescent retinal neurons that were RGCs in which CFP expression was preserved by MS-275 treatment. At three weeks and later, counts of fluorescent retinal neurons in animals that received MS-275 reached a plateau around 33% during weeks 3 through 5 with a decline to 27% at week 6. These values were more than twice the corresponding values for fluorescent retinal neurons present at each time point in the vehicle treated mice. Thus, MS-275 provided substantial protection against loss of Thy-1 promoter activation that persisted to the end of the study.
Within vehicle-treated mice, the loss rate of fluorescent retinal neurons was over 40%/wk during the first 2 weeks, less than 5%/wk during the second two weeks, and negligible during the third 2 weeks. Treatment with MS-275 significantly slowed the rate of RGC loss during the first two weeks while differences in the rate of loss differed insignificantly between the two groups at later time points. First, this indicates that MS-275 slowed the rate of de-differentiation that occurred after optic nerve injury. Second, and perhaps of greater importance, the stable preservation of 2-fold greater numbers of fluorescent retinal neurons in the animals that received MS-275 suggests there is a subset of RGCs that are stably maintained following optic nerve injury that otherwise would de-differentiate and perhaps later die.
Several considerations allow estimation of the effect of MS-275 treatment on RGC survival in the present study. First, the background strain for the Thy1-CFP23Jrs mice used in this study is the C57BL/6 mouse.
24 A previous study showed that 59 ± 4% of GCL neurons in C57BL/6 mice are displaced amacrine cells.
25 Also, though the number of RGCs, though the number of RGCs can vary widely among individual mice, the RGC difference between left and right eyes within the same mouse is typically less than 3%.
21 Thus, as shown in
Table 2, it is reasonable to estimate that the average density of amacrine cells adjacent to the optic nerve at the beginning of the study by calculating 59% of the GCL counts in the sham eyes. This yielded densities of 41.6 amacrine cells/mm in the vehicle group and 47.8 amacrine cells/mm in the eyes of the MS-275 treated mice. Because prior studies indicate that amacrine cell survival is not altered by optic nerve injury,
23,26 the average survival of amacrine cells in the crush eyes of the vehicle and MS-275 mice were likely similar to the average amacrine cell survival in the sham eyes of these experimental groups. Thus, subtracting the estimated amacrine cell density for each group from the observed crush eye GCL densities for each group yields RGC density estimates of 3.2 RGCs/mm in the vehicle-treated group and 9.1 RGCs/mm in the group that received MS-275. The 15% difference in the mean sham eye GCL counts in the vehicle and MS-275 groups, which likely reflects normal variation, can be compensated for as shown in footnote § for
Table 2. This adjustment yielded a final estimate of 7.9 RGCs/mm in the eyes of mice that received MS-275. As shown in the last column of
Table 2, these calculations indicate RGC survival was 147% greater in the crush eyes of mice that received MS-275 than in the crush eyes of mice that received vehicle. Hence, in addition to protection of RGC differentiation, MS-275 treatment also protected RGC survival.
In conclusion, these results indicate that the HDAC-1/HDAC-3 inhibitor MS-275 protects against the loss of RGC differentiation and promotes RGC survival following optic nerve injury. These results justify further studies to determine whether treatment with MS-275 or other histone deacetylase inhibitors may protect vision in pressure-related experimental models of glaucoma and in patients with optic nerve injuries such as ischemic optic neuropathy, optic nerve compression injuries, or glaucoma.