Progressively after this, an increasing proportion of cells exhibited stage III labeling. Imatinib Mesylate By 5 7 days post ONC, 50 60% of the cells in this layer were either stage II or stage III for H2AX, consistent with estimates that RGCs make up 60% of the cells in the GCL. To verify labeling in RGCs, we also retrogradely labeled them with fluorogold prior to ONC. After crush surgery, an estimated 85% of the fluo rogold positive cells exhibited stage II or stage III H2AX labeling. Figure 6 illustrates the change in HDAC3 and H2AX labeling in the retina as RGCs progressed through apop tosis. HDAC3 appeared to be cytoplasmic in control cells, and shortly after ONC, was found co local ized with stage II H2AX labeled cells in the cytoplasm.

In other cells, however, HDAC3 staining was present in the nuclei, while H2AX staining was still localized as a perinuclear ring in the cytoplasm. In mid to late stages post ONC, double labeled cells in the GCL exhibited stage III H2AX labeling and nuclear localization of HDAC3. Because of the strictly nuclear presence of AcH4, the colocalization of AcH4 and H2AX was much different than that observed for HDAC3. In control eyes, the nuclei were strongly labeled for AcH4 and H2AX was only present as a spot that was associated with the nucleoli. Early after ONC, RGCs with stage II H2AX localization also presented with strong nuclear labeling with AcH4. In stage III labeled cells, H2AX and AcH4 colocalization was initially yellow in merged images, but progressed to orange, then red, indicative of both an increase in H2AX intensity and a decrease in AcH4 labeling.

The nuclear localization of this co labeling was confirmed with DAPI staining, which also indicated that cells with nuclear H2AX and decreased AcH4 staining exhibited frag mented and condensed nuclei typical of apoptotic cells. Promoter deacetylation is associated with a downregulation of gene expression in injured RGCs To correlate the observed decrease in histone H4 acetyla tion with gene silencing, we determined if H4 deacetyla tion occurred in transcriptionally significant sites, such as the promoter regions of genes that are downregulated in RGCs in response to ONC. Following ONC, a number of genes are known to decrease in expression. A retrospec tive analysis of the literature indicated that the rapid decrease in transcript abundance for a majority of genes roughly follows an exponential decay curve.

To verify this in a controlled setting, we developed a mini qPCR array of several mRNAs expressed in RGCs and used it to monitor the change in transcript abundance in retinas over the first 7 days post ONC. Similar to the lit erature reports, this prospective study also showed the Entinostat exponential decay of RGC transcripts. Two sets of genes were examined for chromatin immunopre cipitation analysis.