To determine if there was a decrease in acetylation of histones in injured RGCs, concomitant with the increase in HDAC activity and increased nuclear presence, we examined retinal cryosections taken from control and experimental retinas with a polyclonal anti body against pan acetylated histone H4. selleck chemicals Gemcitabine Figure 4A shows low magnification images of the INL and GCL, and higher magnification images of just the GCL. Nuclear labeling was detected in both the INL and GCL of the control retinas. After ONC, the label intensity remained consistent in the INL, but there was an apparent and progressive decrease in labeling of the GCL. High magnification images indicated that the decrease in label intensity was not only due to a loss of cells in this layer, but also to a decrease in the labeling of individual cells within this layer.

To verify the decrease in label intensity of individual nuclei in the GCL, fluorescent pixel inten sity of cells in this layer were measured and normalized to pixel intensity of AcH4 labeling in the INL of the same section. Compared to control eyes, the average AcH4 label intensity of GCL nuclei in experimental retinas pro gressively decreased to 45% by 5 days post ONC. By 7 days post ONC, many RGCs are in the later stages of apoptosis. Consistent with this, we observed an actual increase in the average AcH4 label intensity of cells remaining in this layer at 7 days. Although this average was still significantly below control levels, it may reflect that unaffected amacrine cells made up a larger proportion of nuclei assayed at this time point.

Characterization of HDAC3 translocation and deacetylation of histone H4 in dying cells The increase in nuclear HDAC3 localization and an apparent decrease in histone H4 acetylation in some cells of the GCL following ONC is consistent with the concept of widespread histone deacetylation taking place in dying RGCs, which are the principal cell type affected by the crush procedure. Importantly, we wanted to verify that these changes were characteristic of dying cells. To address this, we first identified dying cells in retinal cryo sections with an antibody against phosphorylated H2AX. The phosphorylation of the histone H2A vari ant, H2AX, is a known marker of apoptosis, and its appearance coincides with early onset DNA damage that precedes mitochondrial involvement in the cell death program.

In the GCL of injured retinas, H2AX underwent a progressive change in localization that allowed us to group the cells into three stages of H2AX labeling. Stage I labeling was principally found in control retinas and in some GCL cells in injured retinas and was characterized by little to no H2AX label ing except for a densely labeled Anacetrapib spot associated with the nucleoli. Stage II was distinguishable by strong perinu clear labeling, while stage III had strong nuclear labeling.