Figure 4

Analysis of cell death phases in a mouse model of Aβ-induced RGC death. (a, b) Wide-angle retinal images showing the Aβ-treated right eye (a) and Aβ+MK801-treated left eye (b) of a single mouse. MK801 reduces the number of annexin V (green)- and PI (red)-positive cells 72 h after intravitreal Aβ(25–35) application. To assess the magnitude of late apoptosis, we studied the co-localisation of PI and annexin V using ImageJ software with the Intensity Correlation Analysis plugin.¹² (c, d) Scatter plots show the distribution of annexin and PI fluorescence intensity in the eyes shown in (a, b), Aβ alone (c) and Aβ+MK801 (d). (e) Thresholds were set automatically by the plugin for each analysis (colour overlay in (c, d)), and quantification of each cell death phase was performed from generated data (e). MK801 treatment reduced the overall levels of cell death (blue, total), mostly by reducing the number of cells in late-phase apoptosis (yellow, Anx+/PI+) and necrosis (red, PI+). (f, g) Wide-angle retinal images showing the Aβ-treated right eye (f) and Aβ+PMA-treated left eye (g) of a single mouse. (h, i) PMA increased the level of annexin V (green) and PI (red) positivity, as shown in the corresponding scatter plots. (j) Quantification of all treated animals shows that PMA significantly (P<0.05) increases the overall levels of cell death (blue, total), most of this being due to a significant (P<0.05) increase in the number of cells in the early phase of apoptosis (green, Anx+). (k) Comparison of cell phases in all Aβ and SSP models using co-localisation scatter plot analysis. At the previously established time points of maximal apoptosis (Aβ=72 h and SSP=2 h), both models show a similar trend, with no significant difference in the magnitude of any phase of cell death. However, both models show significantly more RGCs in the phases of late apoptosis (P>0.05, yellow, Anx+/PI+) and necrosis (P>0.05, red, PI+) than early apoptosis (green, Anx+). Error bars 95% confidence intervals, ^*P<0.05