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. Author manuscript; available in PMC: 2021 Dec 28.
Published in final edited form as: Dev Dyn. 2020 Feb 27;249(6):723–740. doi: 10.1002/dvdy.163

Figure 6. Pharmacological inhibition of P2RY12 signaling results in delayed clearance of apoptotic cells in the developing retina.

Figure 6.

Mpeg1:mCherry embryos were pretreated with PSB-0739 for 1 hour, then imaged with continual immersion in PSB-0739. Acridine Orange (AO, green) was used to label apoptotic cells in vivo. A. Selected z planes and timeframes are shown for imaging in the presence of PSB-0739. Isolated AO+ cells (arrows) are detected, which are ignored by microglia. Time stamp in upper right (hour:min) begins at appearance of the first AO+ cell (arrow, left panel). Scale bar in A applies to all panels. B. Box plots represent the total number of AO+ or PtdSer+ cells detected during 8 hours of imaging in control or PSB-0739 treated retinas (n=6-7 eyes from 6 embryos per group). Individual measurements are shown in circles and are overlaid on the box plots. A one-way ANOVA (p=0.00016) was performed, followed by Tukey’s HSD post-hoc test. P values shown for pairwise comparison with p<0.05; NS=not significant. An increased number of AO+ cells (late stage apoptosis) are detected in PSB-0739 treated retinas, but the total number of PtdSer+ cells detected in control and PSB-0739 treated retinas is not significantly different (NS). C. The phagocytic index (fraction of cells observed to be cleared by microglia) was determined for AO+ and PtdSer+ cells in control and PSB-0739 treated retinas. Box plots, with individual measurements (overlaid circles), are shown. A generalized linear model with binomial family was used, followed by one-way ANOVA (p=2.97×10−12) followed by Tukey’s post-hoc. P values are shown for pairwise comparisons with p<0.05, NS=not significant (n=6-7 eyes from 6 embryos per group). D. The time of PtdSer exposure from onset to engulfment was determined in control and PSB-0739 treated retinas. Box plots are shown, each overlaid circle represents the average of the measurements for each embryo. (n=7 eyes from 6 embryos per group) P value (Mann-Whitney U test) is shown. Results indicate that time from PtdSer exposure to microglial clearance is increased in the presence of PSB-0739.