Fig. 3
From: Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
Inhibition of nSMase increases apoptotic particles that promote degeneration. A Live spinning disk confocal images of tdTomato-expressing hRGCs (orange) labeled with polarity-Sensitive Indicator of Viability and Apoptosis (pSIVA, green) following treatment with DMSO (left), GW4868 with original medium (middle), or GW4869 with fresh medium (right). B Mean intensity of pSIVA over time from control (Ctrl, DMSO or no treatment) cells, cells treated with GW4869 and maintained in original medium, and GW4869-treated cells with fresh medium. DMSO 2 replicates, 15 regions of interest (ROI), GW4869 original medium 4 replicates 18 ROIs, GW4869 fresh medium 2 replicates 15 ROIs. The average intensity of pSIVA plotted over time with linear regression lines of best fit. pSIVA intensity continued to increase in GW4869 original medium cultures (p = 0.006). Exchanging original medium with fresh medium reduced pSIVA intensity and halted apoptosis (p = 0.6363). pSIVA intensity detected from GW4869 original medium cultures was significantly greater than DMSO and GW4869 fresh medium conditions at each time point (p < 0.001). We did not detect a significant difference in pSIVA intensity between control and DMSO conditions (p ≥ 0.31). C Density of pSIVA-positive particles detected for each condition. GW4869 increased the number of pSIVA-positive particles in both original and fresh medium conditions (p ≤ 0.03). Removing the original medium and exchanging fresh medium reduced the number of pSIVA-positive particles in GW4869-treated cells. D Example time-lapse images of an hRGC treated with GW4869 and maintained in original medium with a degenerating putative axon. E Quantification of pSIVA localized to putative axons relative to somas following 48 min of live imaging. A value of 1 indicates equal intensity in somas and axons. pSIVA intensity was enhanced along the axons of cells treated with GW4869 and maintained in original medium (p < 0.001). DMSO: 2 replicates 45 cells. GW4869: 4 replicates 79 cells. Statistics: (B) Linear regression. B Repeated measures Two-Way ANOVA, Tukey’s post hoc comparisons, (C) Kruskal–Wallis test, Dunn’s post-hoc comparisons, (E) Mann–Whitney test