Figure 3

Systemic 5-FU treatment causes cell death in the adult CNS. Cell death was determined using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. The number of TUNEL⁺ cells was analyzed in control animals (that received 0.9% NaCl i.p.) and 5-FU-treated animals and presented as percentage normalized values of controls at each time point. For ease of comparison, data presented in the figures show the control value (mean set at 100% of the day 1 value) and normalized values of 5-FU treatment groups at all time points. Each treatment group and the control group consisted of n = 5 animals at each time point. Figures show apoptosis in animals that received three bolus i.p. injections of 5-FU (40 mg kg^-1 on days -4, -2 and 0 leading up to the analysis, where day 1 of analysis equals 1 day after the last treatment with 5-FU). There was marked and prolonged increase of cell death in the 5-FU treatment group in (a) the lateral subventricular zone (SVZ), (b) the corpus callosum (CC) and (c) the dentate gyrus (DG) at 1, 7, 14 and 56 days and 6 months following treatment. Data are means ± s.e.m.; a two-way ANOVA test was performed on the original un-normalized data set to test the statistical significance of treatment effect and time effect. Bonferroni post-tests were performed to compare the 5-FU-treated group and the control group at each time point. The statistical significance of the Bonferroni post-tests is labeled in the graphs where applicable: ***p < 0.001; **p < 0.01; and *p < 0.05. Two-way ANOVA test results indicate that, in the SVZ, the treatment effect is extremely significant (p < 0.001), the time effect is very significant (p < 0.01); in the CC, the treatment effect is not quite significant (p = 0.06), the time effect is not significant (p = 0.74); in the DG, the treatment effect is extremely significant (p < 0.001), the time effect is significant (p < 0.05). The effect of the interaction between treatment and time is not significant for all three regions. (d) To determine the immediate cellular targets of 5-FU in vivo, we examined co-analysis of TUNEL labeling with antigen expression in animals sacrificed at day 1 after completion of 5-FU treatment. The majority of TUNEL⁺ cells in the SVZ and DG were doublecortin (DCX)⁺ neuronal progenitors. Other TUNEL⁺ cells in these two regions included GFAP⁺ cells (which could be stem cells in the SVZ, or astrocytes in the DG) and Olig2⁺ O-2A/OPCs. There was also a small contribution of NeuN⁺ mature neurons in the DG. In the CC, the majority of TUNEL⁺ cells were Olig2⁺ (which, in this white matter tract, would be oligodendrocytes and O-2A/OPCs), with a small contribution of GFAP⁺ astrocytes. Almost 100% of TUNEL⁺ cells were accounted for by known lineage markers. Each group consisted of n = 4 animals. Data are mean ± s.e.m.