Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line

Abstract

We have used time-lapse imaging to study cisplatin-induced hair cell death in lateral line neuromasts of zebrafish larvae in vivo. We found that cisplatin-induced hair cell death occurred much more slowly than had been shown to occur in aminoglycoside-induced hair cell death. By prelabeling hair cells with FM1-43FX, and assessing hair cell damage, it was established that cisplatin causes hair cell loss in the lateral line in a dose-dependent fashion. The kinetics of hair cell loss during exposure to different concentrations of cisplatin was also assessed and it was found that the onset of hair cell loss correlated with the accumulated dose of cisplatin. These data demonstrate the feasibility and repeatability of cisplatin damage protocols in the zebrafish lateral line and set the stage for future evaluations of modulation of cisplatin-induced hair cell death.

Figure 1

FM1-43 FX labeled neuromast (OC1) in a fixed zebrafish. Zebrafish were prelabeled with 3 μM FM1-43 FX, fixed in 4% paraformaldehyde, then mounted for fluorescence microscopy. Hair cells are easily counted in undamaged (A) and in neuromasts exposed to 1 mM cisplatin for 4 hrs. (B). Scale bar = 10 μm.

Figure 2

Cisplatin dose response relationship. Five day post-fertilization (dpf) zebrafish were prelabeled with FM1-43FX and then exposed to cisplatin for 4 hours. Fish were then fixed and hair cells from four neuromasts (SO1, SO2, O1, OC1) were counted. Hair cell survival was calculated as a percentage of the control group (not exposed to cisplatin). Data bars represent the mean hair cell survival (n=10 fish per cisplatin dose) ± S.D.

Figure 3

Timelapse microscopy of hair cells from single neuromast (OC1) in a living zebrafish exposed to cisplatin. Hair cell nuclei were prelabeled with YO-PRO1, exposed to 1 mM cisplatin, and then imaged at 30 minute intervals. The first timepoint measured was at 5 minutes as this was the time required to anesthetize and prepare the fish for timelapse imaging. Triangles indicate fragmented nuclei. Arrows indicate condensed nuclei. The bottom three panels are images from an unexposed control, demonstrating no significant hair cell loss over 240 minutes of imaging. Scale bar = 10 μm.

Figure 4

Timelapse microscopy of hair cells from single neuromast (OC1) in a living zebrafish exposed to neomycin. Hair cell nuclei were prelabeled with YO-PRO1, exposed to 200 μM neomycin, and imaged at the labeled timepoints. The first timepoint measured was at 5 minutes as this was the time required to anesthetize and prepare the fish for timelapse imaging. Note that as early as five minutes there was already evidence of hair cell damage. Overall, morphologic changes of hair cell death after neomycin treatment were seen much more rapidly than were seen during cisplatin exposure in Figure 3. Triangles indicate fragmented nuclei. Arrows indicate condensed nuclei. Scale bar = 10 μm.

Figure 5

Cisplatin hair cell survival curves with variable cisplatin doses. 5 dpf zebrafish larvae were labeled with FM1-43FX and then exposed to cisplatin at 50 μM (solid line), 100 μM (— — —), 250 μM (---), 500 μM (— - —), and 1000 μM (— - - —). Fish were then removed after 1, 2, 4, 6, 8, and 12 hrs of continuous cisplatin exposure. Hair cell survival was calculated as a percentage of the control group (not exposed to cisplatin). Data points represent the mean value (n=10 fish, per concentration and timepoint) ± S.D.

Figure 6

Linear regression of time required to achieve 50% hair cell loss as a function of cisplatin concentration (t_1/2). Cisplatin concentration is plotted on a logarithmic scale. Data points were calculated from linear regression functions from Table 1. Regression line depicted is represented by the formula t_1/2 = −5.5log(μM cisplatin) + 20 (r² =0.97).

Figure 7

Hair cell survival after treatment with 1 mM cisplatin for 2 hours followed by variable recovery periods. Data bars represent mean hair cell survival (n=10 fish) ± S.D. for each treatment condition. After 2 hours of cisplatin (CDDP) and no recovery, there was minimal hair cell loss. After 4 hours of recovery, there was progressive hair cell loss (one-way ANOVA, p<0.01). Extending recovery for 24 hours resulted in no significant additional hair cell loss. Six hours of continuous cisplatin showed additional damage compared to 2 hrs of cisplatin with 4 hours of recovery (one-way ANOVA, p<0.01).