Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line

Abstract

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells—the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM–1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4–8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.

Cisplatin drives mitochondrial dysregulation in sensory hair cells

“…After establishing that sustained mechanical stimulation increases cumulative lateral-line hair cell mitochondrial activity, we then exposed fish to either media exchange or 250 µM cisplatin for 2 hours followed by 4 hours of recovery in EM (Figure 2A). This protocol produces approximately 30% hair cell death (32). Upon conclusion of the cisplatin exposure protocol, we obtained post-treatment images and counted the number of remaining hair cells in each neuromast compared to baseline (Figure 2, B-G).…”

“…As stated above, the mammalian cochlea demonstrates selective injury of hair cells in high frequency regions following cisplatin exposure. We have previously observed that the zebrafish lateral-line organ also experiences selective hair cell death in response to cisplatin, i.e., exposure to cisplatin appears to target a subset of neuromast hair cells in a dose-dependent manner (32). Since the lateral-line organ does not exhibit tonotopic organization, selective hair cell injury observed within zebrafish is surprising, especially when considering neuromast hair cells diffusely take up cisplatin within 15 minutes of exposure (33).…”

“…Still to be determined is whether modulating mitochondrial calcium transients during a critical window prior to the activation of Caspase-3 could provide protection from cisplatin induced hair cell death. One important difference between neomycin-induced and cisplatin-induced hair cell death in the zebrafish lateral line is that neomycin kills hair cells within minutes, while cisplatin-induced death takes hours (32). A key challenge to performing this experiment in cisplatin-treated zebrafish is determining when and how to inhibit hair cell mitochondrial calcium uptake such that it provides protection while minimally impacting normal hair cell respiration and metabolism (54).…”

Macrophage depletion protects against cisplatin-induced ototoxicity and nephrotoxicity

“…Previous studies have implicated macrophages in the context of cisplatin-induced hair cell loss. Lee et al ( 56 ) reported increased inflammatory responses, macrophage recruitment, and phagocytosis of hair cells in zebrafish larvae treated with cisplatin. However, selective depletion of macrophages did not affect the loss of hair cells in this model.…”

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish

“…FM1-43 experiments were performed with myo6b::gfp fish, raised to 5 dpf as above. Fish were exposed to drug conditions for 30 min, with 4 µM FM1-43 stain added into the drug treatment solution at minute 29, for a 1-min co-treatment stain exposure followed by simultaneous washout of all drugs and stain in fish water and anesthesia in 675 µM tricaine (selection of staining concentration and exposure duration was informed by ( Hernandez et al, 2006 ; Van Trump et al, 2010 ; Thomas et al, 2013 ; Monroe et al, 2016 ; Hailey et al, 2017 ; Majumder et al, 2017 ; Rocha-Sanchez et al, 2018 ; Stawicki et al, 2019 ; Parkinson and Stawicki, 2021 ; Lee et al, 2022 ; Derudas et al, 2023 ; Schrauwen et al, 2023 )). Four to seven fish per condition were transferred to 96-well plates for imaging.…”