ReviewScreening for chemicals that affect hair cell death and survival in the zebrafish lateral line
Highlights
► The zebrafish allows screening for chemicals that protect and damage hair cells. ► Protection screens yield many candidate protective drugs and small molecules. ► Toxicity screens can be used to identify dangerous drugs and drug combinations.
Introduction
Hearing loss has been recognized by the World Health Organization (2010) (WHO) as a growing, serious global health concern. In the United States alone, 15% of adults aged 20–69 report hearing impairment, and the prevalence of hearing impairment of seniors ranges from 60% to 90%, depending on the criteria used. In 2005, the WHO estimated that 278 million people worldwide had moderate to profound hearing loss, with 80% of them living in low- and middle-income countries. While much work has been done to alleviate hearing loss through hearing aids or cochlear implants, prevention should be our ultimate goal. Developing methods to prevent hair cell death is an obvious mission and, when successful, will likely eliminate a large percent of hearing loss worldwide.
Hearing loss prevention can come in a number of ways. Simply educating people worldwide about the dangers of noise exposure, chronic infection, and ototoxic injury would prevent a large percentage of hearing loss worldwide. In fact, the WHO estimates that roughly half of all cases of deafness and hearing loss would be avoidable through exposure prevention and early diagnosis. From a research standpoint, prevention can come by protecting hair cells and other cells of the inner ear from injury, and also by identifying agents that injure hair cells and supporting structures.
The zebrafish lateral line system offers a rapid and efficient model to evaluate the effects of large numbers of drugs on mechanosensory hair cells (Froehlicher et al., 2009; Coffin et al., 2010; Ou et al., 2010). Lateral line organs are composed of hair cells that share most properties with hair cells of the inner ear, including selective susceptibility to known ototoxins such as aminoglycoside antibiotics and chemotherapy agents (Williams and Holder, 2000; Ton and Parng, 2005; Harris et al., 2003; Ou et al., 2007). Their location on the body surface allows rapid analysis of the response to individual drugs and drug combinations. In this review, we will describe our experience using the zebrafish lateral line to screen and study chemicals that protect and damage hair cells of the inner ear.
Section snippets
Why screen for ototoxicity?
Ototoxins are drugs or other small molecules that cause inner ear damage. The number of well-established ototoxins is small for a number of reasons. For Food and Drug Administration (FDA) drug approval, most attention is focused appropriately on toxicity to larger organ systems such as the heart, liver, and kidney, but not the inner ear or hearing. In addition, there is no standard screen for ototoxicity. While drugs that cause dramatic hearing loss are ultimately identified (e.g.
Why screen for protectants?
A large number of drugs and chemicals have been proposed to protect the inner ear against damage. These compounds include agents such as antioxidants, caspase inhibitors, and jun-kinase inhibitors (Matsui et al., 2002; Wang et al., 2003, 2004; Sha et al., 2006; Campbell et al., 2007; Eshraghi et al., 2007; Feldman et al., 2007). In particular, studies with aspirin as well as d-methionine have shown some promising evidence that they may be able to partially prevent hearing loss in humans, and in
Previous chemical screens in zebrafish
The zebrafish larva has numerous qualities that make it an ideal model organism for screening. First, the zebrafish's small size and high fecundity make it useful for examining large numbers of treatment conditions. Second, the optically clear body facilitates phenotypic evaluation of a number of organ systems, including, but not limited to the heart, nervous system, eyes, ears, and lateral line hair cells. The zebrafish genome is also very well characterized and manipulated (Ekker et al., 2007
Hair cells of the lateral line can be rapidly assessed
In all aquatic vertebrates, the hair cells of the lateral line are external and directly exposed to the water surrounding the fish, where they are used to detect direction and changes in flow (Fig. 1). At five days post-fertilization (dpf), the larval zebrafish is approximately 3 mm in length. While a number of investigators had previously demonstrated that lateral line hair cells are susceptible to aminoglycoside exposure (Kaus, 1987; Song et al., 1995), in vivo imaging of individual zebrafish
Fluorescent dyes
Critical to in vivo studies of the zebrafish lateral line is the ability of hair cells to selectively take up a number of fluorescent dyes (Santos et al., 2006). Some of these deserve special mention for particular utility in screening. YO-PRO1 (Invitrogen) is a cyanine dye which binds to DNA and very brightly labels hair cell nuclei (Fig. 2). The staining is easily visualized for over 24 h and thus allows long-term imaging, or longer duration screens.
FM1-43 (Invitrogen) is well known as a
Disadvantages of the zebrafish
It is important to note some critical differences between zebrafish lateral line hair cells and mammalian inner ear hair cells. Hair cells of the lateral line essentially extend their stereocilia and kinocilia into the surrounding media. There is no separation of fluid spaces analogous to those of the inner ear (Fig. 1). Inner ear hair cells also come in several varieties (e.g. inner and outer hair cells) with distinct susceptibilities to ototoxins. Overall, any discovery made in fish must be
Chemical screening for hair cell protection
Our chemical screens have initially focused on protection, particularly against aminoglycoside-induced hair cell death. While different ototoxins likely activate different death pathways, it is also likely that there is overlap and convergence. Our hope is that a chemical or combination of chemicals found to be protective against aminoglycoside-induced hair cell death will also have potential applications against other causes of hair cell injury.
Chemical screening for hair cell toxicity
Screening for chemicals that protect hair cells has provided a number of interesting protective compounds. A simple modification of the protection screen transforms it into a screen for chemicals that are damaging to hair cells of the zebrafish lateral line. While any potential hair cell toxin must then be confirmed in mammalian systems, identifying chemicals that cause selective hair cell toxicity in a living organism is a promising method for identifying potential ototoxins.
Selecting a damage protocol
There are a number of technical considerations to be aware of when using zebrafish lateral line hair cells for chemical screens. Parameters for screening will obviously depend on the purpose of the screen. To identify potential therapeutic drugs aimed at protecting hair cells, a robust and invariant damaging paradigm is needed. Typically the lowest dose of damaging agent that still gives maximum damage without systemic lethality is used. Short incubation times are important to speed sample
Conclusion
The zebrafish lateral line is a unique and promising system that allows rapid and efficient in vivo evaluation of hair cells. As demonstrated in this review, chemical screening for drugs that affect hair cell survival is an evolving and dynamic process. With each new screen come changes and modifications in screening parameters and methodology. Screening for protection against a variety of ototoxic insults is possible, as is screening for other causes of hair cell toxicity. Subsequent testing
Summary points
- 1.
The zebrafish lateral line allows rapid and efficient in vivo screening for chemicals that protect and damage hair cells.
- 2.
Hair cell protection screens have yielded candidate protective small molecules and FDA-approved drugs.
- 3.
The lateral line has been used successfully to screen clinically approved drugs and bioactive compounds for hair cell toxicity and may be a useful platform to identify dangerous drugs and drug combinations in humans.
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