Zebrafish: A Novel Screening Method


zebrafish.png

Zebrafish: Counting hair cells

Work by us and colleagues worldwide has shown that zebrafish hair cells have many properties in common with the inner ear hair cells of humans and other mammals. We have shown that they express common genes and are affected by the same chemical compounds as mammals. Since the zebrafish lateral line hair cells are on the outside of the fish, they are readily accessible to visualization and manipulation. Using a variety of fluorescent dyes, the health of hair cells is directly observable. The pictures (left) show a normal zebrafish with the fluorescent dye staining the hair cells of the lateral line. Normal hair cells appear in stereotyped locations. Treatment with chemicals that kill hair prevents fluorescent staining. Here, a five-day-old zebrafish larva is exposed to a concentration of 200 micromolar neomycin and exhibits almost total loss of hair cells.


Screening hair cell reaction to chemicals

Five-day-old zebrafish larvae can be placed in wells of a 96-well microplate. Each well contains normal zebrafish media, with additional compounds to test for potential toxic damage. In this way, hundreds or thousands of chemicals can be screened in a short time. Our early work demonstrated many important findings:

  • Low False Negative Rate: Screening libraries of FDA-approved drugs revealed that most known ototoxants caused hair cell death.

  • Low False Positive Rate: Screening libraries of FDA-approved drugs identified only a few drugs that were not known to be ototoxins caused hair cell death; however, many of these had case reports of hearing loss side effects.

  • Dose Response Curve: Known ototoxins, such as aminoglycosides antibiotics, showed a clear dose response curve of increasing hair cell death with increased dose of toxant.

screen-hair-cells.png

neomycin.png

Hair cells exposed to aminoglycosides

In the graph to the left, hair cell survival is plotted against the concentration of neomycin. Five-day-old zebrafish larvae are exposed to neomycin, and after one hour of incubation, removed, washed, and stained to count the surviving hair cells. The variability of the assay, shown by the standard deviation (SD), is very low.


protectants.png

Identifying hair cell protectants

Using the same techniques, it is possible to assess the combination of a hair cell toxin (neomycin) with a chemical to protect hair cells against the toxin. We screened a library of more than 10,000 chemicals looking for any that would protect the hair cells. Hits were examined in great detail with a full dose response curve to determine the range of dose that was effective and the HC50 (the dose that protected 50 percent of the hair cells). We also examined the effectiveness of the protectant against a range of doses of the toxin. The graph (left) shows the result of using various concentrations of our best protectant to protect against the full range of effective neomycin concentrations. The compound ORC-0001 (aka BPN-0001) was the most effective of the 10,000 chemicals in protecting zebrafish hair cells exposed to neomycin.