Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 175

14.2.8 Drug Treatment

For drug treatment, 7dpf zebrafish were deposited into 6-well plates containing 3mL
of sterile fish water per well. Drugs were dissolved in DMSO and diluted by fish
water to 1, 10, and 100 M for testing; final DMSO concentration was 0.1%. Animals

were treated with drugs 1 h before injecting rho-HRP. To ensure zebrafish remained
in either control or drug-treated condition during dye injection and image capture,
zebrafish were placed on slides with 0.32mM tricaine mixed with either fish water
(control condition) or drug solution (drug-treated condition) to immobilize animals,
and image acquisition began 15 min after rho-HRP injection, T0. Zebrafish were
maintained in drug solution throughout image acquisition at T0, T1, T2, and T4.
0.1%DMSO was used as vehicle control. Mean % retention at the optimal time point
for 10 zebrafish in each condition was then determined and used to assess
drug effects.

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14.2.9 Statistics

ANOVAwas used to determine if drug effects were significant ( P 0.05), followed by

Dunnett's test (pairwise comparison) to identify which concentrations that caused
significant effects.

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14.3 RESULTS

The overall aim of this research was to develop a zebrafish bioassay to identify
potential Pgp efflux inhibitors. Pgp has been shown to be the most important efflux
transporter, affecting drug absorption, distribution, metabolism, and excretion
(ADME). Design of drugs that are not Pgp substrates is one strategy for increasing
drug retention in the brain (Mahar Doan et al., 2003). Alternatively, a Pgp efflux
inhibitor can be coadministered with drug to enhance retention in the brain (Kemper
et al., 2001).

14.3.1 Identification of Optimal Zebrafish Stage
for Assessing Pgp Efflux

To optimize assay conditions, we first determined the stage at which Pgp efflux is fully
functioning. We injected rhodamine 123, a fluorescent Pgp substrate shown to cross
BBB in rats and cows (Wang et al., 2005; Fontaine et al., 1996; Rose et al., 2005), into
zebrafish circulation at 3, 4, 5, 6, and 7dpf stages. Lateral images of zebrafish head
were captured 2 h post injection (hpi). Fluorescence was detected in zebrafish brain
(yellow boxed region) at 3, 4, and 5dpf (Fig. 14.3, yellow lines), indicating that dye
crossed BBB and was retained in the brain region. However, no fluorescence was
observed in 6 and 7dpf zebrafish brains (blue arrows), confirming that Pgp efflux
system is fully functioning by 7dpf.