Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 178

Using this quantitative data, efflux kinetic curves were plotted (data not shown).
Based on these kinetic curves, at T2, the difference in percent dye retention between
DMSO control and verapamil-treated zebrafish was significant (nonoverlapping error
bars). After T2, percent dye retained in the brain plateaued (the higher percent
retention observed at T4 compared with T2 may be due to drug toxicity). Percent dye
retained in verapamil-treated zebrafish was higher than in DMSO control zebrafish.
Based on these results and taking into consideration (a) higher experimental vari-
ability at T2 and (b) the potential for drug toxicity at T4, we decided to use T0 and T3
to quantify rho-HRP efflux for all subsequent studies.

14.3.3.1 Validation of Assay Conditions Using an Additional Pgp
Inhibitor To further validate optimum conditions for drug screening, we
assessed an additional Pgp inhibitor, phenytoin. As shown in Fig. 14.5, left
panels, using the same gain and exposure time we acquired fluorescence images
of ROI (25

) in each animal for each treatment condition. Next, we applied a constant
threshold to brain images, which were pseudocolored red (Fig. 14.5, right panels). As
previously described, to eliminate artifacts, the threshold was set automatically by the
software and adjusted manually, when necessary. We then calculated percent
fluorescence retention at each time point. As shown in Fig. 14.5, percent

Figure 14.5 Morphometric analysis
of fluorescent images. Fluorescent
images of ROI in individual zebrafish
were captured at 25 . T0 and T3

images of DMSO control and 100

m M

phenytoin-treated zebrafish were
acquired, as shown in left panels. The
same threshold was applied to images
that were then pseudocolored red
(right panels). Total fluorescence
intensity (area of ROI average
fluorescence intensity) in the
pseudocolored ROI was quantitated
using ImageJ software. Dye retention
was 15.8% (2,211,762/
14,132,040100%) for DMSO-
treated animals and 77.4%
(13,618,250/17,597,328100%) for
phenytoin-treated animals. Anterior
to the left; white scale bar is 100 mm.
(See the color version of this figure in
Color Plates section.)