Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 177

Pgp substrate, rho-HRP. After injecting rho-HRP into the midline of the optic tecta
bordering the cerebellum, we assessed Pgp efflux by measuring active efflux of rho-
HRP from the brain tissue into the lumen of brain microcapillaries. Since rho-HRP
was injected directly into brain tissue, the concentration was higher than in brain
microcapillaries, creating a concentration gradient that facilitated active rho-HRP
efflux from brain tissue to the lumen of microcapillaries (Fig. 14.2a). Moreover, since
rho-HRP does not cross the BBB (Jeong et al., 2001), after it is effluxed, it cannot
diffuse back into brain tissue. To monitor variability in the brain microinjection
procedure, initially we injected rho-HRP into various sites in the brain and examined
level of fluorescence in ROI, which included the midbrain and hindbrain. In these
studies, we determined that the optimum injection site was the midbrain-hindbrain
junction (Fig. 14.2b, red arrow).
Since rho-HRP efflux kinetics can vary depending on treatment conditions, in
order to select the optimal time point for drug assessment, we next treated 7dpf
animals with DMSO or verapamil, a known Pgp inhibitor, and compared efflux
kinetics (Choo et al., 2000; Potschka and Loscher, 2002; Hung et al., 1996). As shown
in Fig. 14.4, dorsal view images of ROI were captured at T0, T1, T2, and T4.
Compared to signal at T0, level of fluorescence decreased at T1 and T2 for both
control and verapamil-treated zebrafish. At T4, low fluorescence intensity was
detected in 0.1% DMSO control animals; however, fluorescence intensity remained
strong in verapamil-treated animals, indicating inhibition of Pgp efflux.

14.3.3 Optimization of Quantitative Morphometric
Analysis

Although inhibition of rho-HRP efflux could be assessed visually, quantitative
analysis was expected to increase accuracy. Using quantitative morphometric
analysis, fluorescence image was quantified from threshold-processed brain images
using the automated measurement function of ImageJ software, as described in
Section 14.2. We quantified total fluorescence in ROI for DMSO control and
verapamil-treated zebrafish (Fig. 14.4). Images for 10 animals from each group
were analyzed and mean and standard error (SE) of the mean were calculated and
compared (Table 14.1).

Table 14.1 Quantitation of Rho-HRP Efflux in Control and Verapamil-Treated Zebrafish

Percentage of dye retention

Time point 0.1% DMSO Verapamil (100 mM)

T0 100 100

T1 48 6 61 7

T2 29 5 44 6

T4 39 6 46 10

Data expressed as meanSE ( n ¼10).