Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 238

Figure 18.5 ROS staining in MD zebrafish. Live 3dpf zebrafish, stained with H 2 DCFDA, are
shown at 3.2magnification; anterior, left. Extensive staining was observed inMD zebrafish. In contrast,
no staining was detected in uninjected and KD control animals. Note that fluorescence is detectable in the
gut (white arrows) because animals ingested dye. Scale bar indicates 200 mm.

18.3.5 Increased ROS Level in MD Zebrafish

ROS have been shown to play a critical role in muscular dystrophy pathogenesis
(Whitehead et al., 2006). Rando et al. provided evidence that in mdx mice, muscle is
more susceptible to ROS-induced damage (Rando et al., 1998) and increased ROS
production causes lipid peroxidation during the stage preceding necrosis (Disatnik
et al., 1998). There is also evidence indicating oxidative stress increases inMDpatients
(Rodriguez and Tarnopolsky, 2003; Grosso et al., 2008) and elevated levels of ROS
indicate oxidative stress, which can lead to inflammation (Whitehead et al., 2006). As
shown in Fig. 18.5, at 3dpf, ROS staining in MD zebrafish muscles, specifically at the
boundaries of the myotomes, was observed. In comparison, no staining was detected
in muscle tissue in uninjected and KD control zebrafish. Note that fluorescence is
detectable in the gut (Fig. 18.5, white arrows) because animals ingested the dye.

18.3.6 Decreased Motility in MD Zebrafish

Muscle strength is an important parameter for evaluating MD progression. During
development, zebrafish demonstrate stereotypical motility patterns; weak muscle
strength can decrease motility. In related studies, we observed that zebrafish movement
differs during alternating light and dark photoperiods and the dark period serves as a
clear trigger for increased activity (Emran et al., 2007; MacPhail et al., 2009).
To quantitate zebrafish movement, we used the VideoTrack System. Total
distance ( D ) traveled by individual zebrafish was recorded automatically for 60min
in alternating 10min light and dark photoperiods. We compared total distance
traveled in the dark period by MD, uninjected, and KD control animals (Fig. 18.6).
The difference between uninjected (726.8

245.5 mm) and KD (1049.5 187.5

mm) control zebrafish was insignificant ( P ¼ 0.3062). Distance traveled by MD

zebrafish (131.4 48.1 mm) was 18% (131.4/726.8 100%) of uninjected control

and 12% (131.4/1049.5 100%) of KD control zebrafish, significantly lower

( P ¼ 0.0310 and 0.0003, respectively).

18.3.7 Characterization of Zebrafish MD Disease
Progression by Histology

Since treatment at different stages of disease progression can result in different
therapeutic outcomes, we next elucidated progression of MD pathology in zebrafish