Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 239

Total distance traveled in the dark period

1400
1200
1000
800
600
400
200
0

Uninjected KD MD

Figure 18.6 Total distance traveled decreased in MD zebrafish. Mean distance traveled in a
10min dark period for uninjected, KD control, and MD zebrafish. Each bar represents meanSE
( n ¼15).

using histology. In mdx mice, presence of small muscle fibers exhibiting centralized
nuclei, which indicates regeneration of muscle cells subsequent to necrosis (Karpati
et al., 1988; Gibson et al., 1995; Rando et al., 2000), is a prominent MD phenotype,
characterized by histology (Karpati et al., 1988; Gibson et al., 1995; Rando
et al., 2000). As shown in Fig. 18.7, H&E staining of sagittal sections in 2-5dpf
zebrafish confirmed MD phenotypes.
By 2dpf, muscle fibers were small and exhibited centralized nuclei (short black
arrows). Myotome shape was irregular or boundaries were missing (Fig. 18.7, blue
arrows). Although in uninjected and KD control zebrafish, midlines were identifiable
at the tip of the V-shaped myotome structure (Fig. 18.7, long black arrows), in MD
zebrafish they were unidentifiable. By 3dpf, large vacuole spaces (Fig. 18.7, yellow
arrows) between muscle fibers, indicating detached sarcolemma and muscle degen-
eration, were frequently observed. By 5dpf, vacuole spaces were more prominent and
muscle structure was severely disrupted. In contrast, uninjected and KD control
zebrafish exhibited normal V-shaped myotomes (Fig. 18.7, white arrows), muscle
fibers appeared to be tightly packed, and nuclei were not centralized, indicating
normal structure of muscle fiber. Note that staining of uninjected and KD control
samples was performed in batches, which contributed to variable color in images.
These results indicate that KD control did not exhibit MD phenotypes, whereas MD
zebrafish exhibited phenotypes similar to those reported for both mdx mice and the
zebrafish sapje mutant (Bassett and Currie, 2004).

18.3.8 Number of Muscle Fibers Increased
in MD Zebrafish

We also analyzedmuscle structure on histology slides containing cross sections of KD
control and MD zebrafish (data not shown). In 1dpf MD zebrafish, number of muscle
cells exhibiting a thin cytoplasm layer and prominent, centered nuclei (regenerated
muscle fiber) increased and muscle fibers were smaller. In 2dpf MD zebrafish, muscle
fibers remained small with centered nuclei andmuscle fibers appearedmore abundant,