Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 241

at 3 and 4dpf, respectively, clearly indicating muscle cell regeneration subsequent to
necrosis (Karpati et al., 1988; Gibson et al., 1995; Rando et al., 2000).

18.3.9 Drug Effects on MD Zebrafish

To validate use of ourMD zebrafishmodel for drug screening, we next assessed effects
of five drugs, prednisone, EGCG, MG132, TSA, and dantrolene, shown to reduce
symptoms in MD patients and animal models. To assess drug effects on myotome
length, we analyzed animals after treatment with the highest nonlethal concentration.
To determine drug effects on ROS level using fluorescence staining and quantitative
morphometric analysis, we assessed all three concentrations for each drug and
established concentration-response curves. Since myotome length was longer and
level of ROS was lower after prednisone treatment, we subsequently performed
histology on samples treated with this drug.

18.3.10 Quantitation of Myotome Length After
Drug Treatment

We next assessed drug effects on myotome length. Because myotome boundaries in
themajority of 4 and 5dpfMD zebrafishwere disrupted, using ImageJ software (NIH),
we measuredmyotome length in 3dpf animals. Data are presented as mean standard

error (SE) of the mean ( n 10). Drug effects were normalized to percent of vehicle

control (0.1%DMSO-treatedMD zebrafish) and results showed that, after prednisone
treatment, myotomes were significantly longer (125.1 3.8%) than vehicle-treated
control MD zebrafish ( P <

¼

0.0001) (Table 18.2). Although higher drug concentrations
were tested, increased toxicity often induced severe deformity or death.

18.3.11 Drug Effects on ROS Level

Next, we performed ROS staining as described previously. We focused on assessing
ROS level in the tail region, where muscle degeneration was observed. As shown in
Fig. 18.8, prednisone and EGCG treatment significantly reduced ROS staining,

Table 18.2 Drug Effect on Myotome Length

Drug Myotome length (percent of vehicle control) ( n ¼ 10) P

Prednisone 125.13.8

< 0.0001

EGCG 109.72.6 0.1136

Dantrolene 105.54.3 0.6305

MG132 104.73.5 0.3881

TSA 102.41.3 0.1485