Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 202

assessed effects in the zebrafish CNV model. Thirty 1dpf zebrafish were incubated
simultaneously with 0.1mg/mL CoCl 2 and test drug continuously in 6mL of fish
water for 4 day. Drugs were dissolved and serially diluted in DMSO before adding to
microwells; final DMSO concentration was 1%. After drug treatment, zebrafish were
washed with fish water and then processed for either whole mount immunostaining or
ELISA processing.

16.2.10 CNV ELISA

Whole mount immunostaining and eye extraction were performed as described
above; however, for the CNV ELISA, Phy-V directly conjugated with HRP
(Phy-V-HRP) was used as the antibody. Isolated intact eyes were distributed into
96-well micro/plates. To generate chemiluminescence signal, 150

L of PS-atto
(Beckman Coulter/Lumigen, Inc.) was added to wells and signal was measured
immediately using a Synergy HT microplate reader (Bio-Tek Instruments, Inc.,
Winooski, VT). A linear relationship was observed between number of eyes/well
and chemiluminescence signal 4 eyes/well was found to be optimal for ELISA
processing and drug assessment.

m

16.2.11 Statistics

Two-way ANOVA was performed for all drug-treated zebrafish. Drug effect was
considered significant if P <0.05 (95% confidence). All calculations were performed
using Excel (Microsoft Corporation, Seattle, WA).

16.3 RESULTS

In this research, we optimized conditions for generating a zebrafish CNV model for
drug screening by treatment with CoCl 2 . Next, we developed a method for
dissociating intact eyes from whole zebrafish. Then, we optimized a CNV ELISA
for drug screening. Since reducing oxygen level failed to upregulate VEGF
expression in zebrafish (Ton et al., 2003), we decided that it was unlikely that a
CNV model could be developed using this approach. CoCl 2 , an hypoxia mimetic, is
known to upregulate VEGF expression (Van Lieshout et al., 2003) and we hypoth-
esized that a CNV phenotype can be induced in zebrafish by adding CoCl 2 directly to
fish water.
To develop a reproducible zebrafish CNV model, we initially determined
optimum conditions for CoCl 2 treatment including compound concentration and
embryo stage. Recent research showed that anoxic and hypoxic atmosphere treatment
using zebrafish older than 1dpf stage resulted in rapid death (Padilla and Roth, 2001).
In addition, in early experiments, we observed that CoCl 2 treatment at 2dpf caused
instantaneous death. We therefore focused on finding the optimal CoCl 2 treatment
conditions using 1dpf zebrafish.