Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 206

Figure 16.3 Zebrafish eye dissociation. Zebrafish were treated with CoCl 2 from 1dpf stage to 5dpf.
Control and compound-treated 5dpf zebrafish were fixed and whole mount stained with Phy-V. Intact
eyes were removed by gentle shaking after treatment with collagenase (150U/mL, at 37 C for 40min).
Dissociated eyes were then distributed into 96-well microplates to perform the CNV ELISA.

Since individual zebrafish eyes are considerably smaller than whole animals, a
sensitive method was required to assess vessels and we used a chemiluminescence
HRP substrate to develop the CNVELISA. An overview of the zebrafish CNVELISA
is shown in Fig. 16.3.
We initially determined the optimal number of eyes/well for the ELISA by
establishing a linear relationship between chemiluminescence signal and number of
eyes per well (data not shown). Four eyes/well generated signal in the middle of the
linear portion of the curve, which permitted detection of both increase and decrease in
signal within the range. Next, using the CNV ELISA, we quantitated effects of co-
treatment of CoCl 2 and Celebrex, genistein, or SU5416. In order to compare results
from different assays performed on different days, drug effects were normalized to
percent inhibition of eye angiogenesis. Relative luminescent units (RLUs) for Phy-V-
HRP-stained zebrafish eyes were used to calculate drug effects. Background signal
from 5dpf zebrafish eyes with no antibody incubation was assessed and mean value
was subtracted from each RLU measurement. Mean RLU of CoCl 2 þ

DMSO control
was used as 100% control. Drug effect was calculated using formula (16.1):

RLU ð drug-treated Þ

percent inhibition ¼ 1 100

% :

ð 16 : 1 Þ

RLU ð control Þ

A typical dose response was observed for Celebrex and SU5416, whereas
genistein inhibited CNV only at a low concentration, 0.0001 mM. ELISA results
agreed with visual results. Based on dose-response curves (Fig. 16.4), optimum drug
concentrations were 20

M
(SU5416). These results demonstrated that the ELISA accurately quantitated angio-
genesis level in drug-treated eyes.

m M (Celebrex), 0.0001 m M (genistein), and 2.5 m

16.3.7 Confirm Drug Effects by
Immunohistochemistry

Since CVP in normal zebrafish forms from 2 to 5dpf, it is possible that inhibitory drug
effects quantitated by ELISAwere due to arrested eye development. To address this