Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 205

CoCl 2 -treated zebrafish eyes were at the 5dpf stage and that CoCl 2 treatment did not
arrest zebrafish embryogenesis. Although under normal conditions, there are no
vessels in the zebrafish retina, immunohistochemical staining using Phy-V mAb
demonstrated that CoCl 2 -treated zebrafish exhibited abnormal vessels in the retina
(Fig. 16.2b, black arrows). Edema (Fig. 16.2b, green arrows), a phenotype present in
human CNV, and degenerated photoreceptors (blue arrows) were also observed.
These results show that CoCl 2 treatment induced abnormal vessels that penetrated
deep into the retinal structure.

16.3.4 Confirmationof HIF-1 a and VEGF Upregulation

by RT-PCR and In Situ Hybridization

Since CoCl 2 is known to upregulate HIF-1 , which can affect downstream VEGF

gene expression and induce abnormal angiogenesis, we assessed HIF-1

a

and VEGF
gene expression at varying time points after CoCl 2 treatment. RT-PCR results showed
that HIF-1

a

was upregulated 4, 6, and 48 h after CoCl 2 treatment (hpt). VEGF
upregulation was observed at 48 and 72 hpt, which, as expected, was later than
HIF-1

a

and VEGF gene expression for
untreated and CoCl 2 -treated eyes was the same (data not shown), possibly because
expression of both genes had plateaued. In situ hybridization confirmed HIF-1

a upregulation. At 96 hpt, level of HIF-1 a

a
upregulation in CoCl 2 -treated zebrafish eyes, which was particularly prominent in the
ganglion cell layer (data not shown). Using whole mount TUNEL staining, apoptosis
was also detected in CoCl 2 -treated zebrafish retinas (data not shown).

16.3.5 Inhibitory Effect of Antiangiogenic
Compounds on New Vessel Formation in Zebrafish

Next, we assessed effects of three compounds on CoCl 2 -induced CNV: Celebrex, a COX-
2 inhibitor, genistein, a natural isoflavone kinase inhibitor, and SU5416, a VEGF receptor
inhibitor. Celebrex and genistein have been shown to exhibit both anti-inflammatory and
antiangiogenic properties (Wei et al., 2004; Wang et al., 2005). In contrast, SU5416 is
antiangiogenic but not anti-inflammatory (Shaheen et al., 1999). To assess compound
effects, we explored different drug treatment regimens: pre-treatment, co-treatment, and
post treatment. We determined that co-treatment with CoCl 2 and antiangiogenic com-
pounds induced optimal effects. Using visual assessment, co-treatment with CoCl 2 ,
Celebrex, and genistein inhibited a higher level of CoCl 2 -induced angiogenesis than
SU5416. Furthermore, Celebrex and genistein also inhibited edema associated with
abnormal angiogenesis, whereas SU5416 did not (data not shown).

16.3.6 Development of a Quantitative Eye-Specific
ELISA

Taking advantage of (a) specificity of Phy-V mAb and (b) ability to dissociate intact
eyes, we developed a quantitative eye ELISA to assess drug effects on angiogenesis.