Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 199

16.1.4 Advantages of Zebrafish Ocular
Neovascularization Model

Development of the ocular vasculature in zebrafish is indistinguishable from devel-
opment in other vertebrates (Fouquet et al., 1997). Two days post fertilization (dpf),
both the choroid vasculature located at the back of the eye and the hyaloid vasculature
surrounding the lens are present. By 5dpf, the choroidal vascular plexus (CVP) is well
formed and hyaloid vessels appear to have regressed (Isogai et al., 2001). Since
choroidal vessels can supply nutrients to the entire retina, zebrafish do not require
retinal vessels. A number of genes involved in vertebrate angiogenesis, including
VEGF , Flk-1 , Ang-1 , Ang-2 , Tie-1 , and Tie-2 , have been identified and shown to
perform the same functions in zebrafish and mammals (Fouquet et al., 1997; Liang
et al., 1998; Lyons et al., 1998). Because early-stage zebrafish are completely
transparent and they develop in 96-well microplates in 100 Lmedia with no feeding,

assessing effects of chemicals on all aspects of vascular formation is straightforward.
Recent research showed that early-stage zebrafish (

m

24 h post fertilization (hpf))
can survive in anoxic or hypoxic atmospheres for 24 h (Padilla and Roth, 2001; Ton
et al., 2003). Although maintenance in a low oxygen atmosphere for 24 h increased
HIF-1

<

and other oxygen homeostasis-related gene expression, these conditions did
not increase expression of genes involved in vascular development and remodeling,
including VEGF (Christopher Ton, personal communication). An alternative method
to induce hypoxia response that can lead to changes in vascular development in
zebrafish was required. To generate a zebrafish CNV model, we then assessed use of
CoCl 2 as a hypoxia induction agent. Using this method, we demonstrated that
compound treatment upregulated HIF 1 a

a

and VEGF expression and induced abnor-

mal angiogenesis in CVP.

16.2 MATERIALS AND METHODS

16.2.1 Zebrafish Breeding

Zebrafish were generated by natural pairwise mating according to The Zebrafish Book
(Westerfield, 1993) and maintained in embryo water (5 g of Instant Ocean Salt in 25 L
of distilled water) at 28 C. Since embryos receive nourishment from an attached yolk
sac for the first 5-6 days of development, no additional maintenancewas required. For
ELISA experiments, Phylonix AB zebrafish embryos were used. For imaging
experiments, Phylonix albino zebrafish were used.

16.2.2 Chemicals and Reagents

All chemicals and reagents were purchased from Sigma (St. Louis, MO), except
Celebrex was purchased from Sequoia Research Product Ltd (Oxford, UK) and
SU5416 was purchased from Calbiochem, a division of EMD Biosciences Inc.
(San Diego, CA). Phy-V antibody, which labels activated endothelial cells