Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 74

VEGF activates these two pathways differentially is unknown (Lawson and
Weinstein, 2002; Covassin et al., 2006). Once the dorsal aorta is specified, angiogenic
sprouting of the intersomitic vessels (ISVs) can occur. After ISVs are created, new
vessels sprout off the PCV. Other vessels develop throughout the body as well,
providing blood flow to all organ systems (Baldessari and Mione, 2008).

7.3 MORPHOLOGICAL AND FUNCTIONAL ASSAYS
TO ASSESS TOXICITY

There are many assays available to test the zebrafish hematopoietic and vascular
systems. Here we describe methods to look at blood cell morphology and function,
as well as vascular function. If a compound induces a toxic effect on the blood or
vascular systems, the function and morphology will likely be affected. When
treating zebrafish embryos with drugs for these experiments, treatment should
begin at one to five somites, when hematopoietic and endothelial progenitors are
first specified. Drug-treated animal samplesshouldalsobecomparedtountreated
and vehicle controls.

7.3.1 Embryonic

During early zebrafish development, there are several methods of assessing devel-
opment of the hematopoietic and vascular systems. By 24hpf, the first erythroblasts
enter circulation (de Jong and Zon, 2005) and can be visualized in a standard
dissection scope. The number of blood cells in circulation increases as definitive
hematopoiesis begins. To assay the morphology of these cells, peripheral blood can
be removed from the embryo for visualization. Cells can then be stained with
May-Grunwald and Giemsa stains to allow cell identification by morphology.

Isolating Peripheral Blood from the Zebrafish Embryo (Chan et al., 1997)

1. Submerge the zebrafish embryo in 0.9 phosphate-buffered saline (PBS).

2. Using a sharp scalpel or razor, cut across the tail.
3. Allow the blood cells to collect in PBS.
4. To collect, use a glass pipette.

Staining with May-Grunwald and Giemsa

1. Stain with May-Grunwald for 10min.
2. Rinse with water.
3. Stain with Giemsa diluted 1:5 for 15min.
4. Rinse with water. Look at slides to confirm that the staining is visible. If not,
repeat step 3.
5. Allow the slides to dry before analysis, which may include differential cell
counts to determine percent of total for each blood cell type.