Development of a Hypoxia-Induced Zebrafish Choroidal Neovascularization Model - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 204

Figure 16.1 Full view of intact CVP in the back of isolated eyes. Untreated (ND5, a) and
CoCl 2 -treated (b and c) 5dpf zebrafish were whole mount immunostained with Phy-V-Alexa 568. After
extensivewashing, zebrafishwere then incubatedwith 50 U/mL collagenase at 37 C for 40min and eyes
were dissociated from whole animals by gentle shaking. Isolated eyes were then placed on depression
slides with the CVP side facing up (8). Yellow arrows indicate individual vessels in the CVP of CoCl 2 -
treated eyes. Red arrows mark intense fluorescence staining, indicating highly activated ECs in CoCl 2 -
treated zebrafish eyes. Green box (c) indicates a large, abnormal, highly activated, EC aggregate. (d)
High-magnification image (100) of area defined by the green box in (c) clearly shows an abnormal EC
cluster. (See the color version of this figure in Color Plates section.)

differentiated outer segment cells in the photoreceptor layer (PC, red arrow).
Although CoCl 2 -treated 5dpf eyes (Fig. 16.2a, right panel, CoCl 2 D5) exhibited the
distinctive five-layer retinal structure, abnormalities included few cells in ganglion
layer (green arrow), narrow inner plexiform layer (short black bar), large vacuoles in
inner nuclear layer (INL, yellow arrows), and oval-shaped, less differentiated outer
segment cells in the photoreceptor layer (red arrow). Since 3 and 4dpf zebrafish do not
exhibit a distinct five-layer retinal structure, presence of this structure indicated that

Figure 16.2 Histological assessment of CoCl 2 -treated zebrafish eyes. One dpf zebrafish
were treated for 4 days with 0.1mg/mL CoCl 2 . (a) Histology showed that untreated 5dpf
eyes (ND5) exhibited a normal five-layer retinal structure comprised of densely packed cells in the
ganglion cell layer, wide inner plexiform layer (long black bar), and differentiated outer segment cells in
the photoreceptor layer (red arrow). CoCl 2 -treated 5dpf eyes (CoCl 2 D5) exhibited the distinctive five-
layer retinal structure. However, few cells in the ganglion layer (green arrow), short inner plexiform
layer (short black bar), large vacuoles in inner nuclear layer (yellow arrows), and oval-shaped,
undifferentiated outer segment cells in the photoreceptor layer (red arrow) were observed, indicating
retinal degeneration. (b) In severe cases, edema (green arrows), degenerated photoreceptors (blue
arrows), and abnormal vessels (black arrows) were observed (CoCl 2 ). Scale bar in (a) ¼ 50 m m.

(See the color version of this figure in Color Plates section.)