Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 255

specified for particle analysis. Five animals for each treatment were quantified and
intensity of fluorescent signals was averaged.

19.2.5 Statistics

All data were presented as mean standard deviation (SD). Student's t-test was used

to compare vehicle-treated and drug-treated zebrafish. Significance was defined as
P

0.05, n ¼ 5.

<

19.2.6 Determination of General CNS
Neuroprotective Activity Against 6-OHDA-Induced
Neuronal Apoptosis

19.2.6.1 Microinjection of 6-OHDA 6-Hydroxdopamine (6-OHDA) is
extremely unstable in solution; it was prepared fresh for each injection. Three-day
zebrafish were anesthetized inMESAB and 500 mM6-OHDAwas microinjected into
the midbrain region. The estimated amount of injection was about 1-2 pM per
compound. Zebrafish were rapidly transferred to fresh fish water after injection,
allowed to recover for 30 min, and incubatedwith neuroprotective antioxidants.Water
alone was injected into zebrafish brain region as a vehicle control.

19.2.6.2 Treatment of Embryoswith1-12 Embryoswere exposed towater-

soluble fullerenes at 1, 10, 100, and 250 M for 24 h, and then microinjected with 6-

OHDA. Twenty embryos were treated with each concentration of each compound.

m

19.2.6.3 Morphometric Analysis Fluorescent signals (SS) were quantified

as SS staining intensity of neuromasts by particle analysis (Scion

Image, Scion Corporation, Frederick, MD). Images of dorsal sides in each animal
were obtained by the same exposure time and fluorescent gain. The positive staining
was defined by size and fluorescent intensity evaluation, and specified for particle
analysis. Five animals for each treatment were quantified and intensity of fluorescent
signals was averaged.

¼ staining area

19.2.6.4 Fluorescence Microscopy and Image Analyses All fluorescence
microscopy studies were performed using a Zeiss M2Bio fluorescence microscope
(Carl Zeiss Microimaging Inc., Thornwood, NY) equipped with a rhodamine cube, a
green FITC filter (excitation: 488 nm, emission: 515 nm), and a chilled CCD camera
(AxioCam MRm, Carl Zeiss Microimaging Inc., Thornwood, NY). Screens were
routinely done using 1.6 eye piece. The

system was also equipped with a z-motorized stage, deconvolution software, and 4D
reconstruction software (AxioVision, Carl Zeiss Microimaging Inc.), which permits

,10 , and 20 objective archromats and 10