Biomedical Engineering Reference
In-Depth Information
concentration-response curves. The data were averaged from multiple experiments.
Best-fit concentration-response curves were generated using KaleidaGraph software
(Synergy Software, Reading, PA) using the equation Y¼M
1
þ
{(M
2
M
1
)/
[1
maximum Y value (100% in this case), M
2
¼
minimum Y value (0% in this case), M
3
¼
þ
(X/M
3
)^M
4
]}, where M
1
¼
the concentration corresponding to the
value midway between M
1
and M
2
(LC
50
), M
4
¼
slope of the curve at M
3
(best fit,
generating R
2
closest to 1), X
¼
concentration of compound, and Y
¼
percent lethality.
19.2.3.3 Visual Toxicity Assessment of Developing Embryos At 5dpf,
organs in five randomly selected embryos were inspected by light microscopy.
Body morphology, liver, intestine, and heart were assessed. Since drug treatment
was initiated at 24hpf, which is after circulation and heartbeat are present, but
before the liver and intestine are developed, observations are for effects on
developing organs.
19.2.3.4 BodyMorphology Defects in the development of midline structures,
including notochord and floor plate, often result in abnormal body shape in zebrafish.
To assess the potential drug effects on midline development, abnormalities in body
shape, including small body size and bent or missing tail, were examined.
19.2.4 Determination of Otoprotective Activity
19.2.4.1 Hair Cell Assessment
To damage inner hair cells, 5-day zebrafish
were treated with gentamicin (2.5
M) for 24 h to induce
apoptosis in inner hair cells. To test drug effects on protection of hair cell damage, test
compounds were administered at 0, 0.1, 1, 10, 100, and 250
m
g/mL) or cisplatinum (10
m
M concentrations with
either the gentamicin or cisplatinum treatment. The inner hair cells in the lateral
neuromasts were examined by 2-[4-(dimethylamino)styryl]-N-ethylpyridinium
iodide (DASPEI or 2,4-Di-Asp) staining.
m
19.2.4.2 DASPEI Staining Zebrafish were incubated with DASPEI solution
(1mM) for 2 h and rinsed thoroughly in fish water. Zebrafish were anaesthetized with
MESAB (0.5 mM 3-aminobenzoic acid ethyl ester, 2 mMNa
2
HPO
4
), and mounted in
methylcellulose in depression slide for observation.
19.2.4.3 Morphometric Analysis
Fluorescent signals (SS) were quantified
as SS
staining intensity of neuromasts by particle analysis (Scion
Image, Scion Corporation, Frederick, MD). Images of lateral sides in each animal
were obtained by the same exposure time and fluorescent gain (anterior on the left;
posterior on the right; dorsal on the top). The size of neuromasts was defined and
¼
staining area
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