Biomedical Engineering Reference
In-Depth Information
maintained at 28
C in fish water (200mg Instant Ocean Salt (Aquarium Systems,
Mentor, OH) per liter of deionized water; pH 6.6-7.0 maintained with 2.5mg/L
Jungle pH Stabilizer (Jungle Laboratories Corporation, Cibolo, TX); conductivity
670-760
S). Embryos were cleaned (dead embryos were removed) and sorted by
developmental stage (Kimmel et al., 1995) at 6 and 24hpf. Because the embryo
receives nourishment from an attached yolk sac, no feeding is required for 7dpf.
m
4.4.2 Compounds
Compounds included clozapine, erythromycin, quinidine, terfenadine, astemizole,
amiodarone, lidocaine, verapamil, haloperidol, and clomipramine. Stock solutions
for each compound were made in DMSO and stored at
20
C before use. Fish water
containing 0.1% DMSO was used as vehicle control, terfenadine was used as a
positive control for assessing heart rate, and Celebrex was used as a positive control
for assessing heart function and morphology.
4.4.3 Determination of Concentration Range
Thirty, 2dpf hatched zebrafish embryos were distributed into six-well microplates
in 3mL fish water containing varying compound concentrations and exposed for
24 h. Untreated and 0.1% DMSO-treated zebrafish were used as controls. Five
concentrations, one log apart, were tested initially: 0.1, 1.0, 10, 100, and 500
M. If
no lethality was observed in the initial trial, additional concentrations were tested.
Best-fit concentration-response curve was generated using JMP statistical soft-
ware; Y ¼M
1
þ
m
{(M
2
M
1
)/[1
þ
(X/M
3
)^M
4
]}, where M
1
¼
maximum Y value
(100% in this case), M
2
¼
the concen-
tration corresponding to the value midway between M
1
and M
2
(i.e., LC
50
),
M
4
¼
minimum Y value (0% in this case), M
3
¼
slope of the curve at M
3
(best fit, generating R
2
closest to 1), X
¼
concen-
tration of compound, and Y
percent lethality. LC
0
and LC1
10
were estimated
using JMP statistical software (SAS Institute, Cary, NC). The appropriate con-
centrations were then used to perform for following assays.
¼
4.4.4 Heart Rate
Ten hatched, 2dpf zebrafish embryos were distributed into six-well microplates in
3mL fish water containing the compound at desired concentrations. Embryos were
exposed for 4 h prior to heart rate assessment. After drug treatment, zebrafish were
visually examined under a dissecting microscope (Zeiss Stemi 2000-C, Zeiss,
Thornwood, NY) equipped with a SPOT Insight 10ZNC digital camera
(Diagnostic Instruments, Inc., Sterling Heights, MI) at room temperature
(22-24
C). To facilitate observation, zebrafish were placed in 2%methylcellulose.
Atrial and ventricular rates (beats per min; bpm) were counted for 30 s with a
stopwatch and a counter.
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