Pharmacokinetics in Zebrafish Embryos (ZFE) Following Immersion and Intrayolk Administration: A Fluorescence-Based Analysis

Immersion treatment: zebrafish embryos at the two-cell stage were immersed in Danieau’s medium containing the compound. A compound concentration of 10 µM and DMSO concentration of 0.1% (v/v) in a volume of 5 mL was used per well (6-well-plates). At 72 h, the zebrafish medium containing the compound was removed, and the animals rinsed 3 × with Danieau’s medium. Next, the embryos were kept for another 48 h in Danieau’s medium supplemented with DMSO (0.1%, v/v) in the absence of compound. In the case of control experiments, the embryos were exposed to Danieau’s medium supplemented with DMSO (0.1%, v/v). Intrayolk microinjection (IY): zebrafish embryos at the two-cell stage were positioned in a Petri dish at room temperature. IY microinjection was performed using glass needles fitted to a micromanipulater connected to a gas pressure microinjector. Needles were filled with compounds dissolved in the vehicle (DMSO/saline (1:1)), placed under the microscope, and using forceps, the tip was cut off in a manner to allow for a consistent volume to be injected. Afterwards, the embryos were transferred to 6-well plates. Control embryos were exposed to vehicle only. Previous to fluorescence imaging, the embryos were dechorionated (up to 72 hpf) and immobilized by hypothermia, rinsed three times with Danieau’s medium, and positioned latero-lateral (right lateral recumbency) on a single cavity glass slide and covered by a drop of agarose (0.1%). Then, the fluorescence in the selected area was quantified as integrated fluorescence intensity (RFU). The RFU values of the non-yolk compartment (i.e., RoB: rest of body) was assessed by subtracting the yolk results from the corresponding integrated fluorescence intensities found in the WB.