Modeling Drug-Induced Hemorrhage in Zebrafish Larva

Start with a culture dish containing fertilized zebrafish embryos in an embryo medium.

Position the dish under a stereo microscope and use dissection forceps to remove the chorion, the outer membrane surrounding the embryos.

This enables the embryos to be exposed to the surrounding medium.

Transfer these dechorionated embryos into a dish containing embryo medium with a drug that inhibits the synthesis of cholesterol, which is crucial for maintaining the brain's blood vessel integrity.

Incubate at a controlled temperature until the embryos develop into larvae.

Over time, the drug permeates the developing larvae, inhibiting cholesterol synthesis.

Cholesterol deficiency weakens the brain's blood vessels, causing them to rupture and leak blood into the brain, developing hemorrhagic spots.

Observe the larvae under a stereo microscope.

Separate the hemorrhagic larvae, with distinct red spots, from a non-hemorrhagic population.

Transfer these hemorrhagic larvae to a new dish for further studies.

At 24 hours post-fertilization, under a brightfield stereomicroscope, use sharp ultrathin dissection forceps to de-chorionate embryos for atorvastatin treatment. Then, add 30 milliliters of E3 embryo medium to two clean Petri dishes, one for treatment and the other for control. Remove 60 microliters of embryo water from the treatment dish, and add 60 microliters of 0.5 millimolar atorvastatin to achieve 80% of larvae hemorrhaged.

Using a Pasteur pipette, transfer 100 embryos in as little water as possible to each dish. Incubate the two dishes at 28 degrees Celsius. At any time after 50 hours post-fertilization, under the microscope, use a Pasteur pipette to carefully separate hemorrhaged fish from non-hemorrhaged populations and transfer the larvae to new dishes containing fresh E3 media.

To make it easier to separate hemorrhage-positive larvae, you could use fish without pigment or fish that express fluorescent protein in red blood cells.