This method provides a complimentary pre-clinical approach for studying the immediate cellular response to blood in the brain following a hemorrhagic stroke, a very serious condition for which we have no specific medications available for patients. Unlike rodent models, the transparency of zebrafish larvae allow us to observe cellular responses within the brains of live intact animals in real time using fluorescent microscopy. To begin, use a tea strainer to collect all fertilized embryos from natural spawning in breeding boxes produced from one male and one to two female adult zebrafish.
Transfer 100 embryos to each Petri dish containing standard E3 embryo medium. Incubate at 28 degrees Celsius and stage according to standard guidelines. At six hours post-fertilization, remove dead and unfertilized embryos from the dish using a Pasteur pipette and put the dishes back into the incubator.
At 24 hours post-fertilization, under a bright-field stereo microscope, use sharp ultra thin dissection forceps to decorionate 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. On the third day, under a fluorescent microscope screen the larvae to ensure the expression of fluorescent protein. Then fill the light sheet mounting chamber with E3 media containing 0.2%MS-222 for anesthetization.
Using a Pasteur pipette, transfer a single droplet containing one to six larvae to a dry Petri dish surface for mounting. Use a pipette to remove as much liquid as possible. Add a drop of 1.5%low melt agarose from the 45 degree Celsius heat block to the larvae and use an 800 micrometer mounting capillary to draw the larvae up head first.
If positioning is not accurate, expel the larvae from the agarose and mount again. Leave the capillary to cool and then insert into the light sheet chamber. On the ZEN imaging software, press continuous to orientate the larvae and press acquire to acquire z-stack images of the head between the eye lenses.
In the processing tab, generate a maximum intensity projection image from each z-stack. To randomly select for motility assay, transfer 24 larvae after anesthesia into fresh E3 media and allow the animals to recover from anesthesia. After larvae have fully recovered from anesthesia, using a pipette with the end cut transfer recovered larvae into E3 medium without methylene blue.
Plate one larva in one milliliter per well of a 24 well plate. Load the plate into the camera chamber. In the EthoVision XT tracking software, adjust the experiment settings to set up a white light startle routine to increase spontaneous swimming and assay motion for 10 minutes.
Repeat the locomotion assay at 96 and 120 hours post-fertilizatin. Assessment of brain cell death using a transgenic ubiquitin secreted Annexin V M venous reporter line results in clear definite clusters of dying cells in hemorrhaged larvae that are absent in all non-hemorrhaged larvae indicated by green fluorescence Bright-field images demonstrate the presence of brain bleeds. Dying cells were observed in both Atorvastatin and bubble head models for hemorrhaged larvae.
The morphology of the MPEG1 positive macrophages changes in intracerebral hemorrhage positive larvae as the cells adopt in active rounded amoeboid shape. These activated rounded cells were monitored over time to show an increased phagocytic response of the ubiquitin secreted Annexin V M venous expressing dying cells in intracerebral hemorrhage positive larvae. Brain hemorrhage is associated with a significant decrease in motility at 72 and 96 hours post-fertilization in comparison to intracerebral hemorrhage negative sibling controls.
Motility at 120 hours post-fertilization recovers to near baseline levels. The most important aspect of this procedure is to be sure to thoroughly examine the larvae for the presence or absence of brain hemorrhages before proceeding to the phenotyping assays. This model can also be used for drug screening to determine if phenotype severity can be improved upon after a bleed, an approach which may lead us to identify new drug candidates in the future.
This technique allows us to explore the cellular responses immediately after a bleed in the brain during a time point which has been so notoriously difficult to study before now. Although none of the reagents or instruments described within this protocol are specifically hazardous, standard care and precautions must be taken throughout whenever using chemicals, sharps, or lasers.
