The overall goal of this procedure is to create a system that lacks cerebral spinal fluid or CSF within the embryonic brain ventricular system. This is accomplished by first manually draining embryonic CSF or ECSF from the zebrafish brain ventricles. Next, the ECSF is collected for further analysis of content.
Then potential factors are reintroduced into the drained brain ventricles. Finally, the effective removal of ECSF and reintroduction of factors on brain development is analyzed. Ultimately, the function of zebrafish ECSF can be shown through monitoring aspects of brain development, such as cell division, death, and neurogenesis.
Additionally, ECSF content can be determined by mass spectrometry. Using this technique, we can begin to study the function of embryonic cerebral spinal fluid during development. It can also be applied to other systems to further understand diseases which alters CSF composition and volumes such as hydrocephalus.
To begin, fill an oil injector apparatus with mineral oil according to the manufacturer's instructions. After pulling microinjection needles mount a needle on the micro manipulator connected to the oil injector. Carefully break the tip of the needle for uniform size.
Measure with a micrometer or compare to a reference needle. Fill the needle with oil using the oil injector to push oil down the length. Use a 200 microliter pipette tip to poke holes into a 1%agros coated dish, and remove the agros plugs.
Fill the dish with embryo medium under a stereo microscope. Use forceps to decco the embryos and to transfer them to an agros coated dish. Anesthetize the embryos by adding trica to the dish to drain the ECSF.
Orient the embryos with their tails in the holes of the agros and their posterior sides closest to the micro manipulator, allowing for visualization of the dorsal side of the brain. Position the needle at Therom OMERE zero slash one hinge point, or the widest point of hindbrain ventricle. Next, carefully pierce the roof plate of the hindbrain ventricle, being sure not to go through the depth of the brain into the yolk.
Use the oil injector apparatus to drain the ECSF and collect the fluid in the microinjection needle. Be careful to avoid any cells. Once the ECSF is collected in the needle, carefully stop suction from the oil injector.
Drain the replenished ECSF every two hours during the desired time interval between drainage time points. Remove the needle and store the embryos at 28.5 degrees Celsius or desired temperature. To collect the ECSF for further analysis.
First, move the dish out from under the needle and carefully position the needle into a tube containing the desired buffer. Using the oil injector drain the ECSF out of the needle into the buffer, trying to avoid contaminating the solution with oil to isolate cells from the ECSF. Spin it at 10, 000 G.Collect uncontaminated supernatant, and store it at negative 80 degrees Celsius until ready for further use.
To reintroduce selected factors, load a second needle with a test factor and inject one to two nanoliters into the brain ventricles shown here is an example of ventricles before and after drainage. The drained ventricles are collapsed due to lack of ECSF. These dorsal images show that the hind brain neuro epithelium does retain its characteristic morphology and appears to be open despite lack of ECSF likely due to robust hinge points.
However, lateral views demonstrate that the hindbrain ventricle has been drained consistent with the presence of a thin, flexible roof plate epithelium, which collapses ventrally in wild type embryos. ECSF is continually produced and refills the brain ventricles two to three hours post draining. Therefore, CSF needs to be continually depleted over the time of interest.
ECSF has a different protein profile than whole embryo extract and analysis on an SDS page, protein gel demonstrates that a detectable amount of protein can be collected from zebrafish ECSF. The gel shows that the collected protein contains higher levels of ECSF specific proteins indicated by asterisks than neuro epithelial cellular contaminants, and that a substantial amount of protein can be collected sufficient for mass spectrometry analysis. After watching this video, you should have a good understanding for how to manually drain ECSF from embryonic zebrafish brain ventricles for further analysis of its content and function of specific factors within the fluid.
