The overall goal of this procedure is to evaluate cerebrovascular structures in embryonic zebra fish. This is accomplished by first collecting fertilized zebra fish eggs, and setting up treatment conditions. After a growth period of three days, the zebra fish are sacrificed fixed, and an eye is carefully removed before mounting the specimen on cover glass.
Then the cerebro vasculature is imaged by confocal microscopy. Finally, 3D renderings of the vasculature are generated using open source software. Ultimately, analysis of the reconstructions is used to determine effects of the treatment conditions on blood vessel branching and density.
In the developing zebra fish brain. This technique evaluates the entire vascular system of embryonic zebrafish. It's allowed us to study the biology of amyloid beta at an organ systems level.
Visual demonstration of this method is critical as the dissection and image analysis are difficult to learn because they're technically challenging. To begin, raise a transgenic zebrafish strain expressing enhanced green fluorescent protein in the desired tissues at 28.5 degrees Celsius with a 14 hour light, 10 hour dark cycle. In this example, transgenic fish that express EGFP in vascular endothelial cells are raised the night before egg collection set up males and females in mating tanks with mesh bottoms The following morning, remove the divider and allow the fish to mate checking for eggs every 15 minutes.
After using a mesh strainer to collect the eggs and E three medium to wash them, use E three to transfer the eggs to 100 millimeter culture dishes and incubate them at 28 degrees Celsius to alter cerebrovascular branching. Beginning at 24 hours post fertilization, add a chemical such as a gamma secretase inhibitor solubilized in DMSO. If desired, add 0.03%PTU to inhibit pigment formation.
After transferring the embryos in solution to the wells of a 12, well plate incubate at 28.5 degrees Celsius until the embryos reach the desired developmental stage. After sacrificing embryos in trica and fixing them with aldehyde overnight at four degrees Celsius. Store embryos in PBS in the dark at four degrees Celsius.
To remove the eye from the embryo, use a sharpened tungsten needle and cut around the tissue connecting one eye. Then cut the muscles and finally the optic nerve to remove the eye. Once the eye is removed, turn the embryo over onto a cover glass with its empty eye socket facing down and touching the cover glass.
Add a drop of 3%Methylcellulose to hold the embryo in place and to cover the entire embryo to prevent desiccation. During imaging using an inverted confocal microscope with a 20 x plan APO objective image, the embryo immediately, many imaging systems will only be able to resolve vascular structures halfway through this specimen. If this is the case, resus, suspend the embryo in PBS and remount it on the contralateral side for further imaging to carry out 3D reconstruction of cerebrovascular images using image J, which is optimized for 3D renderings.
Import confocal stacks by going to plugins, loci, bio formats importer, and then select the confocal file for Nikon stacks select view stack with hyper stack and set the color mode to gray scale. Then check auto scale and split channels. These selections will open four separate channel panels.
Close all but the 16 bit image with the file name followed by c equals one. Adjust the threshold by using the scroll tab along the bottom to scan through to find a slice that has the region or structure of interest. Then go to image adjust threshold.
In the panel that comes up, slide the top bar to the left so that the structure can be seen quite well over background and leave the bottom slide where it is to create a new eight bit image. Select b and w. Then dark background.
Do not select calculate threshold for each image unless different adjustments are required for each slice. Next, go to plugins 3D viewer threshold zero resampling factor one or two, and deselect the red and blue color boxes to make a green 3D output. Otherwise, it will produce a white rendering.
Then select apply. Use the mouse and keyboard controls to rotate, spin, and zoom the 3D image to save still images at any point. Use the capture option in the menu.
The size of the image box on the screen dictates pixel dimensions of the image produced, so if a high resolution image is desired, make the box larger by dragging the bottom right corner. Create a spinning 3D movie by selecting view record 360 degree rotation. Finally, save the file in one of several available formats and use the free open source media player VLC for viewing 3D.
Reconstruction of vascular structures provides a comprehensive and visually interesting perspective of zebra fish development. Shown here are several angles of vascular structures in a six day post fertilization zebrafish embryo that expressed EGFP in endothelial cells with monochrome green or white colors. It can be difficult to appreciate signal intensity, but pseudo coloring provides image intensity from a lookup table, which allows for better depth perception of overlapping structures.
For example, this figure shows a pseudo colored 3D image of the vasculature in a six day post fertilization zebra fish After its development. This technique allowed us to explore the vascular effects of amyloid beta in a vertebrate brain. After watching this video, you should have a good understanding of how to visualize the vascular networks in embryonic zebrafish, which provides an economical and accessible system to study angiogenesis.
