Our group is interested in the neurodevelopmental as well as the environmental factors that influence whether or not brain tumors will form in a child. We are addressing how to obtain both brains and serum from the same embryonic mice, so we can reduce the total number of animals needed for a given study. The brains allow us to study neurodevelopment, and then the serum allows us to link those observations to systemic factors.
This protocol allows us to collect both brains and serum reproducibly from embryonic mice. We are able to obtain higher volumes of serum, and the serum is non-hemolyzed. We have recently identified that maternal obesogenic diet exposure affects neurodevelopmental phenotypes, it increases the risk of optic pathway glioma formation in neurofibromatosis Type 1-related mirroring models.
We developed this protocol in order to determine whether these findings are related to changes in the macronutrient profiles. We will focus on the mechanism by which obesogenic diet affects neurodevelopment, and increases the risk of NF1 optic pathway gliomas. To remove the fetus from the properly euthanized dam, place the animal ventral side up in a 100 millimeter dish.
Pinch the skin covering the abdomen, and with dissection scissors, make a midline incision. Make additional incisions perpendicular to the midline incision to create skin flaps, exposing the uterus containing the fetuses. Pull the uterus out of the abdominal cavity.
Using scissors, disconnect the uterus from the dam. Transfer the intact fetal sack containing the fetuses, resembling pearls on a string, into cold, sterile PBS in a dish. Place the dish of fetuses on ice, to eventually harvest fetal serum and brain.
Proceed to harvest the fetal serum from the fetal sac previously isolated from the dam. Using a number five forceps, carefully dissect the fetal sac to remove the fetus for serum collection. With curved forceps, transfer the fetus to a dish contain fresh, cold, sterile PBS.
Gently blot the fetus on a paper towel to remove residual amniotic fluid and PBS from the specimen. Hold the fetus by the abdomen, partially decapitate the fetus with micro scissors by making an incision at the ventral neck. Ensure the blood vessels are cut, but the skin keeping the head is still attached.
Hold the body at a 45-degree angle with the incision facing down. Hold a 50-microliter minovet or capillary tube parallel to the incision site where the blood forms a droplet. Carefully allow the capillary tube to fill with blood.
Place the head in a fresh 60-millimeter dish containing cold, sterile PBS for further tissue collection. After the blood is collected within the capillary tube, press the plunger quickly into a 1.5-milliliter tube Flick, tap, or quickly spin the tube to ensure the blood is in the bottom. Centrifuge the blood samples at four degrees Celsius and 16, 000 G.Using a 200-microliter pipette, transfer the serum to fresh 500-microliter tubes without disturbing the blood cell palate at the bottom of the tube.
Proceed to collect the brain from the previously isolated head of the fetus under a dissecting microscope. Using curved forceps, hold the snout and use a number five forceps to carefully peel off the skin from the skull cap, keeping it attached to the snout for ease of holding. Holding at about 75 degrees relative to the brain, run the forceps along the midline until the clear membrane of the developing skull parts to each side.
Shift the forceps parallel to the cranium, insert it underneath the skull cap, and pinch, and peel to one side. After one side is removed, grasp the remaining side with the forceps and peel to the other side. Using a micro spoon, gently scoop the brain out of the skull.
Transfer the brain to a fresh 60-millimeter dish containing cold PBS. Using the curved forceps, hold the brain at the junction of the hindbrain and forebrain, with the non-dominant hand. With the dominant hand, hold the scalpel with the blade parallel to the surface of the brain, then, press down firmly at the mid-cortical region.
Still holding the hindbrain, make a second incision in front of the hindbrain to separate it from the forebrain. In the middle section, look for a small linear space on the ventral surface, which is the third ventricle. Using number five forceps, carefully pinch off the lining on either side of the ventricular surface, then, transfer it to a fresh, clean, 1.5-milliliter micro centrifuge tube.
Place the tube quickly into a CryoSafe container of liquid nitrogen.
