The overall goal of this procedure is to prepare sex stratified hippocampal, primary dissociated neuron cultures. The first step of the procedure is to sex and dissect embryos on embryonic day 18, to obtain sex stratified primary neuronal cultures. The next step is to prepare associated neurons from the neuronal tissue.
The final step is to plate the neurons at the appropriate density and grow them for experimentation. Ultimately, this method in conjunction with other protocols can be used for any study in which sex is thought to be an important determinant of outcome. This method can help answer key questions in the gender research field, such as whether male and female rodents respond differently to neuronal ischemia Of the several required preparations, there were a few worth reviewing, such as the tissue culture plates.
Preparation dilute poly de lycine to 0.1 milligrams per milliliter. Then under a laminar flow tissue culture hood generously cover the bottom of each well of a multi-well culture treated sterile tissue culture plate with the diluted poly D lycine seen incubate these plates overnight at 37 degrees Celsius. Second, be sure to wipe down the dissection area the counter and scope with 70%ethanol.
Third, fill a large flat dish with ice label and fill enough 60 millimeter dishes with cold dissection solution to keep the male and female heads brains and hippo campi separated. Also, fill two larger 100 millimeter culture dishes with cold dissection buffer. Fourth, prepare two 15 milliliter sterile tubes.
Label one male and the other female to each of these tubes add the appropriate volume of dissection buffer. When harvesting E 18 embryos, it is important to carefully remove each pup from the uterine horns and the embryonic sack. Because the pups are fragile, carefully cut the uterine tissue between each embryo to expose the embryonic sack.
Then very carefully slice the sack on the surface with the edge of a small narrow scissors. The embryonic fluid and the embryos can then be gently pushed through the opening without damaging the embryos. When dissecting the pups, use curved forceps with repeated sweeping motions to very gently push the intestines and liver cran.
This gradually exposes the kidney's bladder, and most importantly, the internal sex organs. When determining the sex of the fetus, it is very important that you dissect gently so as not to disturb the very fragile internal sex organs. Male pups are identified by the presence of striated testes found just below the kidneys.
The testes are approximately one third to one half the size of the kidneys and will occasionally have dropped to sit just above the bladder. Females are most readily identified by a lack of testes. They can also be identified by the small uterus that sits above the bladder and fallopian tubes that extend up toward the kidneys.
Under higher magnification, the ovaries can be identified as small dark organs at the end of the fallopian tubes immediately below and sometimes underneath the kidneys to harvest the brains. After splitting the skull, gently push the skin and skull aside. Then very gently extract the brain by carefully scooping it from the forebrain towards the brainstem with the base of the microsurgical scissors or with a closed angled forceps.
Carefully use the forceps to transfer the extracted brain to a clean labeled dish filled with cold dissection.Buffer. Keep the dish on ice. Repeat this process until all brains have been harvested.
This step should yield two plates of whole brains in dissection buffer, one for male and one for female tissue. Before dissecting the hippocampus, very carefully remove the meninges from each hemisphere. It is the pinkish membrane rich with blood vessels.
Delicately pinch, lift and pull it. The membrane being careful not to pierce the brain tissue beneath. Then pull the meninges away from the brain.
The brain can roll a bit beneath the forceps, but should stay in place. The mouse brain may then be dissected according to which area of the brain is being cultured and studied. Placed the dissected brain pieces in the correctly labeled dish filled with dissection, buffer, and kept on ice.
After dissecting out the last brain, transfer them to appropriately labeled 15 milliliter conical tubes. Next, gently wash the tissue. With cold dissection buffer, the tissues can be lost to a vacuum, so allow the tissue to settle to the bottom of the tube and remove as much liquid as possible from the tube.
With a pipette, replace the removed solution with fresh clean dissection buffer. Then repeat the wash step after treating the hippo campi with pappa and changing to neuro basal medium, gently dissociate the tissue with gentle tation using a five milliliter serological pipette set on the slowest speed. Avoid introducing bubbles by always ejecting onto the side of the tube.
After the eighth tri, allow the tissue to settle. Then add two milliliters of neuro basal medium to the unassociated tissue and repeat the tri process. Repeat this process adding up to 10 milliliters.
Some unassociated tissue will remain, but further tation results in poor neuronal health. Determine the cell density and plate them on the prepared plates. Gently swirl the cell suspension and pipette the desired volume of suspended cells.
In this example, 150 to 200, 000 cells per square centimeter are plated. Before loading each plate, be sure to always swirl the cell suspension. Once a plate is filled, gently swirl it to disperse the cells and then immediately transfer it to the incubator.
Leave the cells undisturbed for a minimum of 24 hours. The next day, warm, complete neuro basal feeding media to 37 degrees Celsius. After 24 hours of growth, aspirate the plating media from each well one well at a time and replace it with feeding media.
Then return the plate to the incubator every four to seven days. Carefully replace half the medium with fresh feeding medium, the cell cultures can survive two weeks while gradually losing density. DNA was isolated from the harvested tissue of each embryo.
Sex PCR was performed using primers for the male specific gene, SRY, and the universal marker. Myo genin. The results confirm the sex specificity of the two tissue pools in male primary hippocampal neuron cultures pharmacological inhibition of PARP one with PJ 34 was found to be neuroprotective against oxygen and glucose deprivation, but was not seen in female embryos.
Male neuron cultures prepared from PARP one knockout mice were also protected against oxygen and glucose deprivation or OGD induced cell death treatment of wild type male neurons with the TPM two channel inhibitor.Two. A PB in addition to PJ 34 had no further neuroprotective effect against OGD two. A PB alone is neuroprotective to OGD in wild type males, but two.
A PB is not protective in male or female PARP one knockout mice. These data support the hypothesis that PARP one activity exacerbates neuron cell death in male but not female neurons through the activation of the TRP M two channel. Following this procedure, other methods such as oxygen, glucose deprivation, western blotting or QPCR can be performed in order to answer additional questions.
