The ultimate research question of my project is to figure out how we can manipulate the innate immune responses of the central nervous system to protect against viral infections. Currently we research their response to the profoundly antiviral protein, interferon beta. An experimental challenge is generating enough cells to allow for detailed investigation.
Each result needs to be validated using multiple techniques, and this can, unfortunately, result in many animals being culled. Due to the large number of cells generated by this novel culture technique, many different experimental conditions can be investigated from one pregnant mouse. Additionally, using in vitro alternatives where possible also greatly decreases animal suffering.
This method reduces and refines animal use closely following the principles of the three Rs.This culture method can be used to address many different research questions using a variety of readout methods such as Eliza, QPCR, microscopy, and flow cytometry. This technique could help other researchers to reduce and refine their animal usage. We hope to improve our understanding of the innate antiviral response, modulating it to protect against opportunistic viruses such as human polyomavirus two or John Cunningham virus, as it is commonly known, the primary agent of PML, which can be a severe side effect of immune suppressive drugs used to treat MS and other diseases.
Begin by placing a sacrificed pregnant mouse on its back on a dissection board. Using forceps, pinch the abdominal midline. With a pair of sharps scissors, cut the abdomen through the skin and the peritoneum from the genitalia to the rib cage over the midline.
Remove the uterus containing the embryos and place it immediately on ice. Carefully cut through the yolk sack on the placental sides and remove the embryos. Then place the decapitated embryonic heads into an iced dish containing calcium and magnesium-deficient HBSS.
Place the head into a 35 millimeter dish facing left. Pierce one eye with the edge of the forceps, firmly holding the chin with the other. Gently tear the scalp skin from the nape along the midline towards the snout.
Use the angled forceps to enter through the white oval spinal cord and crack the skull open along the midline, exposing the brain. Gently peel the skull away from the sides. Then lift the brain out of the skull and dispose of the skull.
Use forceps to remove the meninges. Place the brains in a bijou containing two milliliters of calcium and magnesium-deficient HBSS. Add 250 microliters of 10 times trypsin to the bijou.
Triturate the brains by shaking the bijou and then incubate it for 15 minutes at 37 degrees Celsius. Next, add two milliliters of soybean trypsin inhibitor to each bijou. Shake to disperse the inhibitor evenly.
Without centrifuging, transfer two milliliters of the supernatant from each bijou into a 15 milliliter centrifuge tube. Using a 19 gauge needle attached to a five milliliter syringe, aspirate the suspension twice to triturate the remaining cells in the bijou. Repeat aspiration twice with a 21 gauge needle.
Using a 23G needle, transfer the cells from the bijou into the 15 milliliter centrifuge tube containing the cell supernatant. And centrifuge at 200G for five minutes at room temperature. Using a five millimeter serological pipette, transfer all the supernatant into a new 15 milliliter centrifuge tube without disturbing the pellet.
Repeat centrifugation. Add 10 milliliters of the plating media to a tube containing the combined pellets from both centrifugation steps. And mix well to create a whole suspension.
Stain the cells with trypan blue and count using a hemocytometer or a cell counter. Plate the cells by adding the required volume of the murine E17 embryonic brain cell suspension into a well plate. Incubate the cells at 37 degrees Celsius under five to 7%carbon dioxide for two to four hours.
After removing the media, add new differentiation media to each well. Press down any floating cover slips using a sterile pipette tip. Maintain cultures by removing part of the supernatant and replacing it with fresh differentiation media three times each week.
Cultures were visualized by immunofluorescence staining for NG2 and nestin as developmental markers, SMI31, MBP, and NeuN as neuronal markers, and CNP, GFAP, and Iba1 as glial markers. Infection of cultures with neurotropic Semliki Forest virus affected the oligodendrocytes and the neurons. qRT-PCR investigations of the infected cells demonstrated upregulation of the chemotactic cytokine Ccl5 mRNA in cultures treated with interferon beta.
ELISA studies displayed increased Ccl5 in the supernatant, thus displaying a correlation between mRNA and protein expression. Flow cytometric studies of single-cell suspension showed that 70%of the cells remained viable. A large number of microglia, neurons, and astrocytes were present, while oligodendrocytes were the least abundant.
