This method can help answer key questions in the bacterial pathogenesis field about whether bloodborne bacteria can cross the blood-brain barrier to infect the central nervous system and brain. With this technique, bacterial numbers within the blood and brain tissue of infected animals can be determined while excluding the number of bacteria residing within the blood vessels of the brain. Demonstrating the procedure will be Pallab Ghosh, a research associate from my laboratory.
At 72 hours post-infection, set an automated perfusion system to a flow rate of four milliliters of PBS supplemented with 10 millimolar of EDTA per minute. And place the euthanized animal in the supine position in a dissection pan. Confirm euthanization by a lack of response to paw pinch.
And wet the abdomen with 75%ethanol. Use scissors to make an abdominal wall incision to just below the rib cage to expose the diaphragm and visceral organs. And cut the diaphragm to open the thoracic cavity.
Cut the rub cage bilaterally to expose the left ventricle and use forceps to gently grasp the heart. Insert a 21-gauge butterfly needle connected to the perfusion system into the left ventricle and carefully cut the right atrium to collect about 200 microliters of blood into a two-milliliter tube containing 10 millimolar EDTA to prevent coagulation. Then perfuse the animal for four minutes with 15 to 20 milliliters of PBS plus EDTA.
After complete perfusion, the brain and liver will appear blanched ensuring that remaining bacteria are from the harvested organs and not the circulating blood. Following perfusion, carefully transfer the organs of interest into individual sterile, 15-milliliter conical tubes containing five milliliters of sterile four degrees Celsius PBS per tube. To harvest the brain after decapitation, use scissors to cut the scalp skin down the midline between the eyes, keeping the tips pressed against the skull to trim any excess tissue as necessary.
Gently insert one tip of the scissors into the foramen magnum and cut laterally into the skull on both sides. Carefully cut the skull between the eyes down the midline in the skull, taking care to apply lateral pressure and to avoid perturbation of the brain. Using forceps, open the skull to expose the brain and place a spatula between the underside of the brain and the base of the skull to carefully transfer the brain into a 15-milliliter tube containing five milliliters of four degrees Celsius PBS.
In a biosafety cabinet, insert the tip and run a tissue homogenizer for 10 seconds in sequential 5%bleach, sterile water, 75%ethanol, and sterile water solutions in separate 15-milliliter conical tubes. When the tip has been thoroughly sterilized, homogenize the infected brain in its tube. When no visible tissue fragments remain, re-sterilize the tip to prevent bacterial carryover to the next organ sample, and homogenize the next organ.
When all of the organs have been homogenized, prepare 10-fold serial dilutions of each organ homogenate in sterile PBS and plate the dilutions on brain-heart infusion agar plates. Then incubate the homogenate dilution cultures at 37 degrees Celsius overnight and count the number of colonies on each plate to determine the total number of bacteria per organ or milliliter of blood. Calculation of the bacterial burdens in L.monocytogenes-infected mouse organs as just demonstrated suggests that perfusion post-infection does not significantly alter the bacterial burden in the blood or organs of infected mice that were examined in this representative study.
Following this procedure, Listeria monocytogenes-infected organs can be further processed for additional analysis such as histopathological processing to answer additional questions about the infiltration of inflammatory cells into infected mouse organs compared to uninfected control animals. While attempting this procedure, it is important to remember to avoid perturbation of the brain and to maintain minimal contact between the scissors and the brain tissue.
