The overall goal of this procedure is to isolate adherent inflammatory leukocytes from brain blood vessels of plasmodium burge eye anca infected mice to induce experimental cerebral malaria. Mice are infected with plasmodium berge eye anca parasitized red blood cells by injection. After the mice begins showing signs of disease, the circulating cells of malaria infected mice are removed by intraoral perfusion.
The brains are then harvested and the tissue is digested with enzymes to allow isolation of inflammatory leukocytes. Multicolor flow cytometry is then performed to immuno phenotype. The brain sequestered leukocytes analysis of the resulting data reveals the percentage and absolute numbers of leukocytes migrating to the brain of malaria infected mice.
This method can help answer key questions in the malaria pathogenesis field, such as what type of inflammatory cells migrate to the site of parasite sequestration in the brain of infected animals under different experimental conditions. Visual Demonstration of this method is critical. The perfusion steps are difficult to learn as they require both speed and care not to damage any significant blood vessels Demonstrating some parts of this procedure will be Leanna Mcic, an animal technician, Ms.Victoria, Rick, a PhD student, and Dr.Lisa Aez, a postdoc from my laboratory.
Begin this protocol by infecting the mice with p berge eye anca defrost a cryopreserved aliquot of P berge eye anca parasitized red blood cells or pbcs by allowing it to come to room temperature. Remove an eight to 12 week old donor mouse from its cage and place it on a workstation. Grasp the scruff of the neck between the forefinger and thumb.
Turn the mouse over and inject 100 to 200 microliters of plasmodium berge eye into the peritoneal cavity. Routinely two donor mice are injected with pbcs. After injecting, place the mice in their cage four to five days post-infection.
Remove a donor mouse from its cage and place it on a workstation. Place a drop of blood from the mouse's tail vein near the end of a frosted end microscope slide. Place a second slide at a 45 degree angle and back it into the drop of blood.
Once the blood spreads along the edge, push the spreader slide evenly across the microscope slide to make the blood smear. Allow the smear to air dry to fix the blood smears. Submerge the slides in 100%methanol for 30 seconds.
Then stain slides in freshly prepared GIM ZA for 10 minutes after staining with gim za, rinse the slide with running water for 30 seconds. Allow the slide to air dry, then examine it under the microscope using the 100 x oil immersion lens. Enumerate the PRBC within a thousand erythrocytes and calculate percent para if the donor mouse has reached the required levels of 2.5 to 5%Parsit Collect blood from the donor mouse by cardiac puncture under anesthesia or retroorbital bleeding using a heparinized micro hematocrit capillary tube.
Dissolve the blood in RPMI medium at a final concentration of one times 10 to the six PRBC per 0.2 milliliters per mouse. Consider that a normal mouse hematocrit is about six times 10 to the ninth red blood cells per milliliter. In fact, eight to 12 week old experimental C 57 black six mice by injecting one times 10 to the sixth PRBC in 0.2 milliliters intraperitoneal as before.
To monitor para levels of infected mice, prepare a blood smear as before. Para should be determined every two to three days starting on day two or three post infection from day five. Post infection onwards.
Mice are monitored daily for signs of severe malaria once para levels are greater than 5%and mice display neurological symptoms at around day six post-infection. The infected mice are euthanized by CO2 inhalation and immediately perfused with PBS as described in the accompanying document. The most difficult aspect of this procedure is the intracardiac perfusion.
A series of troubleshooting tips are outlined here With the mouse pinned on a styrofoam dissection board. Within a dissection tray. Wipe the ventral side with 70%ethanol, then use large scissors and forceps to open skin along the midline to expose the thoracic cavity, fold and pin skin to the sides.
Hold the sternum with fine forceps and cut the diaphragm and along both sides of the sternum, severing the ribs. Take care not to damage any large blood vessels. Pin the rib cage by the sternum loosely next to the head.
Hold ventricles with fine forceps and carefully incise right atrium with fine scissors. Insert 23 gauge needle attached to gravity perfusion system into left ventricle towards the ascending aorta. While PBS is running, insert only 0.5 centimeters of the needle tip perfuse the mouse for five minutes or until effluent is clear.
Following interal perfusion, dissect the brain and place it in a 10 milliliter centrifuge tube containing RPMI.Medium. Place the freshly harvested brain on top of a 40 to 60 mesh size stainless steel cell strainer in a Petri dish containing three to five milliliters of RPMI tissue culture medium. Cut the brain tissue into small pieces.
Then using a crystal plunger push small pieces of brain tissue through the cell strainer. Transfer the brain homogenate to a 10 milliliter tube and centrifuge it at 250 times G for 10 minutes at four degrees Celsius. Following the spin, discard the supernatant and dissolve pellet in three milliliters of RPMI.
Medium containing collagenase D and DNA swan. Rotate the mixture for 30 minutes at room temperature. Remove any cell debris by pushing the mixture through a 70 micron nylon cell strainer.
Then incubate on ice for five minutes following the incubation in a 10 milliliter tube. Carefully overlay the brain homogenate on a seven milliliter 30%per call cushion centrifuge at 400 times G for 20 minutes at room temperature with the break off. After discarding the supernatant, resuspend the pellet in one milliliter of red blood cell lysis, buffer, and incubate on ice for five minutes.
To lyse adherent pbcs, which are not removed from the brain by intraoral perfusion. Add nine milliliters of RPMI medium wash cells and centrifuge at 250 times G for 10 minutes at four degrees Celsius. Resus suspend the brain sequestered leukocytes or BSL containing pellet in 50 to 100 microliters of RPMI medium and transfer cells to a micro centrifuge tube count viable cells under the microscope using a hemo cytometer and trian blue exclusion.
Six days post-infection, 20, 000 to 100, 000 BSLs can be recovered once the viable cell counts have been obtained. The cells are prepared for analysis by flow cytometry. The details of this procedure are given in the accompanying document, but are summarized here first.
All of the cells isolated from each brain are centrifuged in a 1.5 milliliter micro fuge tube and are resuspended in staining buffer with anti CD 16 and CD 32 blocking antibodies. After a 10 minute incubation on ice, the cells are washed with staining buffer centrifuged again and resuspended in staining buffer containing anti CD four, anti CD eight anti TCR and anti NK 1.1 fluorescent antibodies. The cells are incubated on ice for one hour washed with staining, buffer and centrifuge.
After the spin, the cells are resuspended in PBS and at least 5, 000 to 10, 000 events are acquired on a flow cytometer. The resulting data is analyzed using appropriate flow cytometry software. BSL populations were recovered from brains of perfused or UN perfused, malaria infected and naive control mice and stained with pe, anti NK 1.1 and a PC anti TCR beta antibodies.
These dot plots depict percentages of NK cells, T lymphocytes, NK 1.1 positive TCR positive cells and double negative cells. NK 1.1 negative TCR negative in perfused or unfused, malaria infected and naive control mice consistent with previous findings. Alpha beta TCR positive T cells comprised a high proportion of the BSL pool in brains of perfused malaria infected mice on day six post infection.
This population appeared to be significantly underrepresented in brains of non perfused animals. The apparent reduction of T-cell frequencies in non perfused brains was associated with a considerably higher percentage and total number of double negative cells in these animals. High percentage and numbers of double negative cells in un perfused brains were not only detected in malaria infected mice, but also in naive controls suggesting that these cells are non-inflammatory leukocytes present in brain blood vessels that are recovered together with inflammatory cells if intracardiac perfusion is not performed.
While attempting this procedure, it's important to remember to perform extensive intra cardio profusion of infected mice prior to organ extraction to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells. After watching this video, you should have a good understanding of how to isolate and analyze brain sequestered leukocytes from plasmodium virga infected mice by flowy geometry.
