The overall goal of this procedure is to study the mechanisms by which the bacteria neer meningitis infects human blood vessels in vivo. This is accomplished by first grafting human skin onto an immunodeficient mouse, thereby introducing human dermal blood vessels. In the second step, the humanized mice are infected with the bacteria.
The infection is then allowed to proceed for a predetermined time period. In the final step, blood and tissue samples are obtained for evaluating the outcome of the infection. Ultimately, a combination of histology, immunofluorescence, and blood and tissue analysis is used to study the progression of the infection in the human vessels.
The main advantage of this technique over existing techniques such as cell culture models or animal models, is that for the first time, we can observe the typical clinical pathology that takes place during these infections. As soon after collection as possible, clean the human skin surface with 70%ethanol, and then lie it flat on a well clean surface, preferably under a flow hood. Next, use a dermatome with a preset thickness to cut 200 to 400 micrometer slices from the top of the skin.
Check the slices for integrity, and then trim them further into two by two centimeter squares. Place the squares into PBS and then clean the shaved area of a surgically prepped anesthetize six to eight week old, severe combined immunodeficient beige mouse with 70%ethanol. After applying local anesthetic to the graft site, use curved scissors to carefully remove the superficial layer of mouse skin in an area slightly smaller than the graft pieces, taking care not to disturb the underlying vasculature.
Moving quickly to avoid drying out the graft, place one of the human skin slices over the graft bed, ensuring the epidermal side is facing up. Align the edge of the tissue and fix it with tissue glue. Then carefully fit the rest of the human skin to the graft bed, placing small amounts of tissue glue around each edge.
Always trim the human skin to size rather than make the graft bed larger. After confirming that each corner is fixed, clean the area with an iodine solution and apply a bandaid firmly but not tightly with the cushion side over the graft, taking care that the animal's breathing is not affected, then fix the bandaid with dressing tape and allow the animal to recover on a warm surface. Once a awake return the animal to its cage until the bacterial injection re inoculate an overnight plate culture into liquid culture.
The morning of the infection, when the bacteria reach the desired concentration, dilute the cells to one times 10 to the seventh colony forming units per milliliter. Then after anesthesia, confirm sedation of the grafted mice by toe. Pinch and place the animals on a heat pad.
Place eye ointment on the animal's eyes, and then ip. Inject each mouse with eight milligrams of human transfer in saline. Then for each animal, cut the tip of the tail and spread a small drop of blood from the tail onto an agar plate to confirm that the blood is sterile prior to infection.
Then inject 100 microliters of the bacterial culture intravenously via retroorbital injection. After a few minutes, snip the end of the tail again and spread this blood sample onto fresh agar plates as well. To establish the blood colony forming units immediately following infection, seal the tail with tissue glue and then dilute the bacterial inoculum and spread it onto agar plates with the appropriate antibiotics.
To establish the inoculum colony forming units at six hours post-infection, draw another small amount of blood from each of the mouse tails, and then dilute and plate the blood drops as just demonstrated to establish the colony forming units at six hours. Then incubate all the plates at 37 degrees Celsius in 5%carbon dioxide overnight to perform organ colony forming. Unit counts of the infected animals.
Begin by making a midline incision in each euthanized mouse. Then for each animal, cut through the rib and make an incision in the heart. Use a sterile syringe to withdraw as much blood as possible from the heart and chest cavity, and dispense it into individual sterile tubes for each sample.
Next, remove the organs of interest and then transfer the skin grafts and accompanying sections of mouse skin onto one sterile plate per animal. Allow the collected blood to clot for 15 minutes, and then spin it down. Then remove the plasma from the blood and store it at minus 80 degrees Celsius for later analysis.
Next, weigh homogenizing tubes containing 500 microliters of PBS each. And then use a sterile biopsy punch to take four millimeter biopsy samples from each dissected tissue. Place the biopsies individually into the homogenizing tubes with one two millimeter bead in each tube containing a skin sample.
Store the remaining tissues in 4%para formaldehyde at four degrees Celsius for microscopy, and weigh the homogenizing tubes to establish the biopsy weight. Then homogenize the samples at 6, 000 RPM for 30 seconds, repeating the skin homogenization until the homogenate is turd. Finally, spread the cell slurries from each tube onto fresh agar plates and grow the cultures overnight at 37 degrees Celsius in 5%carbon dioxide.
To establish the organ colony forming unit counts in these representative results. A GFP positive strain of N meningitis. Serogroup C was used.
Blood counts showed that five minutes after IV injection of one times 10 to the six colony forming units of bacteria, there was an average of 1.5 times 10 to the fifth colony forming units per milliliter circulating in the blood. After six hours, the counts average 4.8 times 10 to the fourth colony forming units per milliliter by 24 hours. The average counts were 2.4 times 10 to the fourth colony forming units per milliliter.
But in this group of 10 mice five had no detectable circulating bacteria while the other five had relatively high counts. These bars on these graphs show the median values for each group CFU counts taken from the skin. Samples show the majority of mice having considerable counts in the human skin, averaging 2.1 times 10 to the fourth CFU per milligram of tissue at six hours, and 4.4 times 10 to the second CFU per milligram of tissue at 24 hours.
The contralateral mouse skin samples exhibited no bacterial counts at 24 hours and only a very low number at six hours, demonstrating the strong preference for end meningitis to the human vessels in the grafted tissue. In general, bacterial counts were very low to non-detectable in the other organ sampled, although two animals did show counts, which may be correlated with high circulating bacterial numbers. This model also can be used to determine the role of virulence factors in vivo.
For example, in this experiment, bacterial strains lacking functional type four pill I were used mice infected with these mutant strains exhibited no bacterial adhesion in the human skin grafts, confirming the crucial role type four pill I plays in adhesion in vivo. In these confocal images of a skin graft, two hours after infection, human vessels stained with lectin UEA conjugated romine can be observed. The epidermal dermal border of the skin is clearly identifiable and the infecting GFP positive and meningitis can clearly be observed throughout the vessels.
This h and e stained human skin graft demonstrates inflammation and vascular leaks, as well as the thrombosis concentrated to vessels close to the epidermal dermal border. 24 hours after infection in about 30%of the infections, bacterial adhesions in the skin led to the development of macroscopically detectable purpura. So the development of this technique paves the way for researchers involved in research and host pathogen interactions to study the mechanisms of disease in the context of human vessels.
