Techniques that monitor the bacteria burden and a neutrophil respond simultaneously can provide insight into the mechanisms of Staph aureus persistence and the efficacy of treatment strategies. The main advantage of this technique is that multiple parameters can be measured longitudinally in a live host throughout the duration of infection. It is critical to show where to place the wound on a mouse dorsum, how to excise the skin, and how to inoculate the wound with Staph aureus.
Begin by thawing the bioluminescence Staph aureus strain of interest from negative 80 degrees Celsius storage on ice. Before streaking the culture on a 5%bovine blood agar plate. For an overnight incubation at 37 degrees Celsius.
The next morning, transfer three to four individual colonies from the Staph aureus plate into the tryptic soy broth, or TSB. Supplemented with 10 micrograms per milliliter of chloramphenicol for an overnight shaking incubation at 37 degrees Celsius. The next morning, dilute 120 microliters of culture sample in six milliliters of TSB.
With 10 micrograms per milliliter of chloramphenicol, for a two hour incubation at 37 degrees Celsius, and 200 rotations per minute. When the optical density at 600 nanometers reaches 0.5, transfer three milliliters of bacterial suspension into 10 milliliters ice-cold DPBS. After carefully decanting the supernatant, add additional chilled DPBS to the pellet, and vortex the re-suspended bacteria thoroughly.
After a second centrifugation, re-suspend the bacterial pellet in 1.5 milliliters of PBS on ice. To verify bacteria concentration, serially dilute 100 microliters of the bacterial suspension by a factor of 1 x 10 to the 4th, and 1 x 10 to the 5th, in fresh PBS. And plate 20 microliters of each dilution on individual agar plates.
After an overnight incubation at 37 degrees Celsius, count the number of colony-forming units by gross examination to calculate the bacterial concentration from the previous day. For excisional skin wounding of the recipient animals, confirm a lack of response to toe pinch, before shaving a one by two inch section on the back of the mouse. Gently wipe to remove any stray hairs.
And disinfect the exposed skin, with sequential 10%povidone iodine, and 70%ethanol soaked gauze applications. Moving outward from the center of the surgical area, in a spiral pattern. After allowing the skin to dry for about one minute, firmly press a sterile six millimeter punch biopsy at the center of the prepared surgical area.
Twist the punch biopsy to create a circular outline on the skin, that fully cuts through the skin tissue, and at least one section of the outline. Taking care to not to cut into the underlying fascia or tissue. Then use sterile scissors, and forceps, to cut through the epidermis and dermis, following the circular pattern imprinted by the punch biopsy.
For Staph aureus inoculation, fill a 28 gauge insulin syringe with 50 microliters of the prepared bioluminescent bacterial inoculant. And use a finger to pull the dermis of the wounded animal to the side. Holding the syringe nearly parallel to the tissue, slowly insert the syringe into the tissue until a sudden decrease in resistance is felt.
Indicating piercing of the fascia. With the needle placed in the center of the wound, slowly deliver the entire volume of the inoculant. Confirm that the inoculant forms a bubble at the center of the wound, with minimal leakage or dispersion.
And remove the syringe slowly from the animal. Then return the animal to it's cage with heat and monitoring until full recovery. After wounding and infection, and daily during the experiment, place the anesthetized mouse in a bioluminescent and florescence imager, with the wound as flat as possible.
In the imager software, select luminescence and photograph as the imaging modes. The exposure time should be preset to one minute at small binning, F/Stop 1 for luminescence, F/Stop 8 for photograph, and open for the emission filter. Then click acquire, to record the image.
For in vivo fluorescence imaging, select fluorescence, and photograph, as the imaging modes. The exposure time should be preset to one second at small binning, F/Stop 1 for fluorescence, F/Stop 8 for photograph, and Excitation and Emission wavelengths of 465 over 30 nanometers, and 520 over 20 nanometers, with a high lamp intensity, respectively. Then click acquire to record the image.
After obtaining both sets of images, return the animal to it's cage, with monitoring, until full recovery. For image analysis, open the image to be quantified, in an appropriate image analysis software program, and place the default circular region of interest over the entire wound area including the surrounding skin. Click measure region of interest, and record the values for the mean flux for the wound, to allow the mean flux of each signal to be plotted versus time.
To measure the wound healing, fit a circular region of interest over the wound edge, and measure the area of the wound in centimeters squared, to allow plotting of the fractional change from the baseline versus time. In this representative experiment, conditional EGFP expression myeloid differentiation primary response 88, Or MyD88 knockout mice, demonstrated a greater susceptibility to Staph aureus inoculation, than did non conditional EGFP expressing animals. With an 80%lethality observed during the first eight days of infection.
Both strains of mice lost approximately 5%of their body weights immediately following infection. However, the conditional EGFP expressing mice recovered their original weights by day two. While the MyD88 mice did not.
Wound closure in the presence of Staph aureus infection was not different between the two strains. However, sterilely wounded MyD88 knockout mice, had a significant defect in wound healing, compared to conditional EGFP expressing animals. Whole animal imaging revealed a higher bacterial burden, as evidenced by an increased bioluminescent signal in the wounds of MyD88 knockout animals, compared to conditional EGFP expressing animals.
A 40%decrease in initial neutrophil trafficking to uninfected wounds of MyD88 knockout animals, was also observed compared to conditional EGFP expressing animals. When 1 x 10 to the 7th colony forming units of Staph aureus were introduced immediately after wounding, neutrophil trafficking was significantly attenuated in knockout animals compared to conditional EGFP expressing animals. Before dispensing the bacteria, make sure there are no air bubbles in the syringe, and that the location of the needle is correctly positioned in the center of the wound.
Animal tissues can be harvested at end point to measure the expression of proteins of interest and bacterial dissemination from the wound. Staph aureus is infectious to humans. As such, personal protective equipment should be worn when handling the bacteria, and caution should be exercised, when handling a syringe containing the pathogen.
This technique has helped explore the use of biological treatments for Staph aureus infections that enhance the hosts innate immune response against infection.
