Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

To monitor the recruitment of neutrophils — essential immune cells that facilitate bacterial clearance, begin with an anesthetized, transgenic, immunocompromised mouse with a wound exposing the connective tissue on its back.

The transgenic mouse's neutrophils express an enhanced green fluorescent protein, which facilitates neutrophil tracking. Additionally, their impaired bacteria-killing ability makes the mouse more susceptible to infection.

Take a genetically modified bacterial inoculant possessing bioluminescence — light-emitting characteristics.

Inject the bacterial inoculum at the wounded site, ensuring its delivery between the connective tissue and the fascia — a fibrous tissue layer beneath the connective tissue. At the injection site, the bacteria proliferate and colonize, causing tissue infection.

In response to the infection, the immune cells release chemokines — chemical signaling molecules that attract the neutrophils. The neutrophils rapidly migrate via chemotaxis — chemical-induced movement, and extravasate through the blood vessels into the tissue in the vicinity of the bacterial infection.

Image the mouse for bioluminescence and fluorescence signals, representing the bacterial burden and the recruited neutrophils, respectively.

Over time, an increase in the bioluminescence and fluorescence signals correlates with the increased bacterial burden and continual neutrophil recruitment at the infection site.

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 its 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 fluorescence 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 1 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 1 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 its 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.