Our protocol allows bacterial dissemination and burden tracking in an experimental neonatal sepsis model in real time, and the ability to correlate other markers of disease with the pathogen burden. This protocol allows us to longitudinally analyze sepsis in individual neonatal mice, providing the opportunity to observe and compare infection dynamics and bacterial dissemination in real time. Preclinical testing of interventions for neonatal sepsis can be performed with this method, allowing for the monitoring of the effects of host control of the bacteria.
Begin by placing age-matched pups into either high or low-dose litter groups within a biosafety level two cabinet. On postnatal day three or four, record the weights of all the pups prior to inoculation. Load insulin syringes with PBS or the high or low-dose E.coli lux inoculum in the biosafety cabinet and place the loaded syringes on ice.
Place the neonate to be injected onto a clean surface within the biosafety cabinet and raise the skin at the nape of the neck as if to scruff the pup. Insert the needle bevel up just beneath the skin in the space created between the skin and the muscle. When the needle can be felt under the skin, inject 50 microliters of the inoculant while simultaneously releasing the pinched portion of the skin to prevent backflow, then remove the needle slowly and with care.
When all of the pups have been injected in the same manner, return the pups to their dams. Immediately after the injection and at the appropriate experimental time points thereafter, place the cage with E.coli lux infected neonatal mice and dam into a biosafety level two laminar flow hood and open the software in the micro CT computer. After initializing the system and waiting for the stage temperature to lock at 37 degrees Celsius and the CCD temperature to lock at negative 90 degrees Celsius, place up to four anesthetized pups onto the imaging box in the imaging chamber in the prone position.
Shut the imaging chamber door and select the luminescence imaging option. Select open filter and next and set the excitation filter to block and the emission filter to open. Select 500, 520, 560, 580, 600, and 620 nanometers.
Then image the pups before returning them to the cage with the dam with monitoring until anesthesia recovery. At the appropriate experimental endpoint, in a biosafety cabinet, douse the euthanized neonate with 70%ethanol to prevent contamination and place the animal on its right side. Use forceps to grasp the skin between the abdomen and rear left leg and use fine tip surgical scissors to make a skin incision.
Then continue incision toward the back of the animal until the entire spleen is exposed. Use the forceps and scissors to remove the spleen from the abdomen and place it in the solution appropriate for its downstream application. To obtain the lungs, peel back the skin of the chest completely and entering at the base of the sternum with the scissors held vertically, cut upward until the rib cage is split.
Use forceps to grasp the right and left lungs individually and remove the lungs from the thoracic cavity. Remove the heart from the lung tissue with scissors. Then place the lung in the solution appropriate for its downstream application.
To perform an in vitro bacterial killing assay, place the uninfected spleen into a 40 micrometer nylon strainer within a sterile 60 millimeter Petri dish containing five milliliters of PBS supplemented with 10%fetal bovine serum and use a sterile three milliliter syringe plunger to disaggregate the tissue into a single cell suspension. Transfer the cells to a 15 milliliter centrifuge tube and collect them by centrifugation. Resuspend the pellet in one milliliter of red blood cell lysis buffer per three to four spleens.
After five minutes at room temperature, wash the spleens in PBS and resuspend the pellet in 250 microliters of PBS supplemented with bovine serum albumin and two millimolar EDTA for counting. Next, isolate the Ly6 B2 positive cells with the appropriate immunomagnetic beads according to manufacturer's protocol and seed the enriched Ly6 B2 positive cells at a one times 10 to the five cells per 100 microliters of complete medium per well density in a black or white 96-well plate. Prepare the bacterial inoculum at the desired multiplicity of infection in a final volume of 100 microliters per well and add 100 microliters of bacterial inoculum or medium alone to each well for a one-hour incubation in the cell culture incubator.
At the end of the incubation, replace the medium with 200 microliters of fresh complete medium supplemented with 100 micrograms per milliliter of gentamycin to remove any remaining extracellular bacteria and return the cells to the cell culture incubator for an additional two hours. At the end of the incubation and at each experimental time point thereafter, use a plate reader to measure the luminescence in each well of the lidded culture plate before returning the plate to the cell culture incubator until the next reading. While most animals exhibit high levels of bacteria in the blood at 24 hours post-infection, some pups have low or undetectable bacteria in the blood, suggesting clearance of the infection by this time point.
In addition, neonatal Ly6 B2 positive splenic cells infected with bioluminescent E.coli for one hour before the removal of extracellular bacteria and subsequent gentamycin treatment expressed a high level of luminescence at three hours that decreases over time indicative of bacterial clearance. Live animal imaging of the luminescent bacteria further confirms the increase in bacteria dissemination and growth in neonatal pups over time at 10 and 24 hours post-infection. Intravital imaging in conjunction with micro CT facilitates the identification of infection foci within the brain, lungs, and other peripheral tissues.
The lungs of some highly infected mice demonstrate opaque regions consistent with inflammatory consolidation that co-localized to luminescent bacterial signals. These regions of presumed inflammatory exudate are not observed in uninfected control lungs. A significant increase in inflammatory cytokine expression relative to controls is also observed in both low and high inoculum groups, correlating with a notable thickening of the alveolar wall, increased alveolar hemorrhaging, and inflammatory infiltration in these animals.
The most important things to remember are to execute proper technique when performing the injections and to pay strict attention to the neonates when administering the anesthesia. Using this technique, we can visualize neonatal sepsis in real time to study interventions and host directed therapies aimed at improving the immune response.
