This study investigates the effects of pathogenic bacteria on neonatal mouse pups using bioluminescent imaging. By comparing low and high concentrations of bacteria, the research aims to understand bacterial proliferation and immune response.
Begin with age-matched neonatal mouse pups.
Gently lift the skin at the back of each pup's neck. Subcutaneously inject one pup with a low-concentration suspension of pathogenic bacteria and another with a high-concentration suspension.
These bacteria express a bioluminescent operon that produces the luciferase enzyme and its substrate, enabling autonomous light emission.
A protective polysaccharide capsule surrounds the bacteria and masks their surface antigens, preventing recognition by the pups' immune cells.
Return the pups to their biological mother for postnatal care.
Bacterial proliferation at the injection site triggers inflammation and increased vascular permeability, enabling bacterial entry into the bloodstream and systemic spread.
Place the anesthetized pups under a luminescence imaging system.
Luciferase oxidizes the substrate to produce visible light, allowing visualization of bacteria inside the pups.
Capture images over time to compare bacterial growth and spread in pups inoculated with high and low bacterial concentrations.
Begin by placing age-matched pups into either high or low-dose litter groups within a biosafety level two cabinet. On postnatal day 3 or 4, 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 luminescent 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.
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