This article outlines a method for inducing meningitis in adult mice through the injection of Neisseria meningitidis into the cerebrospinal fluid. The protocol emphasizes the importance of sterilization and proper anesthetic techniques to ensure animal welfare and experimental accuracy.
Begin with a restrained adult mouse and sterilize its abdomen. Inject an exogenous iron source intraperitoneally into the lower-right abdomen, preventing accidental damage to the surrounding organs.
The injected iron source enters the systemic circulation and travels to different organs to supply adequate iron for meningococcal replication.
Place the anesthetized mouse in a prone position, keeping the vertebral column straight, for easier access to the cisterna magna, a specific region within the cerebrospinal fluid, CSF-filled subarachnoid space between the middle and inner layers of the meninges surrounding the brain.
Sterilize the mouse's head. Inject a suspension of Neisseria meningitidis or meningococcus, a gram-negative pathogenic bacteria, through an occipital burr hole into the CSF-filled cisterna magna.
Within hours of injection, meningococci multiply in the CSF, occupying the subarachnoid space, which has inefficient host defense mechanisms.
The bacteria interact with the cells lining the layers of the meninges. This interaction triggers an inflammatory response; the cells release pro-inflammatory cytokines, and immune cells are recruited, leading to meningeal inflammation or meningitis.
Eventually, meningococci may also enter the systemic circulation and colonize other organs.
Before starting the experiment, weigh the animals, and evaluate their body temperature. Scruff the animal from the neck down, and disinfect the abdomen with 70% ethanol. Approximately two to three hours before the infection, using a 25-gauge needle, inject iron dextran intraperitoneally in the lower-right quadrant of the abdomen.
After anesthetizing the animal, check for the depth of anesthesia by confirming the absence of pain response upon pinching the toe. After placing the mouse in the laminar flow cabinet, position it in sternal decubitus, and carefully stretch the limbs in the cervical spine to keep the vertebral column in a straight position.
To maintain a consistent bacterial suspension, gently mix it before loading it in a syringe with an 8-millimeter, 30-gauge needle. Disinfect the head with 70% ethanol. Place the animal in lateral recumbency, hold the ears out of the way, and flex the neck moderately.
Ensure that the midline of the neck and the head are in a perfectly parallel position to the tabletop. Then, touch the atlas wings making sure that they overlap, and feel the natural indentation on the midline, where the needle is most likely to enter the occipital hole.
Fill a syringe with an 8-millimeter, 30-gauge needle with the established sub-lethal bacterial dose of Meningococci or iron dextran-supplemented GC broth as the control in a total volume of 10 microliters. Use the needle to identify the injection point, and then, inject the content of the syringe into the cisterna magna of mice through an occipital Burr hole.
Discard the syringe and needle safely after the injection. Place the animal in the cage under a laminar flow cabinet. Wait for the awakening and full recovery of movement and proceed for the animal survival on the infected animals as described in the protocol.
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