Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis
Transcrição
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.
