Neisseria meningitidis is one of the most common causes of sepsis and meningitidis among the human population all over the world, and the bacteria is restricted to the human host. In this experimental protocol, we will describe the tailored procedure for the induction of meningococcal meningitidis in a mouse model based on the intracisternal inoculation of bacteria. This murine model reproduced the characteristic pathological events of meningococcal meningitis as a severe human disease.
So, it represents a useful tool to evaluate the host-pathogen interaction and to analyze the pathogenetic mechanism responsible for this lethal disease. The infection model could be useful not only to evaluate innovative therapeutic strategy to prevent bacteria replication directly into CSF, but also to analyze the efficacy of possible passive immunotherapy against human pathogens. The technique requires the training phase to guarantee the success not only of the intracisternal inoculum, but more in general the disease induction.
Demonstrating the procedures will be Roberta Colicchio and Chiara Pagliuca as researchers, and Elena Scaglione as PhD student, all by my laboratory. To begin, pick a single colony from a fresh Neisseria meningitidis culture on gonococcal agar plate supplemented with 1%polybitox supplement and inoculate in 10 milliliters GC Broth. All the procedures that involve the use of meningococci must be done under a laminar flow bio safety cube cabinet.
Grow the bacteria at 37 degrees Celsius in an orbital shake incubator at 220 RPM and keep checking the optical density of the culture at different time points with a spectrophotometer. Grow the culture until it reaches OD600 of 0.7 corresponding to the early exponential phase. Once this optical density is obtained, make frozen stocks by adding 10%glycerol and dispensing one milliliter of the culture in the cryo vials.
Store the vials at 80 degrees Celsius until use. Before the infection, thaw frozen bacteria at room temperature, then transfer into a centrifuge tube in centrifuge for 15 minutes at 1500xG Remove the supernatant and re-suspend at one milliliter of fresh GC Broth containing five milligrams per kilogram iron dextran. Before starting the experiment, weigh the animals and evaluate their body temperature.
Scuff the animal from the neck-down and disinfect the abdomen with a 70%ethanol. Approximately 2 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 on the laminar flow cabinet position it in sternal decubitus and carefully stretch the limbs and 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 eight millimeter 30 gauge needle. Disinfect the head with a 70%ethanol.
Place the animal in lateral recumbency, pull the ears out of the way, and flex the neck moderately. Ensure that the midline of the neck and the head are in perfectly parallel position to the table top. 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 control in a total volume of 10 microliters. Use the needle to identify the injection point and then inject the content of the syringe to 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. After anesthetizing the mouse, disinfect the chest with a 70%ethanol.
Using a 25 gauge needle withdraw 600 to 700 microliters of blood by cardiac puncture of the chest cavity. Collect the blood in a tube containing 3.8%sodium citrate and store at 80 degrees Celsius for the later viable bacterial cell counts evaluation. Lay the euthanized mouse in the supine position.
Using scissors and forceps cut the fur along the sagittal plane of the body. With pins, fix the skin with the fur on the sides of the body. Use sharp scissors to cut off the peritoneal membrane.
Then with scissors and disposable forceps, excise the organs, such as spleen and liver, and put each in a separate steril Petri dish with one milliliter of GC Broth supplemented with 10%glycerol. Using a plunger of a five millimeter syringe, homogenize the organs mechanically at room temperature for about two to three minutes until single-cell suspension is formed and transfer it to a scurdent cryo vial. Place the tube immediately on the dry ice.
Make GC agar plates with antibiotics and dry them prior to use at 37 degrees Celsius for two to three hours. Prepare 10 full serial dilutions of the samples in GC Broth from each homogenized tissue. Plate them on GC agar plates and incubate overnight at 37 degrees Celsius with 5%carbon dioxide.
With 70%ethanol, disinfect the head of a euthanized mouse. Using small surgical scissors and a fine-tipped steel forceps, resect the fur and the skin of the removed head, then proceed towards the top of the skull in order to clearly see the sutures and to guide the opening of the cranium. To open the skull, insert the tip of tiny scissors through the foramen magnum.
Cut towards the center of the cranium and across the midline of the parietal bone to the opposite side of the skull and gently cut along the lateral limb of the lambdoid suture. Using fine-tipped forceps, starting from the posterior parietal corner, lift the cranium and pull it diagonally upward to discover the brain, making sure that the brain tissue is not attached to any bone of the head when the skull is lifted. Use disposable forceps to remove any connective tissue between the skull and the brain and do not allow the brain tissue to dry out.
Use disposable forceps to immediately place the brain in a Petri dish with one milliliter of GC Broth supplemented with 10%glycerol. Use the plunger of a five milliliter syringe to homogenize the brain mechanically at room temperature for about two to three minutes until a single-cell suspension is formed. Transfer the suspension into a 2 milliliter sterile tube and store at 80 degrees Celsius for the later viable bacterial cell counts evaluation.
Survival of mice infected with 93/4286 wild-type or cssA defective Neisseria meningitidis strains were evaluated. The median lethal dose for the wild-type strain corresponded to the meningococcal challenge of 10 to the 4th CFU. Whereas the mortality rate 10 to the 5th CFU was equal to 83.4%and 100%with 10 to the 5th CFU dose.
In order to obtain the median lethal dose for the cssA defective mutant strain, a dose of 10 to the 8th CFU was necessary and amount 1000 folds higher than the wild-type strain. After the injection, there was a rapid increase of wild-type bacteria in the brain tissue reaching the peak at around 24 hours post-infection. In the brain of mice infected with the mutant strain, the viable counts dropped progressively over time.
Also, 33.3%of the mutant infected mice showed bacterial clearance from the infection site, unlike wild-type infected animals. 48 hours after the infection, the cssA defective mutant was completely cleared in spleen and liver, whereas the animals infected with the wild-type strain exhibited a persistent infection. The described experimental method could be useful to analyze brain damage associated with this lethal disease, by its pathological evaluation, such as immunohistochemistry, immunofluorescence, and cerebral bleeding analysis.
