Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intra-cisternal injection of mice

NOTE: The entire procedure is performed in the laminar flow cabinet to maintain aseptic conditions.

1. House laboratory mice (8-week-old, female Balb/c) under specific pathogen-free conditions. Provide food pellets and water ad libitum.
2. Settle the animals in the new environment for 1 week before starting the experiment.
3. Before starting the experiment, weigh and evaluate their body temperature.
   NOTE: The inbred Balb/c female mice of eight-week-old weigh an average of about 19 g. The average temperature of laboratory mice physiologically ranges from 36-38 °C.
4. Scruff the animal from the neck, clean the abdomen area with the 70% ethanol, and inject i.p. iron dextran (dissolved in 1% phosphate saline buffer, 250 mg/kg) in the lower right quadrant of the murine abdomen by using a 25 G needle 0.5 mm x 16 mm, approximately 2-3 h before the infection.
   NOTE: The intraperitoneal injection was performed in the lower right quadrant of the murine abdomen to avoid damaging abdominal organs such as the urinary bladder, cecum, etc. The administration of an exogenous iron source, in the form of iron dextran, to animals prior to the infection favors bacterial multiplication in the host.
5. After 2-3 h, perform animal anesthesia with ketamine (50 mg/kg) and xylazine (3 mg/kg), and ophthalmic lubricant.
6. Check for the depth of anesthesia by ensuring the absence of pain response upon pinching the toe.
7. Position the mouse in sternal decubitus and carefully stretch the limbs and the cervical spine to keep the vertebral column in a straight position.
8. Gently mix the bacterial suspension to maintain a consistent suspension before loading a syringe of 30 G needle x 8 mm.
   NOTE: Prepare the bacterial suspension as close as possible to the injection time; meanwhile, store it at room temperature.
9. Based on the post-thawing bacterial titer (CFU/mL), proceed with the calculation of the total CFU to be used, with respect to the total number of animals to be infected (CFU bacterial dose per number of animals). Proceed with the calculation of the exact volume to be taken from the vial to obtain the total CFU by applying a proportion between the post-thawing bacterial titer (CFU/mL) and the total CFU useful for the infection of n mice (post-thawing bacterial titer CFU: ml = total CFU: x). Establish the final volume with respect to the total number of animals to be infected.
   NOTE: In these experiments, a wide range of titers from 10⁴ to 10⁹ per animal was used.
10. Clean the surgical area with 70% ethanol.
11. Identify the injection point with the help of a needle and inject the established CFU of meningococci (wild-type strain and isogenic mutant strain), or GC broth supplemented with iron dextran (5 mg/kg) as control, in a total volume of 10 µL into the cisterna magna of mice through an occipital burr hole by using a 30 G needle x 8 mm.
    1. Perform the cisternal inoculum by placing the needle at the craniocervical junction, specifically in the dorsal subarachnoid space. Ventroflex the head to make this space accessible.
    2. Briefly, place the animal in lateral recumbency, hold the ears out of the way, and flex the neck moderately (90 to 100°). Ensure that the midline of the neck and the head (from the nose to the occiput) are in a perfectly parallel position to the tabletop.
    3. Touch the atlas wings and make sure that they overlap, eliminating axial rotation. A natural indentation can usually be touched on the midline where the needle is most likely to enter the occipital hole.
    4. Discard the syringe and needle safely after the injection of mice with Neisseria.
12. Place the animal in the cage and wait for the awakening and full recovery of movement.
13. Keep the cages with infected mice under a laminar flow cabinet.
14. Monitor mice, 24 h post-infection, for clinical signs of coma according to the coma scale. Coma scale: 1 = coma, 2 = does not stand upright after being turned on the back, 3 = stands upright within 30 s, 4 = stands upright within 5 s, minimal ambulatory activities, 5 = normal.
    NOTE: When in animals was recorded pain, analgesia with meloxicam (5 mg/kg i.p. for the duration of the study) was administered.

2. Animal Survival and CFU Counts

1. Animal Survival
   1. Prepare bacterial inocula at different doses (ranging from 10⁴ to 10⁹ CFU per mouse) to infect animals by the i.cist. route.
   2. Inoculate control mice with GC broth, supplemented with iron dextran (5 mg/kg), in the same manner.
   3. Monitor animals for clinical symptoms: ruffled fur, hunched appearance, hypothermia, weight loss, lethargy, or moribund every day throughout the whole experiment for 168 h (7 days) at least twice a day for the duration of the experiment.
   4. Measure the body weight and temperature by using a digital balance and a rectal thermometer, respectively every day.
   5. Record the survival of mice for a week.
      NOTE: Record the natural death of the animal post infection while the animals that reach a coma value of 2 or that survive over 168 h of observation will be euthanized.
   6. Anesthetize mice with a coma value of 2 or that survive over the observation time with ketamine (50 mg/kg) and xylazine (3 mg/kg) and apply ophthalmic ointment.
   7. Check that the pain response is absent by a toe pinch.
   8. Perform euthanasia of mice by cervical dislocation and record them as dead for statistical analysis.
   9. Proceed for the animal survival or CFU counts assay on the infected animals.

Materials

Name	Company	Catalog Number	Comments
Alarm-Thermometer	TESTO	9000530	Device
BD Micro Fine syringe	BD	320837	U-100 Insulin
BD Plastipak syringe 1ml 25GA 5/8in	BD	300014	05x16mm
BD Plastipak syringe 5ml	BD	308062	07 x 30mm
BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET	Bio Air s.c.r.l.	Aura B3	Device
C150 CO2 Incubator	Binder	9040-0078	Device
Diete Standard Certificate	Mucedola s.r.l.	4RF21	Food pellet for animal
Dumont Hp Tweezers 5 Stainless Steel	F.S.T. by DUMONT	AGT5034	0,10 x 0,06 mm tip
Electronic Balance	Gibertini	EU-C1200	Max 1200g, d=0.01g, T=-1200g
Iron dextran solution	Sigma-Aldrich	D8517-25ML
Ketamine	Intervet
Microbiological Safety Cabinet BH-EN and BHG Class II	Faster	BH-EN 2004
Microcentrifuge tubes 1.5ml	BRAND	PP780751	Screw cap PP, grad
Mouse Handling Forceps	F.S.T.	11035-20	Serrated rubber; Gripping surface:15 x 20 mm
Natural Latex Gloves	Medica	M101
Touch N Tuff disposible nitrile gloves	Ansell	92-500
Xylazine	Intervet