The studies were supported in part by PRIN 2012 [grant number 2012WJSX8K]: &ldquo;Host-microbe interaction models in mucosal infections: development of novel therapeutic strategies&rdquo; and by PRIN 2017 [2017SFBFER]: &ldquo;An integrated approach to tackle the interplay among adaptation, stressful conditions and antimicrobial resistance of challenging pathogens&rdquo;.

This protocol was conducted to minimize animal suffering and reduce the number of mice in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). In vivo experiments reported in this study were approved by the Ethical Animal Care and Use Committee (Prot. number 2, 14 December 2012) and the Italian Ministry of Health (Prot. number 0000094-A-03/01/2013). All the procedures should be performed inside the Biosafety Cabinet 2 (BSC2) in a BSL2 room, and the potential infected waste should be disposed in dedicated containers.
1. Infection of Mice with N. meningitidis Serogroup C Strain
CAUTION: N. meningitidis is potentially a harmful pathogen and all necessary precautions must be taken when handling this microorganism. The entire experimentation requires Biosafety Level 2 (BSL2) containment. The researcher involved in animal studies should wear disposable personal protective equipment (PPE) for the duration of the experiment.
<ol>
	<li>Preparation of bacteria for in vivo infection studies
	<ol>
		<li>Pick a single colony from a fresh Neisseria meningitidis culture on GC (Gonococcal) agar plate supplemented with 1% (vol/vol) Polyvitox supplement and inoculate in 10 mL of GC broth.</li>
		<li>Grow the bacteria at 37 &#176;C in an orbital shaker incubator with the speed of 220 rpm. Keep checking the O.D. of the culture with a spectrophotometer. Grow the culture until the early exponential phase at an optical density OD600nm of 0.7, corresponding to &#8776; 7 x 108 CFU/mL.</li>
		<li>Once the required O.D. is obtained, make frozen stocks by adding 10% glycerol. Dispense 1 mL of the culture in the cryovials. Store the vials at -80 &#176;C until use. 
		NOTE: Although it is better to use fresh bacterial culture, frozen stocks were used to simplify and standardize the in vivo experiment. Usually the frozen stocks have been employed within approximately 6 months from the preparation.</li>
		<li>Before the infection, thaw frozen bacteria at room temperature.</li>
		<li>Harvest the bacterial cells by centrifugation for 15 min at 1,500 x g and resuspend in 1 mL of fresh GC broth containing iron dextran (5 mg/kg). 
		NOTE: The GC broth is prepared with the addition of iron dextran (5 mg/kg) in order to favor the replication of meningococci in the host tissue14,18,27.</li>
		<li>Before using, perform viable counts of bacteria to determine the exact number of CFU for infection. To do so, pick up 10 &#181;L of bacterial suspension and proceed with serial dilutions and spread each dilution on GC agar plates and incubate at 37 &#176;C with 5% CO2, for 18-24 h.</li>
	</ol>
	</li>
	<li>Intra-cisternal injection of mice 
	NOTE: The entire procedure is performed in the laminar flow cabinet to maintain aseptic conditions.
	<ol>
		<li>House laboratory mice (8 week-old, female Balb/c) under specific pathogen free conditions. Provide food pellets and water ad libitum.</li>
		<li>Settle the animals in the new environment for 1 week before starting the experiment.</li>
		<li>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 ranging from 36-38 &#176;C24.</li>
		<li>Scruff the animal from the neck,&#160;clean the abdomen area with the 70% ethanol&#160;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 urinary bladder, cecum, etc. The administration of exogenous iron source, in the form of iron dextran, to animals prior to the infection favors the bacterial multiplication in the host14,18,27.</li>
		<li>After 2-3 h, perform animal anesthesia with ketamine (50 mg/kg) and xylazine (3 mg/kg) and ophthalmic lubricant.</li>
		<li>Check for the depth of anesthesia by ensuring the absence of pain response upon pinching the toe.</li>
		<li>Position the mouse in sternal decubitus and carefully stretch the limbs and the cervical spine to keep the vertebral column in a straight position.</li>
		<li>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.</li>
		<li>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 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 titer from 104 to 109 per animal was used.</li>
		<li>Clean the surgical area with 70% Ethanol.</li>
		<li>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 &#181;L into the cisterna magna of mice through an occipital burr hole by using a 30 G needle x 8 mm.
		<ol>
			<li>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 accessible28.</li>
			<li>Briefly, place the animal in lateral recumbency, hold the ears out of the way and flex the neck moderately (90 to 100&#176;). Ensure that the midline of the neck and the head (from the nose to the occiput) are in perfectly parallel position to the tabletop.</li>
			<li>Touch the atlas wings and make sure that they overlap, eliminating axial rotation. A natural indentation can usually be touched on midline where the needle is most likely to enter the occipital hole.</li>
			<li>Discard the syringe and needle safely after the injection of mice with Neisseria.</li>
		</ol>
		</li>
		<li>Place the animal in the cage and wait for the awakening and full recovery of movement.</li>
		<li>Keep the cages with infected mice under a laminar flow cabinet.</li>
		<li>Monitor mice, 24 h post infection, for clinical signs of coma according to the coma scale29. 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. 
		&#160;NOTE:&#160;When in animals was recorded pain, analgesia with meloxicam (5 mg/kg i.p. for duration of study) was administered.</li>
		<li>Proceed for the animal survival or CFU counts assay on the infected animals as detailed in step 2.</li>
		<li>Perform euthanasia of mice with a score of 2 by cervical dislocation and record as dead for statistical analysis.</li>
	</ol>
	</li>
</ol>
2. Animal Survival and CFU Counts
<ol>
	<li>Animal survival
	<ol>
		<li>Prepare bacterial inocula at different doses (ranging from 104 to 109 CFU per mouse) to infect animals by the i.cist. route (see step 1.2).</li>
		<li>Inoculate control mice with GC broth, supplemented with iron dextran (5 mg/kg), in the same manner.</li>
		<li>Monitor animals for clinical symptoms: ruffled fur, hunched appearance, hypothermia, weight loss, lethargy, or moribund24,25,26,30, every day throughout the whole experiment for 168 h (7 days)&#160;at least twice a day for the duration of the experiment.</li>
		<li>Measure the body weight and temperature by using a digital balance and a rectal thermometer, respectively everyday.</li>
		<li>Record the survival of mice for a week. 
		NOTE: Record the natural death of animal post infection while the animals that reach a coma value of 2 or that survive over 168 h of observation will be euthanized.</li>
		<li>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.</li>
		<li>Check that the pain response is absent by toe pinch.</li>
		<li>Perform euthanasia of mice by cervical dislocation and record as dead for statistical analysis.</li>
	</ol>
	</li>
	<li>Evaluation of colony forming units (CFU) counts in peripheral organs
	<ol>
		<li>Use a sub-lethal bacterial dose (5 x 105CFU/mice) on the basis of animal survival results to inoculate animals by the i.cist. route (see subsection 1.2).</li>
		<li>Monitor the rectal temperature every day during the infection and perform anesthesia of animals at 48 h post infection as mentioned in step 2.</li>
		<li>Proceed with the 70% ethanol disinfection of the chest and withdraw 600-700 &#181;L of blood by cardiac puncture of the chest cavity using a 25 G needle 0.5 mm x 16 mm.</li>
		<li>Collect the blood in a tube containing 3.8% sodium citrate and store at -80 &#176;C for the later viable bacterial cell counts.</li>
		<li>Perform cervical dislocation to sacrifice the animals. Confirm the death recording the absence of heartbeat, after sacrificing the mouse according to all relevant institutional and ethical guidelines.</li>
		<li>Lay the mouse in the supine position and use scissors and forceps to proceed with the cut of the fur along the sagittal plane of body. Fix the skin with the fur on the sides of the body with pins.</li>
		<li>Cut off the peritoneal membrane using sharp scissors. Use scissors and disposable forceps to excise the organs (e.g., spleen and liver) and put each one in sterile Petri dish with 1 mL of GC broth supplemented with 10% (vol/vol) glycerol.</li>
		<li>Safely dispose the mouse body as per IACUC guidelines.</li>
		<li>Homogenize the organs mechanically at room temperature with the plunger of a 5 mL syringe for about 2-3 min until a single-cell suspension is formed and transfer it in a tube.</li>
		<li>Put the tube with the homogenized tissue sample immediately on the dry ice. 
		NOTE: Samples can be stored at -80 &#176;C in a 2 mL sterile tube for performing the viable bacterial cell counts evaluation at later points.</li>
		<li>Make GC agar plates with antibiotics when required. Dry the plates prior to the use pre-incubating at 37 &#176;C for 2-3 h.</li>
		<li>Prepare 10-fold serial dilutions of the samples in GC broth from each homogenized tissue and plate onto GC agar plates. Incubate overnight at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Preparation of Brain Tissues for CFU Count
<ol>
	<li>Use a sub-lethal bacterial dose (5 x 105&#160;CFU/mice) on the basis of animal survival results to inoculate animals by the i.cist. route (see subsection 1.2).</li>
	<li>Perform anesthesia of animals at established infection time as mentioned in step 2.</li>
	<li>Perform euthanasia of animals by cervical dislocation.</li>
	<li>Clean the surgical area with 70% Ethanol.</li>
	<li>Cut off the head of mouse using a large scissors.</li>
	<li>Resect the fur and the skin with the help of small surgical scissors and a fine tipped steel forceps, proceeding towards the top of the skull to be able to clearly see the sutures and to guide the opening of the cranium.</li>
	<li>Insert the tip of a tiny scissors through the foramen magnum to open the skull.</li>
	<li>Cut towards the center of the cranium and across the midline of parietal bone to the opposite side of the skull. Gently cut along the lateral rim of the lambdoid suture.</li>
	<li>Lift the cranium starting from the posterior parietal corner and pull it diagonally upward to discover the brain, by using fine tipped forceps.</li>
	<li>Be sure that the brain tissue is not attached to any bone of the head, when the skull is lifted.</li>
	<li>Use disposable forceps to remove any connective tissue between the skull and the brain, to ensure that brain tissue being removed along with the skull.</li>
	<li>Do not allow the brain tissue dry out too much. Place the brain, using disposable forceps, in a Petri dish with 1 mL of GC broth supplemented with 10% (vol/vol) glycerol.</li>
	<li>Homogenize the brain mechanically with the plunger of a 5 mL syringe (see step 2.2.9). Transfer the samples into a 2 mL sterile tube and store -80 &#176;C for the later viable bacterial cell counts evaluation as discussed in step 2.2.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnosis+of+experimental+meningitis+by+bacterial+heat+production+in+cerebrospi-+nal+fluid.">Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospi- nal fluid.</a> BMC Infectious Diseases. 7, 116 (2007).</li><li>Tuomanen, E. I., Saukkonen, K., Sande, S., Cioffe, C., Wright, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+inflammation,+tissue+damage,+and+mortality+in+bacterial+meningitis+in+rabbits+treated+with+monoclonal+antibodies+against+adhesion-promoting+receptors+of+leukocytes.">Reduction of inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes.</a> Journal of Experimental Medicine. 170, 959-969 (1989).</li><li>Goldschneider, I., Gotschlich, E. C., Artenstein, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunity+to+the+meningococcus.+I.+The+role+of+humoral+antibodies.">Human immunity to the meningococcus. I. The role of humoral antibodies.</a> Journal of Experimental Medicine. 129, 1307-1326 (1969).</li><li>Goldschneider, I., Gotschlich, E. C., Artenstein, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunity+to+the+meningococcus.+II.+Development+of+natural+immunity.">Human immunity to the meningococcus. II. Development of natural immunity.</a> Journal of Experimental Medicine. 129, 1327-1348 (1969).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+methods+for+the+diagnosis+of+meningitis+caused+by+Neisseria+meningitidis,+Streptococcus+pneumoniae,+and+Haemophilus+influenzae:+WHO+manual,+2nd+Edition.">Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae: WHO manual, 2nd Edition.</a> World Health Organization. , (2011).</li><li>Chiavolini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+inducing+experimental+pneumococcal+meningitis+in+outbred+mice.">Method for inducing experimental pneumococcal meningitis in outbred mice.</a> BMC Microbiolology. 4, 36 (2004).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+Subarachnoidal+Route+of+Infection+for+Investigating+Roles+of+Streptococcus+suis+Biofilms+in+Meningitis+in+a+Mouse+Infection+Model.">Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model.</a> Journal of Visualized Experiments. (1), e137 (2018).</li><li>Pagliuca, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Bacteroides+fragilis+protection+from+Bartonella+henselae-induced+damage.">Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.</a> PLoS One. 7, 49653 (2012).</li><li>Larson, J. A., Howie, H. L., So, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+accelerates+ferritin+degradation+in+host+epithelial+cells+to+yield+an+essential+iron+source.">Neisseria meningitidis accelerates ferritin degradation in host epithelial cells to yield an essential iron source.</a> Molecular Microbiology. 53, 807-820 (2004).</li><li>Festing, M. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variability+of+inbred+and+outbred+mice.">Phenotypic variability of inbred and outbred mice.</a> Nature. 263, 230-232 (1976).</li><li>Festing, M. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warning:+the+use+of+heterogeneous+mice+may+seriously+damage+your+research.">Warning: the use of heterogeneous mice may seriously damage your research.</a> Neurobiology of Aging. 20, 237-244 (1999).</li></ol>

The authors have nothing to disclose.

In this study, we describe an experimental protocol to induce meningococcal meningitis in adult mice by i.cist. inoculation of meningococcal bacteria. To our knowledge, no other model of meningococcal meningitis has been developed in laboratory mice infected by i.cist. route; in the past, this way has been explored to provide models of meningococcal meningitis in both rat31 and rabbit32. It is well-known that the highest rate of meningococcal disease is found between young ...

Neisseria meningitidis is a Gram negative &#946;-proteobacterium restricted to the human host, well known for being one of the most common causes of meningitis and sepsis in the human population across the world. It colonizes the upper respiratory tract (nose and throat) of healthy and asymptomatic carriers (2-30% of the population), but the bacterium sometimes evades various host immune defenses and spreads from the bloodstream to the brain causing an uncontrolled local inflammation, known as meningococcal meningitis. A combination of host and bacterial factors appears to contribute to the transition from the commensal to the invasive behavior1.
N. meningitidis is specialized exclusively in human colonization and infection. It has a narrow host range and, therefore, has limited in vivo pathogenesis studies due to the lack of suitable animal models that reproduce the human meningococcal disease. As a result, it had led to fundamental gaps in the comprehension concerning the pathogenesis of septicemia and meningitis caused by meningococcus. In the last decades, the development of many in vitro systems allowed the identification of several meningococcal virulence factors2,3,4. Although these valuable studies provided important insights to understand the role of these factors for a successful meningococcal infection, these models did not allow assessment of the consequences of bacterial interactions with the humoral and cellular immune system and even less with the whole tissue. In vivo animal models of infection are of great relevance as well for the evaluation of protection degree conferred by vaccine formulations. As a human-tropic pathogen, meningococcus possess appropriate determinants necessary for successful infection such as surface structures (i.e., type IV pili and opacity proteins) and iron uptake systems for human receptors and transport proteins (i.e., transferrin and lactoferrin)5,6,7 to properly adhere, survive and invade the human host. Finally, the genetic variation abilities of the pathogen to evade and/or block the human immune response further contribute to the high species tropism8,9. Therefore, the absence of specific host factors, involved in the interaction, can block steps of the pathogen&#8217;s life cycle, establishing significant difficulties in the development of small animal models summarizing the meningococcal life cycle.
Over the past decades, several approaches have been developed to improve our understanding of the meningococcal infectious cycle. Infections of two animal model, mouse and rat, either intraperitoneally (i.p.) or intranasally (i.n.), were developed to reproduce meningococcal disease10,11,12,13,14,15,16,17. The laboratory mouse is probably one of the more versatile animals for inducing experimental meningococcal infection.
However, the i.p. way of infection leads to the development of severe sepsis although it does not mimic the natural route of infection, whereas the i.n. route of infection was useful to evaluate meningococcal pathogenesis, even though it may induce lung infection prior to sepsis10,11,12,13,14,15,16,17.
The i.p. mouse model was instrumental to assess the protection from the meningococcal challenge10,11,12. The mouse model of meningococcal colonization based on the i.n. route of infection has been developed with infant mice, as they are more susceptible to meningococci, to reproduce an invasive infection mimicking the course of the meningococcal disease in humans13,14,15,16,17. Moreover, to promote meningococcal replication in the murine host, a growing number of technical strategies were also applied including the administration of the iron to the animals to improve the infection, the use of high bacterial inoculum, mouse-passaged bacterial strain as well as the employment of infant or immunocompromised animal hosts10,13,15,18,19. Expression of specific human factors like CD4620 or transferrin21 has increased the susceptibility of mice to this human-tropic bacterium; the employment of the human skin xenograft model of infection has also been useful to evaluate the adhesion ability of meningococci to human endothelium22,23. Collectively, the recent development of humanized transgenic mice has improved the understanding of the meningococcal pathogenesis and its host interactions.
Previously, we developed a murine model of meningococcal meningitis where the inoculation of bacteria was performed into the cisterna magna of adult mice with mouse-passaged bacteria24. Clinical parameters and the survival rate of infected mice demonstrated the establishment of meningitis with characteristics comparable to those seen in the human host, as well as, the microbiological and histological analyses of the brain. From these infected mice, bacteria were, also, recovered from blood, liver, and spleen, and bacterial loads from peripheral organs correlated with the infectious dose. In particular, this model was employed to evaluate the virulence of an isogenic mutant strain defective in the L-glutamate transporter GltT24. Recently, using our mouse model of meningococcal meningitis based on i.cist. route with serogroup C strain 93/42862,24 and an isogenic mutant defective in cssA gene encoding for UDP-N-acetylglucosamine 2-epimerase25, we have analyzed the role of exposed sialic acid in the establishment of disease in mice.
In this protocol, we describe a straightforward method to induce experimental meningococcal meningitis based on the i.cist. route of infection in Balb/c adult mice. This method is particularly useful for the characterization of meningococcal infection in a murine host, as well as for the assessment of the virulence between wild type reference strains and isogenic mutants. The intra-cisternal route of infection ensures complete delivery of the meningococci directly into the cisterna magna, which in turn facilitates bacterial replication in the cerebrospinal fluid (CSF) and induces meningitis with features that mimic those present in humans2,24,25,26.

<table><tbody><tr><td>1,8 Skirted Cryovial With external thread</td><td>Starlab</td><td>E3090-6222</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube</td><td>Falcon</td><td>352070</td><td>30 mm x 115 mm</td></tr><tr><td>Adson Forceps</td><td>F.S.T.</td><td>11006-12</td><td>Stainless Steel</td></tr><tr><td>Alarm-Thermometer</td><td>TESTO</td><td>9000530</td><td></td></tr><tr><td>BactoTM Proteose Peptone</td><td>BD</td><td>211693</td><td></td></tr><tr><td>BD Micro Fine syringe</td><td>BD</td><td>320837</td><td>U-100 Insulin</td></tr><tr><td>BD Plastipak syringe 1 mL 25 G 5/8 inch</td><td>BD</td><td>300014</td><td>05 mm x 16 mm</td></tr><tr><td>BD Plastipak syringe 5 mL</td><td>BD</td><td>308062</td><td>07 mm x 30 mm</td></tr><tr><td>BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET</td><td>Bio Air s.c.r.l.</td><td>Aura B3</td><td></td></tr><tr><td>BioPhotometer</td><td>Eppendorf</td><td>Model #6131</td><td></td></tr><tr><td>Bottle D</td><td>Tecniplast</td><td>D</td><td>Graduated up to: 400 mL, Total Volume 450 mL, 72 mm x 72 mm x 122 mm</td></tr><tr><td>C150 CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Binder</td><td>9040-0078</td><td></td></tr><tr><td>Cage Body Eurostandard Type II</td><td>Tecniplast</td><td>1264C</td><td>267 x 207 x 140 mm&lt;sup&gt;3&lt;/sup&gt;, Floor area 370 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Cell Culture Petri Dish With Lid</td><td>Thermo Scientific</td><td>150288</td><td>Working Volume: 5 mL</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Microcentrifuge 5415R</td><td></td></tr><tr><td>Cuvetta semi-micro L. Form</td><td>Kartell S.p.A.</td><td>01938-00</td><td></td></tr><tr><td>di-Potassium hydrogen phosphate trihydrate</td><td>Carlo erba</td><td>471767</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate anhydrous ACS-for analysis</td><td>Carlo Erba</td><td>480141</td><td>g1000</td></tr><tr><td>Diete Standard Certificate</td><td>Mucedola s.r.l.</td><td>4RF21</td><td>Food pellet for animal</td></tr><tr><td>Dumont Hp Tweezers 5 Stainless Steel</td><td>F.S.T. by DUMONT</td><td>AGT5034</td><td>0.10 x 0.06 mm&lt;sup&gt;2&lt;/sup&gt; tip</td></tr><tr><td>Electronic Balance</td><td>Gibertini</td><td>EU-C1200</td><td>Max 1200 g, d = 0.01 g, T = -1200 g</td></tr><tr><td>Eppendorf Microcentrifuge tube safe-lock</td><td>Eppendorf</td><td>T3545-1000EA</td><td></td></tr><tr><td>Erythromycin</td><td>Sigma-Aldrich</td><td>E-6376</td><td>25 g</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>F.S.T.</td><td>14084-08</td><td>Stainless Steel</td></tr><tr><td>Filter Top (mini- Isolator), H-Temp with lock clamps</td><td>Tecniplast</td><td>1264C400SUC</td><td></td></tr><tr><td>GC agar base</td><td>OXOID</td><td>CM0367</td><td></td></tr><tr><td>Gillies Forceps 1 x2 teeth</td><td>F.S.T.</td><td>11028-15</td><td>Stainless Steel</td></tr><tr><td>Glicerin RPE</td><td>Carlo Erba</td><td>453752</td><td>1 L</td></tr><tr><td>Graefe Forceps</td><td>F.S.T.</td><td>11052-10</td><td>Serrated Tip Width: 0.8 mm</td></tr><tr><td>Inner lid</td><td>Tecniplast</td><td>1264C116</td><td></td></tr><tr><td>Iron dextran solution</td><td>Sigma-Aldrich</td><td>D8517-25ML</td><td></td></tr><tr><td>Ketamine</td><td>Intervet</td><td></td><td></td></tr><tr><td>Microbiological Safety Cabinet BH-EN and BHG Class II</td><td>Faster</td><td>BH-EN 2004</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL&amp;nbsp;</td><td>BRAND</td><td>PP780751</td><td>screw cap PP, grad</td></tr><tr><td>Mouse Handling Forceps</td><td>F.S.T.</td><td>11035-20</td><td>Serrated rubber; Gripping surface: 15 mm x 20 mm</td></tr><tr><td>Mucotit-F2000</td><td>MERZ</td><td>61846</td><td>2000 mL</td></tr><tr><td>Natural Latex Gloves</td><td>Medica</td><td>M101</td><td></td></tr><tr><td>New Brunswick Classic C24 Incubator Shaker</td><td>PBI international</td><td>C-24 Classic Benchtop Incubator Shaker</td><td></td></tr><tr><td>Petri PS Dishes</td><td>VWR</td><td>391-0453</td><td>90 x 14.2 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Pipetman Classic P20</td><td>Gilson</td><td>F123600</td><td>2-20 &amp;mu;L</td></tr><tr><td>Pipetman Classic P200</td><td>Gilson</td><td>F123601</td><td>20-200 &amp;mu;LL</td></tr><tr><td>Pipetman Classim P1000</td><td>Gilson</td><td>F123602</td><td>200-1,000 &amp;mu;L</td></tr><tr><td>Polyvitox</td><td>OXOID</td><td>SR0090A</td><td></td></tr><tr><td>Potassium Chloride</td><td>J.T. Baker Chemicals B.V.</td><td>0208</td><td>250 g</td></tr><tr><td>Potassium Dihydrogen Phosphate</td><td>J.T. Baker Chemicals B.V.</td><td>0240</td><td>1 kg</td></tr><tr><td>PS Disposible forceps</td><td>VWR</td><td>232-0191</td><td></td></tr><tr><td>Removable Divider</td><td>Tecniplast</td><td>1264C812</td><td></td></tr><tr><td>Round-Bottom Polypropylene Tubes</td><td>Falcon</td><td>352063</td><td>5 mL</td></tr><tr><td>Sodium Chloride</td><td>MOLEKULA</td><td>41272436</td><td></td></tr><tr><td>SS retainer and Polyester FilterSheet</td><td>Tecniplast</td><td>1264C</td><td></td></tr><tr><td>Standard Pattern Forceps</td><td>F.S.T.</td><td>11000-12</td><td>Stainless</td></tr><tr><td>Stevens Tenotomy Scissors</td><td>F.S.T.</td><td>14066-11</td><td>Stainless Steel</td></tr><tr><td>Surgical Scissor - ToughCut</td><td>F.S.T.</td><td>14130-17</td><td>Stainless</td></tr><tr><td>Touch N Tuff disposible nitrile gloves</td><td>Ansell</td><td>92-500</td><td></td></tr><tr><td>Ultra Low Temperature (ULT) Freezer</td><td>Haier</td><td>DW-86L288</td><td>Volume = 288 L</td></tr><tr><td>Wagner Scissors</td><td>F.S.T.</td><td>14070-12</td><td>Stainless Steel</td></tr><tr><td>Xylazine</td><td>Intervet</td><td></td><td></td></tr></tbody></table>

inducing meningococcal meningitis serogroup c in mice via intracisternal delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Survival of mice infected with N. meningitidis wild type and isogenic mutant strains. 
The Neisseria meningitidis strains used in these representative results are the serogroup C reference strain 93/4286 (ET-37) and its isogenic mutant 93/4286&#937;cssA obtained by insertional inactivation of the cssA gene, coding for the UDP-N-acetylglucosamine 2-epimerase, that maps in capsule synthesis locus25. To assess the virulence degree of the c...

Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

inducing-meningococcal-meningitis-serogroup-c-mice-via-intracisternal

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JoVE Journal

Immunology and Infection

7.2K Views.  University of Naples Federico II. Neisseria meningitidis is een van de meest voorkomende oorzaken van sepsis en meningitidis onder de menselijke bevolking over de hele wereld, en de bacteriën is beperkt tot de menselijke gastheer. In dit experimentele protocol beschrijven we de op maat gemaakte procedure voor de inductie van meningokokkenmenitimis in een muismodel op basis van de intracisternale inenting van bacteriën. Dit murinemodel reproduceerde de karakteristieke pathologische voorvallen van meningokokkenmeningitis als ...

Video: Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Neuroscience

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Source: Pagliuca, C., et al., <a href="https://app.jove.com/v/60047">Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the intracisternal delivery of Neisseria meningitidis into a mouse model to generate meningitis infection.

Source: Pagliuca, C., et al., Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery. J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Source: Kawasaki, H., et al. <a href="https://app.jove.com/v/54164">Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.</a> J. Vis. Exp. (2016).
This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup&#39;s brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

	Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 ...

Neuropathology

Infectious Diseases

Intranasal Immunization with BCG in a Mouse Model

Source: Uranga, S. et al., <a href="https://app.jove.com/v/54440">Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunotherapy

Immunogenics and Vaccine Development

Generation of a Murine Model with a Central Nervous System Infection

Source: Shen, T., et al. <a href="https://app.jove.com/v/59132">A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease.</a> J. Vis. Exp. (2019)
This video demonstrates the generation of a mouse model with a CNS infection. A suckling pup is injected with the virus both intracranially and intraperitoneally. The virus spreads, infecting the CNS causing neuronal cell death and inflammation. The pup is assessed for symptoms of infection as the disease progresses.

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

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