Name: 비디오: 수막구균성 수막염을 유발하기 위해 마우스 모델로 수막구균의 수막구균 내 전달(Intracisternal Delivery of meningococci into mouse models to induce meningococcal meningitis) - 개념
Uploaded: 2023-08-31T09:44:29.000+00:00
Duration: 1 min 35 s
Description: 371 조회수. 출처: Pagliuca, C., et al., Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery. J. Vis. 특급 (2019) 이 비디오에서는 Neisseria meningitidis를 마우스 모델로 시추 내 전달하여 수막염 감염을 생성하는 방법을 보여줍니다.

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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.
<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 ranges from 36-38 &#176;C.</li>
	<li>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.</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 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.</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 accessible.</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 a 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 the 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 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.</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.</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 moribund every day throughout the whole experiment for 168 h (7 days) 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 every day.</li>
		<li>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.</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 a toe pinch.</li>
		<li>Perform euthanasia of mice by cervical dislocation and record them as dead for statistical analysis.</li>
		<li>Proceed for the animal survival or CFU counts assay on the infected animals.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alarm-Thermometer</td>
			<td>TESTO</td>
			<td>9000530</td>
			<td>Device</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 1ml 25GA 5/8in</td>
			<td>BD</td>
			<td>300014</td>
			<td>05x16mm</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 5ml</td>
			<td>BD</td>
			<td>308062</td>
			<td>07 x 30mm</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>Device</td>
		</tr>
		<tr>
			<td>C150 CO2 Incubator</td>
			<td>Binder</td>
			<td>9040-0078</td>
			<td>Device</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 tip</td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Gibertini</td>
			<td>EU-C1200</td>
			<td>Max 1200g, d=0.01g, T=-1200g</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 1.5ml</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 x 20 mm</td>
		</tr>
		<tr>
			<td>Natural Latex Gloves</td>
			<td>Medica</td>
			<td>M101</td>
			<td></td>
		</tr>
		<tr>
			<td>Touch N Tuff disposible nitrile gloves</td>
			<td>Ansell</td>
			<td>92-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

intracisternal delivery of meningococci into mouse models to induce meningococcal meningitis

Source: Pagliuca, C., et al., <a href="https://appjove.unicartagenaproxy.elogim.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.

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

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

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&#39;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.

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371 조회수. 출처: Pagliuca, C., et al., Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery. J. Vis. 특급 (2019) 이 비디오에서는 Neisseria meningitidis를 마우스 모델로 시추 내 전달하여 수막염 감염을 생성하는 방법을 보여줍니다. 

비디오: 수막구균성 수막염을 유발하기 위해 마우스 모델로 수막구균의 수막구균 내 전달(Intracisternal Delivery of meningococci into mouse models to induce meningococcal meningitis) - 개념

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

마우스 감염 모델에서 뇌 막 염에 구 이렇게 뵙 Biofilms의 조사 역할에 대 한 감염 intracranial Subarachnoidal 경로

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

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Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes 뇌의 감염

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

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.

내분비주주입을 통한 렙토메닝 세포 변형 유도

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

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

내레이션 대본

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis