This work was supported by U.S. Public Health Service grant AI103806 from the National Institutes of Health.

All animals are to be maintained and handled with maximum care to minimize discomfort during the course of the procedure. The procedure is to be conducted in compliance with the Institutional Animal Care and Use Committee and all federal, state and local laws. Also note that the laboratory experiments are to be conducted in accordance with Biosafety Level 2 guidelines.
1. Growth of L. monocytogenes for Mouse Infection Studies
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	<li>Autoclave 500 mL of Brain Heart Infusio...

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A review of 493 episodes.</a> The New England Journal of Medicine. 328 (1), 21-28 (1993).</li><li>Disson, O., Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+central+nervous+system+by+Listeria+monocytogenes.">Targeting of the central nervous system by Listeria monocytogenes.</a> Virulence. 3 (2), 213-221 (2012).</li><li>Daneman, R., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier.">The blood-brain barrier.</a> Cold Spring Harbor Perspectives in Biology. 7 (1), a020412 (2015).</li><li>Becavin, 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=Comparison+of+widely+used+Listeria+monocytogenes+strains+EGD,+10403S,+and+EGD-e+highlights+genomic+variations+underlying+differences+in+pathogenicity.">Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity.</a> MBio. 5 (2), e00969-e00914 (2014).</li><li>Kirchner, M., Higgins, D. 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The authors declare no competing financial interests.

L. monocytogenes is able to cause life-threatening meningoencephalitis in humans. Prior studies have demonstrated the ability of bacteria to cross the blood-brain-barrier (BBB) and to colonize the brain. Three routes of brain invasion have been proposed during infection: direct penetration of the BBB by bacteria, stealth transport by bacteria contained inside of mononuclear cells3, and axonal migration by L. monocytogenes strains that cause rhombencephalitis15</...

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen and one of the most deadly food-borne pathogens worldwide. Ingestion of L. monocytogenes contaminated food can lead to listeriosis in humans, a severe invasive disease targeting mostly pregnant women, newborns, the elderly, and immunocompromised individuals1. L. monocytogenes is among the leading causes of death by a food-borne pathogen in the U.S. and case fatality rates from listeriosis are as high as 20&#8211;30%, the highest for all food-borne pathogens2. No vaccine currently exists for L. monocytogenes and the ability of bacteria to effectively spread to distal organs and the brain by crossing the blood-brain barrier (BBB) may lead to life-threatening meningitis and colonization of the brain3,4,5,6. Bacterial meningitis is typically severe; while most people who receive treatment recover, infections can cause serious complications, e.g., brain damage, hearing loss, or learning disabilities in children. L. monocytogenes is predicted to account for at least 10% of all community acquired meningitis in the U.S.7.
A major route for bacterial dissemination to the brain and meninges is through the bloodstream. Bacteria circulating in blood vessels in the brain are able to cross the BBB to cause brain infection. The BBB is a highly vascularized barrier system that protects the brain from foreign particles circulating in the blood. Endothelial cells constitute a layer that lines the interior surface of the blood vessels8,9. In addition to L. monocytogenes, multiple bacterial pathogens such as Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, and Haemophilus influenzae type b (Hib) are capable of colonizing the brain by crossing the BBB3,4,5,6. However, when examining bacterial burdens in the brain of infected mice, it is important to determine whether bacteria have crossed the BBB, otherwise the bacterial burden in the brain may represent bacteria that are still in the blood vessels of the brain. Thus, it is necessary to perfuse the mice of all blood prior to determining colony-forming units (CFU) of brain homogenates.
In this study, we describe in vivo methods to examine L. monocytogenes infection of the brain. For the methods described here, we used L. monocytogenes strain 10403S. L. monocytogenes&#160;10403S is one of the most widely used laboratory strains to study systemic listeriosis in the mouse model of infection10. This protocol is based on standard intravenous injection of L. monocytogenes followed by perfusion of the mice. A schematic outline of the infection protocol in mice is shown in Figure 1. L. monocytogenes-infected brains and other organs from non-perfused or perfused mice were collected and the bacterial organ burden determined. These methods are useful for not only determining total bacterial colonization of the brain in infected animals, but are also beneficial for determining whether bacteria have penetrated the BBB in vivo to mediate invasion of the brain. It is important to highlight that this laboratory protocol should be conducted following consultation with the relevant institutional biosafety committee and animal facility management.

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			<td height="21" style="height:21px;width:261px;">Brain Heart Infusion media</td>
			<td style="width:119px;">Becton Dickinson</td>
			<td style="width:119px;">237200</td>
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			<td>0382-50G</td>
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			<td>VWR</td>
			<td>25384-342</td>
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			<td>97062-832</td>
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			<td height="42" style="height:42px;width:261px;">IKA T18 ULTRA-TURRAX Basic Homogenizer</td>
			<td>IKA</td>
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			<td>Beckman Coulter</td>
			<td>DU 800 series</td>
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			<td height="21" style="height:21px;">BALB/c mice</td>
			<td>Jackson Laboratory</td>
			<td>Model #000651</td>
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			<td height="21" style="height:21px;">1 mL syringes</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
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			<td height="21" style="height:21px;">26-gauge needles</td>
			<td>Becton Dickinson</td>
			<td>305115</td>
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			<td height="21" style="height:21px;">21-gauge butterfly needles</td>
			<td>Becton Dickinson</td>
			<td>367281</td>
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			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>60004</td>
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			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
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			<td height="21" style="height:21px;">Round-bottom test tubes</td>
			<td>VWR</td>
			<td>60819-546</td>
			<td></td>
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			<td height="21" style="height:21px;">Phosphate-buffered saline&#160;</td>
			<td>Corning</td>
			<td>46-013-CM</td>
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			<td height="21" style="height:21px;">Stainless steel spatula</td>
			<td>VWR</td>
			<td>82027-520</td>
			<td></td>
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			<td height="21" style="height:21px;">Stainless steel scissors (6.5 in)</td>
			<td>VWR</td>
			<td>82027-592</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel scissors (4.5 in)</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel blunt forceps&#160; (4.5 in)</td>
			<td>VWR</td>
			<td>82027-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel fine tip forceps&#160; (6 in)</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
	</tbody>
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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.

The brain is highly vascularized and L. monocytogenes is known to infect cell types present in the blood3,13. The described protocol is used to demonstrate the ability of L. monocytogenes to cross the blood-brain barrier (BBB) leading to infection of the brain in mice. To determine if bacteria have crossed the BBB in vivo, perfusion of blood in the mouse is performed prior to determining bacterial burden...

Watch this Scientific Journal Video about Listeria monocytogenes Infection of the Brain at JoVE.com

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 Infection of the Brain

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

This method can help answer key questions in the bacterial pathogenesis field about whether bloodborne bacteria can cross the blood-brain barrier to infect the central nervous system and brain. With this technique, bacterial numbers within the blood and brain tissue of infected animals can be determined while excluding the number of bacteria residing within the blood vessels of the brain. Demonstrating the procedure will be Pallab Ghosh, a research associate from my laboratory.At 72 hours post-infection, set an automated perfusion system to a flow rate of four milliliters of PBS supplemented with 10 millimolar of EDTA per minute. And place the euthanized animal in the supine position in a dissection pan. Confirm euthanization by a lack of response to paw pinch.And wet the abdomen with 75%ethanol. Use scissors to make an abdominal wall incision to just below the rib cage to expose the diaphragm and visceral organs. And cut the diaphragm to open the thoracic cavity.Cut the rub cage bilaterally to expose the left ventricle and use forceps to gently grasp the heart. Insert a 21-gauge butterfly needle connected to the perfusion system into the left ventricle and carefully cut the right atrium to collect about 200 microliters of blood into a two-milliliter tube containing 10 millimolar EDTA to prevent coagulation. Then perfuse the animal for four minutes with 15 to 20 milliliters of PBS plus EDTA.After complete perfusion, the brain and liver will appear blanched ensuring that remaining bacteria are from the harvested organs and not the circulating blood. Following perfusion, carefully transfer the organs of interest into individual sterile, 15-milliliter conical tubes containing five milliliters of sterile four degrees Celsius PBS per tube. To harvest the brain after decapitation, use scissors to cut the scalp skin down the midline between the eyes, keeping the tips pressed against the skull to trim any excess tissue as necessary.Gently insert one tip of the scissors into the foramen magnum and cut laterally into the skull on both sides. Carefully cut the skull between the eyes down the midline in the skull, taking care to apply lateral pressure and to avoid perturbation of the brain. Using forceps, open the skull to expose the brain and place a spatula between the underside of the brain and the base of the skull to carefully transfer the brain into a 15-milliliter tube containing five milliliters of four degrees Celsius PBS.In a biosafety cabinet, insert the tip and run a tissue homogenizer for 10 seconds in sequential 5%bleach, sterile water, 75%ethanol, and sterile water solutions in separate 15-milliliter conical tubes. When the tip has been thoroughly sterilized, homogenize the infected brain in its tube. When no visible tissue fragments remain, re-sterilize the tip to prevent bacterial carryover to the next organ sample, and homogenize the next organ.When all of the organs have been homogenized, prepare 10-fold serial dilutions of each organ homogenate in sterile PBS and plate the dilutions on brain-heart infusion agar plates. Then incubate the homogenate dilution cultures at 37 degrees Celsius overnight and count the number of colonies on each plate to determine the total number of bacteria per organ or milliliter of blood. Calculation of the bacterial burdens in L.monocytogenes-infected mouse organs as just demonstrated suggests that perfusion post-infection does not significantly alter the bacterial burden in the blood or organs of infected mice that were examined in this representative study.Following this procedure, Listeria monocytogenes-infected organs can be further processed for additional analysis such as histopathological processing to answer additional questions about the infiltration of inflammatory cells into infected mouse organs compared to uninfected control animals. While attempting this procedure, it is important to remember to avoid perturbation of the brain and to maintain minimal contact between the scissors and the brain tissue.

listeria-monocytogenes-infection-of-the-brain

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Immunology and Infection

12.5K Views.  Harvard Medical School. This method can help answer key questions in the bacterial pathogenesis field about whether bloodborne bacteria can cross the blood-brain barrier to infect the central nervous system and brain. With this technique, bacterial numbers within the blood and brain tissue of infected animals can be determined while excluding the number of bacteria residing within the blood vessels of the brain. Demonstrating the procedure will be Pallab Ghosh, a research associate from my laboratory.At 72 ho...