This work was supported by grants from the National Key Research and Development Program of China [2017YFD0500102]; the National Natural Science Foundation of China [31572544]; the State Key Laboratory of Veterinary Etiological Biology [SKLVEB2016KFKT005]; the Shanghai Agriculture Applied Technology Development Program, China [G2016060201].

The mouse infection experiments were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province, China and performed in the Laboratory Animal Center of Nanjing Agricultural University (Permit number: SYXK (Su) 2017-0007).
1. Preparation of Bacteria
Note: S. suis serotype 2 virulent strain P1/7 was isolated from a diseased pig with meningitis19. Strain P1/7 was grown in Todd-Hewitt broth (THB, formula per liter of THB: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g) and plated on Todd-Hewitt agar (THA, formula per liter of THA: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g; agar, 15.0 g) at 37 &#176;C and 5% CO2.
<ol>
	<li>Collection of strain P1/7 planktonic cells
	<ol>
		<li>Collect 5 mL of planktonic cells from mid-log phase culture (OD600=0.6), centrifuge for 3 min at 8000 &#215; g, and then wash 3 times with PBS.</li>
		<li>Resuspend the cells with 5 mL of 25% glycerol in THB, aliquot into 5 tubes, and then store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Collection of strain P1/7 biofilm state cells
	<ol>
		<li>Take 20 mL of an overnight culture and add to 180 mL of fresh THB; divide the diluted culture into 10 round culture plates equally, and then put the plates in an incubator at 37 &#176;C in 5% CO2 for 24 h.</li>
		<li>After incubation,&#160;shake the plate gently to resuspend the bacteria that have not adhered to the plate, and then discard the supernatant by aspiration. 
		NOTE: Shake the plate gently and avoid resuspending the sediment.</li>
		<li>Add 5 mL of PBS to harvest biofilm state cells, resuspend the sediment completely, and then transfer the sample to a new tube.</li>
		<li>Sonicate the biofilm state cells from the last step with the following parameters: 60 W, 4 cycles, 5 s on and 10 s off.</li>
		<li>Centrifuge for 3 min at 8000 &#215; g and store the supernatant at -80 &#176;C. 
		NOTE: The supernatant may contain biofilm components and it can be used to resuspend the biofilm bacteria before infection as described in the animal experiments (Step 3).</li>
		<li>Resuspend the sediment with 10 mL 25% glycerol in THB and then store the biofilm state cells at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Scanning Electron Microscopy (SEM) Analysis
<ol>
	<li>Fix the collected biofilm state cells and planktonic cells using 4% paraformaldehyde for 12 - 24 h at 4 &#176;C.</li>
	<li>Rinse the samples quickly with distilled water and dehydrate the samples sequentially with an increasing concentration of ethanol (25%, 50%, 70%, 90%, and 100%) for 30 min in each solution.</li>
	<li>Adhere the dried samples to metal holders with double-sided tape and finally coat them in an evaporator with gold and palladium.</li>
	<li>Observe the samples by a scanning electron microscope using the following parameters: EHT (Extra High Tension) = 10 kV; WD (Working Distance) = 7.0 or 8.0 mm; Magnification = 5000 &#215;; Signal A= SE1.</li>
</ol>
3. Animal Experiments
<ol>
	<li>Use SPF 6-week-old female BALB/c mice in this study. Infect all mice through the intracranial subarachnoidal route of infection whose injection site is located 3.5 mm rostral from the bregma. The stereotaxic coordinates of the injection site were clearly described by Chiavolini et al17.
	<ol>
		<li>For histopathological analysis, infect two groups of mice (5 mice per group) using planktonic cells or biofilm state cells at a dose of 3 &#215; 107 colony-forming units (CFU), and another 5 mice were injected with PBS as a control.</li>
		<li>For detection of mRNA expression of TLR2 and cytokines in brain, infect two additional groups of mice (6 mice per group) using planktonic cells or biofilm state cells at a dose of 3 &#215; 107 CFU.</li>
		<li>Determine the number of viable bacteria by plating serial dilutions on to THA.</li>
		<li>Euthanize all mice at 12 h post-infection for further research. 
		&#8203;NOTE: Glycerol in the stock medium must be removed before infection. Both planktonic cells and biofilm state cells recovered from -80 &#176;C are washed 3 times with PBS. Then, planktonic cells are resuspended with PBS and diluted to the appropriate dose for infection; biofilm state cells are resuspended with the stored supernatant (from step 1.2.5) and diluted to the appropriate dose for infection. The volume for infection should not be more than 50 &#181;L.</li>
	</ol>
	</li>
	<li>Detecting the mRNA expression of TLR2 and cytokines in brain tissue
	<ol>
		<li>Extract the total RNA from brain tissue using a RNA extraction kit.
		<ol>
			<li>For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into 5 tubes. Take up to 100 mg brain tissue and add 1 mL of lysis solution to each tube containing the lysing matrix provided by the kit.</li>
			<li>Process the tube in a homogenizer for 40 s at a setting of 6.0, and then centrifuge the tube at 12000 &#215; g and 4 &#176;C for 5 min.</li>
			<li>After centrifugation, transfer the upper phase to a new microcentrifuge tube.</li>
			<li>Incubate the transferred sample for 5 min at room temperature; add 300 &#181;L of chloroform, vortex for 10 s, and then incubate for 5 min at room temperature.</li>
			<li>Centrifuge the tube at 12000 &#215; g and 4 &#176;C for 5 min. Transfer the upper phase to a new tube.</li>
			<li>Add 500 &#181;L of cold absolute ethanol to the tube, before inverting it for 5 times, and then store the sample at -20 &#176;C for at least 1 h.</li>
			<li>Centrifuge the tube at 12000 &#215; g and 4 &#176;C for 15 min and remove the supernatant. Wash the pellet with 500 &#181;L of cold 75% ethanol in RNase-free H2O.</li>
			<li>Remove the ethanol, air dry the pellet for 5 min at room temperature, and resuspend the RNA in 100 &#181;L of RNase-free H2O.</li>
			<li>Incubate the RNA for 5 min at room temperature and determine the RNA concentration using a RNA bioanalyzer.</li>
		</ol>
		</li>
		<li>Perform cDNA synthesis, including elimination of genomic DNA (gDNA), using a thermocycler with a reverse transcription (RT) reagent kit.
		<ol>
			<li>Add 1 &#181;g of RNA to a tube containing 2 &#181;L of 5x gDNA elimination buffer, 1 &#181;L of gDNA elimination enzyme and RNase-free H2O until the volume reaches 10 &#181;L. Incubate for 2 min at 42 &#176;C.</li>
			<li>Add 10 &#181;L of master mix (1 &#181;L of RT enzyme Mix I, 1 &#181;L of RT Primer Mix, 4 &#181;L of 5x Buffer 2, and 4 &#181;L of RNase-free H2O) to the reaction solution from the last step and then mix gently. Proceed immediately with the RT reaction: 37 &#176;C for 15 min and then 85 &#176;C for 5 s.</li>
			<li>Perform the quantitative real-time PCR (RT-qPCR) analysis using a Real-Time PCR machine with a SYBR RT-qPCR kit. Add 10 &#181;L of 2x enzyme, 0.8 &#181;L of forward primer, 0.8 &#181;L of reverse primer, 0.4 &#181;L of 50x ROX Reference Dye II, 2 &#181;L of cDNA template, and RNase-free H2O until a total volume of 20 &#181;L.</li>
			<li>Run each sample in triplicate using the following thermal parameters: 30 s at 94 &#176;C, followed by 40 cycles of 5 s at 95 &#176;C, and 34 s at 60 &#176;C, with a final stage of 15 s at 95 &#176;C, 1 min at 60 &#176;C, and 15 s at 95 &#176;C. Primers for RT-qPCR are listed in Table 1. Housekeeping genes B2m and 18s rRNA were used as the internal controls.</li>
		</ol>
		</li>
		<li>Calculate the relative fold change based on the 2-&#916;&#916;Ct method.</li>
	</ol>
	</li>
	<li>Observation of histological sections
	<ol>
		<li>After 24 h fixation with 10% formalin, reconstruct the fixed brain tissue into appropriate sizes of tissue pieces at 2 - 3 mm.</li>
		<li>Put the tissue pieces in an embedding cassette and immerse in a new fixative solution for dehydration with an automatic vacuum dehydrator.</li>
		<li>Take out the fixed tissue and dehydrate it sequentially in increasing concentrations of ethanol (70%, 80%, 95%, 95%, and 100%) for 30 min in each solution.</li>
		<li>Transparentize the sample with xylene for 15 min, dip it in liquid paraffin at 60 &#176;C for 90 min, and then embed using an embedding machine.</li>
		<li>Cut the paraffin block embedded tissue into 3 &#181;m slices, tile the slice on water at 45 &#176;C, and then dry it in the air.</li>
		<li>Incubate the section for 3 h at 60 &#176;C, stain it using the haematoxylin and eosin (HE) method, and seal it with the neutral gum.</li>
		<li>Observe the histopathological changes of the brain with an optical microscope. A four-point grading system was used to evaluate histological changes. Use an unpaired t test for statistical analysis. 
		Congestion/haemorrhage: absent, score 0; small area of focal congestion/haemorrhage, score 1; large area of focal congestion/haemorrhage, or multiple sites of small area of focal congestion/haemorrhage, score 2; up to three large focal congestion/haemorrhage sites, score 3; diffuse congestion/haemorrhage, score 4. 
		Necrosis: absent, score 0; focal necrosis, score 1; diffuse occurrence of 1 - 10 necrotic cells, score 2; foci of confluent necrosis, score 3; extensive confluent necrosis, score 4. 
		Inflammatory cell infiltration: absent, score 0; few and focal infiltrations, score 1; multifocal infiltration or infiltrations with formation of aggregates, score 2; up to three aggregates, score 3; and more than four aggregates, score 4.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The intracranial subarachnoidal route of infection described here has obvious advantages over other routes of infection. It allows investigators to study the host-bacterium interaction and the effect of bacterial components on host immune responses directly in the brain, which mimic bacterial entrance into the central nervous system. Thus, 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 ...

Streptococcus suis (S. suis) is a major bacterial pathogen of pigs worldwide, causing severe diseases including meningitis, pneumonia, septicaemia, endocarditis, and arthritis1. It is also an emerging zoonotic agent. So far, it has been reported that nine serotypes can cause infection in humans, including serotypes 2, 4, 5, 9, 14, 16, 21, 24, and 312,3,4. In humans and pigs, meningitis is one of the major clinical signs of S. suis infections. In Vietnam and Thailand, S. suis is the major cause of meningitis in adults5. Microbial biofilms are microorganisms that adhere to each other and are concentrated at an interface; they are essential for bacterial virulence, survival in diverse environments, and antibiotic resistance5. Biofilms are typically surrounded by an extracellular matrix that generally contains polysaccharides, proteins, and DNA6. The latter is able to elicit host inflammatory responses and cytokine production7. Biofilm formation has been reported to be involved in streptococcal meningitis in previous studies. Biofilms contribute to Streptococcus agalactiae meningitis in a tilapia fish model and biofilm formation has been revealed within brain tissues and around meningeal surfaces in vivo through intra-abdominal inoculation8. During meningitis, Streptococcus pneumoniae is in a biofilm-like state and bacteria in such a biofilm state were more effective in inducing meningitis in a mouse infection model9. In addition, in our previous study, the biofilm state associated with S. suis in mouse brain contributes to bacterial virulence by survival analysis10. However, direct evidence for biofilm involvement in S. suis meningitis requires further investigation.
Animal models of S. suis infection have been developed in mice using the intraperitoneal (i.p.)11, intranasal (i.n.)12, intravenous (i.v.)13, and the intracisternal (i.c.) routes of infection14,15,16. However, the i.p., i.n., and i.v. routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain. These include extracellular matrix from biofilms. Although the i.c. inoculation was used for S. suis infection, the precise injection site has not been described in those papers. In contrast, the stereotaxic coordinates of the injection site for intracranial subarachnoidal inoculation has clearly been described in a previous study17. This allowed easy recognition of the inoculation point and more simplistic experimental protocol. In addition, the intracranial subarachnoidal route of infection mimics bacterial entrance into the central nervous system from the sinuses or the middle ear17, and the relationship between the middle ear and meningitis caused by S. suis has been demonstrated by Madsen et al18. Moreover, by applying the intracranial subarachnoidal route of infection in mice, we have demonstrated that S. suis small RNA rss04 contributes to meningitis in our previous study10.
In the present study, the intracranial subarachnoidal route of infection was used in mice to investigate the roles of biofilms in S. suis meningitis. Mice were infected with planktonic cells or biofilm state cells of S. suis by this route of infection. Histopathological analysis and increased mRNA expression of TLR2 and cytokines from brain tissue of mice injected with biofilm state cells clearly indicated that S. suis biofilm contributes to meningitis.

<table><tbody><tr><td>Todd Hewitt Broth(THB)</td><td>Becton, Dickinson and Company</td><td>DF0492078</td><td>Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min.</td></tr><tr><td>Agar</td><td>DSBIO</td><td>16C0050</td><td>Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&amp;deg; for 15 min.</td></tr><tr><td>Milli-Q Reference Water Purification System</td><td>Merck KGaA</td><td>Z00QSVCUS</td><td>Without Dnase/ Rnase</td></tr><tr><td>NaCl</td><td>Tianjin Kemiou Chemical Reagent Co., Ltd</td><td>10019318</td><td>Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min. Use KOH to adjust pH to 7.4.</td></tr><tr><td>Na2HPO3</td><td>Xilong Scientific Co., Ltd</td><td>9009012-01-09</td><td>Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min. Use KOH to adjust pH to 7.4.</td></tr><tr><td>KCl</td><td>Xilong Scientific Co., Ltd</td><td>9009017-01-09</td><td>Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min. Use KOH to adjust pH to 7.4.</td></tr><tr><td>KH2PO4</td><td>Xilong Scientific Co., Ltd</td><td>9009019-01-09</td><td>Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min. Use KOH to adjust pH to 7.4.</td></tr><tr><td>KOH</td><td>Xilong Scientific Co., Ltd</td><td>9009014-01-09</td><td>Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&amp;deg; for 15 min. Use KOH to adjust pH to 7.4.</td></tr><tr><td>Glycerol</td><td>Sionpharm Chemical Reagent Co., Ltd</td><td>10010618</td><td>Diluted with equal volumu of purified water, autoclave at 121&amp;deg; for 15 min</td></tr><tr><td>4% paraformaldehyde</td><td>Sionpharm Chemical Reagent Co., Ltd</td><td>80096675</td><td></td></tr><tr><td>25% Glutaraldehyde</td><td>Sionpharm Chemical Reagent Co., Ltd</td><td>30092436</td><td>10-fold diluted with purified water for fixation.</td></tr><tr><td>Ethanol</td><td>Sionpharm Chemical Reagent Co., Ltd</td><td>10009218</td><td></td></tr><tr><td>Chloroform</td><td>Sionpharm Chemical Reagent Co., Ltd</td><td>10006818</td><td></td></tr><tr><td>Spctrophotometre</td><td>DeNovix Inc.</td><td>DS-11+</td><td></td></tr><tr><td>Ultrasound cell crusher</td><td>NingBo Scientz Biotechnology Co.,Ltd</td><td>JY96-IIN</td><td></td></tr><tr><td>Centrifuge</td><td>Hitachi Koki Co., Ltd</td><td>CT15RE</td><td></td></tr><tr><td>Refrigerator</td><td>Aucma Co., Ltd</td><td>DW-86L500</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Zeiss</td><td>EVO-LS10</td><td></td></tr><tr><td>FastRNA Pro Green Kit</td><td>MP Biomedicals</td><td>#6045-050</td><td></td></tr><tr><td>FastPrep-24 Instrument</td><td>MP Biomedicals</td><td>116005500</td><td></td></tr><tr><td>Instrument for PCR</td><td>SensoQuest GmbH</td><td>1124310110</td><td></td></tr><tr><td>QuantStudio 6 Flex</td><td>Thermo Fisher Scientific</td><td>4485689</td><td></td></tr><tr><td>SYBR Premix Ex Taq II</td><td>Takara Biomedical Technology (Beijing) Co., Ltd</td><td>RR820A</td><td></td></tr><tr><td>PrimeScript RT reagent kit with gDNA Eraser</td><td>Takara Biomedical Technology (Beijing) Co., Ltd</td><td>RR047A</td><td></td></tr><tr><td>Fully Enclosed Tissue Processor</td><td>Leica Biosystems Nussloch GmbH</td><td>ASP200S</td><td></td></tr><tr><td>Heated Paraffin Embedding Module</td><td>Leica Biosystems Nussloch GmbH</td><td>EG1150H</td><td></td></tr><tr><td>Semi-Automated Rotary Microtome</td><td>Leica Biosystems Nussloch GmbH</td><td>RM2245</td><td></td></tr><tr><td>Water bath for paraffin sections</td><td>Leica Biosystems Nussloch GmbH</td><td>HI1210</td><td></td></tr><tr><td>Autostainer XL</td><td>Leica Biosystems Nussloch GmbH</td><td>ST5010</td><td></td></tr><tr><td>Agilent 2100</td><td>Agilent Technologies</td><td>G2939A</td><td></td></tr><tr><td>Optical microscope</td><td>Olympus</td><td>BX51</td><td></td></tr></tbody></table>

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.

SEM analysis was performed to examine biofilm formation under the experimental conditions. As shown in Figure 1, there is a significant difference in biofilm formation between planktonic cells (Figure 1A) and biofilm state cells (Figure 1B). SEM analysis showed that biofilm bacteria were in clumps and multiple layers and they were encased in the extracellular matrix,...

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

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.

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 ...

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Universidad de Cartagena UDEC

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Immunology and Infection

7.2K Views.  Nanjing Agricultural University. 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.

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

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 ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Medicine

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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.

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. ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

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.

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

Generation of a Murine Model with a Central Nervous System Infection

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

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

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

Summary

Explore More Videos

Chapters in this video

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

A Murine Model of Subarachnoid Hemorrhage

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

In Vivo Mouse Model of Spinal Implant Infection

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Generation of a Murine Model with a Central Nervous System Infection