The authors thank Dr. Feng Li and Mark Dittmar (OUHSC P30 Live Animal Imaging Core, Dean A. McGee Eye Institute, Oklahoma City, OK, USA) for their assistance. Our research has been supported by National Institutes of Health grants R01EY028810, R01EY028066, R01EY025947, and R01EY024140. Our research has also been supported by P30EY21725 (NIH CORE grant for Live Animal Imaging and Analysis, Molecular Biology, and Cellular Imaging). Our research has also been supported by the NEI Vision Science Pre-doctoral Trainee program 5T32EY023202, a Presbyterian Health Foundation Research Support grant, and an unrestricted grant to the Dean A. McGee Eye Institute from Research to Prevent Blindness.

All procedures were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center (protocol numbers 15-103, 18-043, and 18-087).
1. Sterile glass needles
<ol>
	<li>Turn On the needle p...

Even with the availability of potent antibiotics, anti-inflammatory drugs, and vitrectomy surgery, bacterial endophthalmitis can blind a patient. Clinical studies have been useful in studying endophthalmitis; however, experimental models of endophthalmitis provide quick and reproducible results that can be translated to progress in standard of care, resulting in better visual outcome for patients.
The vitreous volume of the mouse eye is approximately 7 &#181;L40. This s...

Bacterial endophthalmitis is a devastating infection that causes inflammation, and, if not treated properly, can result in loss of vision or blindness. Endophthalmitis results from the entry of bacteria into the interior of the eye1,2,3,4,5. Once in the eye, bacteria replicate, produce toxins and other noxious factors, and can cause irreversible damage to delicate retinal cells and tissues. Ocular damage can also be caused by inflammation, due to the activation of inflammatory pathways leading to inflammatory cell influx into the interior of the eye1,5,6. Endophthalmitis can occur following intraocular surgery (post-operative), a penetrating injury to the eye (post-traumatic), or from metastatic spread of bacteria into the eye from a different anatomical site (endogenous)7,8,9,10. Treatments for bacterial endophthalmitis includes antibiotics, anti-inflammatory drugs, or surgical intervention3,4,11. Even with these treatments, vision or the eye itself may be lost. The visual prognosis after bacterial endophthalmitis generally varies depending upon the treatment effectiveness, the visual acuity at presentation, and the virulence of the infecting organism.
Bacillus cereus (B. cereus) is one of the major bacterial pathogens that causes post-traumatic endophthalmitis7,12. A majority of B. cereus endophthalmitis cases have a rapid course, which can result in blindness within a few days. The hallmarks of B. cereus endophthalmitis include quickly evolving intraocular inflammation, eye pain, rapid loss of visual acuity, and fever. B. cereus grows rapidly in the eye compared to other bacteria which commonly cause eye infections2,4,12 and possesses many virulence factors. Therefore, the window for successful therapeutic intervention is relatively short1,&#160;2,&#160;3,&#160;4,&#160;5,&#160;6,&#160;7,&#160;11,&#160;12,&#160;13,&#160;14,&#160;15,&#160;16,&#160;17,&#160;18,&#160;19,&#160;20,&#160;21,&#160;22,&#160;23,&#160;24,&#160;25. Treatments for this infection are usually successful in treating endophthalmitis caused by other less virulent pathogens, but B. cereus endophthalmitis commonly results in greater than 70% of patients suffering from significant vision loss. About 50% of those patients undergo evisceration or enucleation of the infected eye7,16,22,23. The destructive and rapid nature of B. cereus endophthalmitis calls for immediate and proper treatment. Recent progress in discerning the underlying mechanisms of disease development have identified potential targets for intervention19,26,27. Experimental mouse models of B. cereus endophthalmitis continue to be useful in discerning the mechanisms of infection and testing potential therapeutics that may prevent vision loss.
Experimental intraocular infection of mice with B. cereus has been an instrumental model for understanding bacterial and host factors, as well as their interactions, during endophthalmitis28. This model mimics a post-traumatic or post-operative event, in which bacteria are introduced into the eye during an injury. This model is highly reproducible and has been useful for testing experimental therapies and providing data for improvements in standard of care1,6,19,29,30. Like many other infection models, this model allows for independent control of many parameters of infection and enables efficient and reproducible examination of infection outcomes. Studies in a similar model in rabbits over the past few decades have examined the effects of B. cereus virulence factors in the eye2,4,13,14,31. By injecting B. cereus mutant strains lacking individual or multiple virulence factors, the contribution of these virulence factors to disease severity can be measured by outcomes such as the concentration of bacteria at different hours of postinfection or the loss of visual function13,14,27,31,32. In addition, host factors have been examined in this model by infecting knockout mouse strains lacking specific inflammatory host factors26,29,33,34,35. The model is also useful for testing potential treatments for this disease by injecting novel compounds into the eye after infection30,36. In this manuscript, we describe a detailed protocol which includes infecting a mouse eye with B. cereus, harvesting the eye after infection, quantifying intraocular bacterial load, and preserving specimens to assay additional parameters of disease severity.

<table>
	<tbody>
		<tr>
			<td>2-20 &#181;L pipette</td>
			<td>RANIN</td>
			<td>L0696003G</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>37oC Incubator</td>
			<td>Fisher Scientific</td>
			<td>11-690-625D</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>90003-032</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Cell Microinjector</td>
			<td>MicroData Instrument, Inc.</td>
			<td>PM2000</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Fine tip forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-000-122</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Glass beads 1.0 mm</td>
			<td>BioSpec</td>
			<td>11079110</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>NB-I2400</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microcapillary Pipets 5 Microliters</td>
			<td>Kimble</td>
			<td>71900-5</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Micro-Pipette Beveler</td>
			<td>Sutter Instrument Co.</td>
			<td>BV-10</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microscope Axiostar Plus</td>
			<td>Zeiss</td>
			<td></td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microscope OPMI Lumera</td>
			<td>Zeiss</td>
			<td></td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-16</td>
			<td>BioSpec</td>
			<td>Model 607</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Multichannel pipette 30-300 &#181;L</td>
			<td>Biohit</td>
			<td>15626090</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Multichannel pipette 5-100 &#181;L</td>
			<td>Biohit</td>
			<td>9143724</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Needle/Pipette Puller</td>
			<td>Kopf</td>
			<td>730</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>1897315</td>
			<td>Molecular grade</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Molecular grade</td>
		</tr>
		<tr>
			<td>Reverse action forceps</td>
			<td>Katena</td>
			<td>K5-8228</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

intravitreal injection and quantitation of infection parameters in a mouse model of bacterial endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 &#181;L sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 &#176;C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

Generating a reproducible inoculum and accuracy of the intravitreal injection procedure are key steps in developing models of microbial endophthalmitis. Here, we demonstrated the intravitreal injection procedure using Gram-positive Bacillus cereus. We injected 100 CFU/0.5 &#956;L of B. cereus into the mid-vitreous of five C57BL6 mice. After 10 h postinfection, we observed intraocular growth of B. cereus to approximately 1.8 x 105 CFU/eye. Figure 1 demons...

Watch this Scientific Journal Video about Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis at JoVE.com

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Our protocol allows for the reproducible introduction of microorganism and therapeutic agents into the mouse eye to study the pathogenesis of intraocular infection and novel treatment effectiveness. Our technique allows intraocular infections to be established in a mouse model and facilitates the quantitation of microorganisms, host immune responses, infection related host and pathogen gene expression, and novel treatment effectiveness. Visualization of the injection site, needle angle, and delivery are critical for successful execution of this technique and a proper eye harvest is essential for quantification of the infection parameters.In a biosafety level two facility, add five milliliters of BHI broth to a 10 milliliter snap cap tube and use a sterile loop to transfer a single B.cereus colony from an auger plate culture into five milliliters of broth. After brief vortexing, incubate the sample in a 37 degrees Celsius rotating incubator at 200 revolutions per minute for 18 hours. At the end of the incubation, dilute the culture to a 200 colony forming units per microliter concentration in 10 milliliters of fresh BHI, and after vortexing, add one milliliter of the freshly diluted culture to a 1.5 milliliter microcentrifuge tube on ice.For intravitreal injection, turn on the microinjector and open the gas valve on the compressed air tank attached to the microinjector. Press Mode on the microinjector until the screen shows Balance. Press Balance and place the computer mouse on the operating table.Connect the stainless steel pipette holder to the tubing attached to the fill output on the injector and tightly screw the pipette holder connector around a beveled glass capillary pipette tip needle to allow insertion of the capillary needle into the other end of the pipette holder. Turn on the ophthalmic microscope and set the light intensity to 50%Position the microscope over the procedure site and adjust the microscope to the desired focus. After confirming a lack of response to pedal reflex, place the anesthetized mouse on its left side on the medical underpads with the nose pointed to the right.Locate the right eye through the ophthalmic microscope objective and place the tongs of reverse action forceps on either side of the eye to expose the injection site. Left click the mouse connected to the microinjector to fill the prepared capillary needle with the diluted bacteria solution, and securing the head with the left hand, place the tip of the needle, bevel side up, at the limbus of the eye. With the needle at a 45 degree angle, puncture the eye, taking care that only 0.5 millimeters of the sharp tip of the needle is inserted.Once the needle tip has been inserted, use the left hand to right click on the mousepad to inject 0.5 microliters of the B.cereus solution. To prevent leakage, leave the needle tip inside the mouse eye for two to three seconds before removing, then release the forceps and the eye will go back into the socket by itself. Transfer the mouse to a recovery cage on a warming pad with monitoring until full recumbency.At the appropriate experimental endpoint, add 400 microliters of PBS supplemented with protease inhibitor in one labeled, autoclaved harvest tube per eye on ice and place open fine tip forceps on either side of the infected eye. Push the tips down towards the head to proptose the eye. Once the tongs are behind the eye globe, squeeze the tongs together and pull the forceps away from the head to detach the eyeball.Then immediately place the eyeball into the appropriately labeled harvesting tube. Within 60 minutes of sample collection, place the closed harvesting tubes into a tissue homogenizer and homogenize the samples for two, one minute homogenization periods with a 30 second period of rest in between. After homogenization, place the samples on ice and use 20 microliter aliquots of homogenate to serially dilute the samples in 180 microliters of PBS per dilution, until a factor of one times 10 to the negative eight is reached.Next, label each row of a square pre-warmed BHI plate with the appropriate dilution concentration, and with the plate tilted at a 45 degree angle, add 10 microliters of each dilution into individual rows at the top of the plate. Let each sample run until it almost reaches the bottom of the plate before laying the plate flat. When the samples have been absorbed into the agar, transfer the plate to a 37 degrees Celsius incubator.Colonies should begin to be visible after eight hours of incubation. For an accurate representation of the concentration in the sample, count the row that has between 10 to 100 colonies. The construction of a beveled glass needle tip is critical for delivering the bacteria into the mid-vitreous of the mouse eye.In these images, Bacillus cereus growing on a blood agar plate and in broth culture tubes are shown. As illustrated in this graph of interocular bacterial counts from five different mouse eyes at 10 hours post-infection between one times 10 to the fifth and one times 10 to the seventh colony forming units of bacteria can typically be recovered from a single infected eye. The most important thing to remember when attempting this procedure is to inject that appropriate volume of bacteria into the mouse eye.Following this procedure, we can study inflammation by histology, inflammatory mediators by ELISA, and gene expression by high throughput RNA sequencing to facilitate a better understanding of the pathogenesis of this disease.

intravitreal-injection-quantitation-infection-parameters-mouse-model

Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of Oklahoma Health Sciences Center. Our protocol allows for the reproducible introduction of microorganism and therapeutic agents into the mouse eye to study the pathogenesis of intraocular infection and novel treatment effectiveness. Our technique allows intraocular infections to be established in a mouse model and facilitates the quantitation of microorganisms, host immune responses, infection related host and pathogen gene expression, and novel treatment effectiveness. Visualization of the injection site, needle angle, and d...

Video: Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene...

bacterial infection quantitation assay: a method to quantify intraocular bacterial load in mouse model of bacterial endophthalmitis

This video describes the technique of quantifying bacterial infection from eye homogenates obtained from a murine model of bacterial endophthalmitis caused by Bacillus cereus. This technique helps study the host immune response and its dependency on the host and ocular bacterial gene expression in murine...

Bacterial Infection Quantitation Assay: A Method to Quantify Intraocular Bacterial Load in Mouse Model of Bacterial Endophthalmitis

bacterial endophthalmitis mouse model: a method for generating bacterial endophthalmitis via intravitreal injection of bacterial suspension into eye of mouse

This video describes a method for generating a mouse model that mimics bacterial endophthalmitis &#8211; an intraocular infection resulting from a bacterial invasion into the eye&#8217;s vitreous cavity &#8211; via intravitreal injection of a bacterial suspension into the eye. This mouse model helps study the disease mechanism and evaluate therapeutics for...

Bacterial Endophthalmitis Mouse Model: A Method for Generating Bacterial Endophthalmitis via Intravitreal Injection of Bacterial Suspension into Eye of Mouse

Turn on the ophthalmic microscope and set the light intensity to 50%. Position the microscope over the procedure side and adjust the microscope to the desired focus. After confirming a lack of response to pedal reflux, place the anesthetized mouse on its left side on the medical underpads with the nose pointed to the...

Once the tongs are behind the eye globe, squeeze the tongs together, and pull the forceps away from the head to detach the eyeball. Then, immediately place the eyeball into the appropriately labeled harvesting tube. Within 60 minutes of sample collection, place the closed harvesting tubes into a tissue homogenizer, and homogenize the samples for two one-minute homogenization periods, with a 30-second period of rest in...

evaluation of vaccine-induced immunity against bacterial infection in a mouse model

In this video, we describe a protocol to test the efficacy of an acellular pertussis vaccine against Bordetella pertussis infection in mice. Post-vaccination, the mice were subjected to infection, and their tissue homogenate was cultured to analyze the bacterial colonies as a readout of the vaccine-induced protective...

Evaluation of Vaccine-Induced Immunity Against Bacterial Infection in a Mouse Model

Keep the needle inserted in the muscle for 5 to 10 seconds. Then, rotate the syringe 180 degrees so that the bevel is facing down to create a seal, and to prevent vaccine leakage before slowly retracting the needle from the injection site. Then, return the mouse to its cage with monitoring until full...

isolation of splenic microvesicles in a murine model of intraperitoneal bacterial infection

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

subcutaneous injection of bacterial antigen to induce systemic inflammation in a murine model

This video demonstrates the induction of systemic inflammation in an anesthetized mouse. The injection of mycobacterial antigen-adjuvant depots triggers cytokine release and localized inflammation. Lymphatic vessels open, enabling immune cell entry into lymph nodes and activation of T cells, which release pro-inflammatory cytokines, leading to widespread...

Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model

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

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...

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

a technique to establish a bacterial infection model in insect larvae

This video demonstrates a method to establish a Mycobacterium bovis infection model in the larvae of the greater wax moth, Galleria mellonella. A bioluminescent strain of Mycobacterium bovis BCG is injected into the hemolymph of the larva to establish infection, and the bioluminescence is measured at predefined intervals to study the progress of...

A Technique to Establish a Bacterial Infection Model in Insect Larvae

influenza a virus studies in a mouse model of infection

Influenza A Virus Studies in a Mouse Model of Infection

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of...

in vivo mouse model of spinal implant infection

In Vivo Mouse Model of Spinal Implant Infection

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...

tumor implantation and injection of a tumor vaccine in a mouse model

This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune...

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

deep dermal injection as a model of candida albicans skin infection for histological analyses

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

monitoring neutrophil recruitment dynamics and bacterial burden at the infection site in a murine model

This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

porcine ex vivo cornea model of bacterial keratitis: a technique to establish bacterial infection in corneal epithelial cells

This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo...

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

a periprosthetic joint candida albicans infection model in mouse

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...

A Periprosthetic Joint Candida albicans Infection Model in Mouse

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

a catheter-related candida albicans infection model in mouse

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI,  laying a foundation for clinical...

A Catheter-Related Candida albicans Infection Model in Mouse

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

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.

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.

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 ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

Establishing a Mouse Model for &lt;em&gt;Candida albicans&lt;/em&gt; Biofilm-Related PJI

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.

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 ...