Name: urinary tract infection in a small animal model: transurethral catheterization of male and female mice
Uploaded: 2017-12-01T13:00:00
Description: Watch this Scientific Journal Video about Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice at JoVE.com

We thank Dr Matthieu Rousseau for critical reading of the manuscript and the Laboratory of Dendritic Immunobiology for their helpful insights during the development of this protocol and project. This work was supported in part by funding from the European Union Seventh Framework Programme Marie Curie Action (PCIG11-GA- 2012-3221170, and the Immuno-Oncology LabEx (MAI).

Mouse experiments were conducted in accordance with approval of protocol number 2012-0024 by the Comit&#233; d&#39;&#233;thique en exp&#233;rimentation animale Paris Centre et Sud (the ethics committee for animal experimentation), in application of the European Directive 2010/63 EU.
1. Preparation of Catheters
<ol>
	<li>Prepare one pediatric intravenous-access cannula for each group of mice to be infected. Using the inbuilt spring mechanism, divest each cannula of its needle, as instructed b...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+uncomplicated+urinary+tract+infection.">Diagnosis and treatment of uncomplicated urinary tract infection.</a> Infect Dis Clin North Am. 11 (3), 551-581 (1997).</li><li>Harper, M., Fowlis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+urinary+tract+infections+in+men.">Management of urinary tract infections in men.</a> Trends in Urology, Gynaecology &amp; Sexual Health. 12 (1), 30-35 (2007).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+urinary+tract+infection.">The epidemiology of urinary tract infection.</a> Nat Rev Urol. 7 (12), 653-660 (2010).</li><li>Lipsky, B. 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A., Kline, K. A., Nielsen, H. V., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-CSF+induction+early+in+uropathogenic+Escherichia+coli+infection+of+the+urinary+tract+modulates+host+immunity.">G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity.</a> Cell Microbiol. 10 (12), 2568-2578 (2008).</li><li>Mora-Bau, G., 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=Macrophages+Subvert+Adaptive+Immunity+to+Urinary+Tract+Infection.">Macrophages Subvert Adaptive Immunity to Urinary Tract Infection.</a> PLoS Pathog. 11 (7), e1005044 (2015).</li><li>Oliveira, P. 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M., 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=Intravesical+delivery+of+rapamycin+suppresses+tumorigenesis+in+a+mouse+model+of+progressive+bladder+cancer.">Intravesical delivery of rapamycin suppresses tumorigenesis in a mouse model of progressive bladder cancer.</a> Cancer Prev Res (Phila). 2 (12), 1008-1014 (2009).</li><li>Hagberg, L., 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=Ascending,+unobstructed+urinary+tract+infection+in+mice+caused+by+pyelonephritogenic+Escherichia+coli+of+human+origin).">Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin).</a> Infect Immun. 40 (1), 273-283 (1983).</li><li>Olson, P. D., Hruska, K. A., Hunstad, D. 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Transurethral instillation of male mice offers many new research opportunities into the influence of sex on bladder mucosal disease, but also presents several challenges. Foremost, one limitation is that the instillation may initially prove to be technically difficult, resulting in excessive inflammation during catheter insertion. Improvement in technique can be achieved by the instillation of dead mice with a colored solution, such as Evans blue dye. To confirm that the instillation is successful, bladders should be vis...

Urinary tract infections (UTI) are one of the most common infections in developed countries1. Infection rates are similar between females and males among neonates and the elderly2. Premenopausal adult women, however, have a greatly increased incidence of community-acquired UTI compared to men2,3. Given that this disease primarily impacts women, fundamental and clinical research has overwhelmingly focused on UTI in females. However, UTI in men is a significant and understudied health care challenge4. Indeed, because UTI in men are associated with higher morbidity, these infections are routinely defined as complicated4,5.
As our understanding of the central role of sex biases in physiology and pathology evolves, new methods are required to explore this previously neglected aspect of disease. Sex differences play an essential role in immunity and infection; females have higher incidence of autoimmune disease, while males are more susceptible to certain infections, such as tuberculosis, malaria, and HIV6,7. Bladder cancer, another urologic pathology, is significantly more prevalent in men than in women, and several studies have shown a role for androgens in the development of malignancy8,9,10,11,12. Notably, however, investigation of intravesical therapies for bladder cancer is performed exclusively in female animals, due to the inability to repeatedly catheterize male mice13.
The study of UTI pathogenesis relies heavily on rodent models of infection (e.g., mice and rats). Murine models of UTI may employ uropathogens originally isolated from human infections, such as uropathogenic E. coli (UPEC), Klebsiella, Enterococcus, Staphylococcus, or Proteus14. Typically, bacteria are introduced into the bladder via catheterization of the urethra. Following infection, bladders and kidneys can be removed to assess specific parameters of infection, such as bacterial colonization, tissue damage, or host immune response15,16. Universally, however, only female animals are used for UTI research. Indeed, many studies have noted that, due to anatomical reasons, catheterization of male mice is not possible13,17,18,19. More recently, a surgical approach to male instillation has been described, in which the abdomen is opened, the bladder is externally displaced by applying gentle pressure to the opening in the abdomen, and bacteria are injected into the bladder20. This approach enables male infection, at the cost of surgical intervention. Thus, a major caveat of this approach includes the influence of inflammation, on the outcome of infection such as the potential to induce an anti-inflammatory wound-healing response to the incision21. As our interests include understanding sex bias in response to disease, we developed a method of bacterial intravesical instillation in male mice that more closely matches the long-established non-surgical transurethral approach used in female rodents22.
Our model builds upon an established methodology and provides the ability to directly compare the host immune response to UTI in female and male animals. This method will permit dissection of sex-based differences in infection, and potentially offer molecular and cellular clues to the pronounced differences in susceptibility and response to infection between the sexes. Additionally, this model has value beyond UTI studies, allowing the establishment of models to investigate other bladder-associated diseases, such as bladder cancer, prostatitis, under- or over-active bladder syndrome, and interstitial cystitis.

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	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:283px;">BD Insyte Autoguard Shielded IV Catheters 24G, 0.7 mm external diameter, 14 mM long</td>
			<td style="width:131px;">BD Medical</td>
			<td style="width:135px;">381811 or 381411*</td>
			<td style="width:260px;">* catalog number is country-dependent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">inoculating loop</td>
			<td style="width:131px;">Greiner Bio-One</td>
			<td style="width:135px;">731171</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller broth</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244620</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller agar</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244520</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cuvettes</td>
			<td style="width:131px;">Bio-rad</td>
			<td style="width:135px;">223-9955</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">syringes</td>
			<td style="width:131px;">B Braun</td>
			<td style="width:135px;">9166017V</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Thumb (Adson) forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11006-12</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes, polypropylene</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352063</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tissue Ruptor disposable probes</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">990890</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">15 mL flip cap tubes</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:135px;">362694</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">DNAse</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">18047019</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Liberase TM</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">5401119001</td>
			<td style="width:260px;">should be aliquotted and stored at -20 &#176;C to avoid repeated freeze-thaw</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;M MACS SmartStrainers&#160;</td>
			<td style="width:131px;">Miltenyi</td>
			<td style="width:135px;">130-098-463</td>
			<td style="width:260px;">compatible with 15 mL tubes, preferred</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;m cell strainers&#160;</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352360</td>
			<td style="width:260px;">compatible with 50 mL tubes, if MACS Smart Strainers are not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">96 well plate</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">353077</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fc block</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">553142</td>
			<td style="width:260px;">anti-CD16/CD32</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">anti-mouse CD45 antibody</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">561487</td>
			<td style="width:260px;">many different fluorescent conjugation options are available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes polystyrene with filter cap</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352235</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">insulin syringe</td>
			<td style="width:131px;">Terumo</td>
			<td style="width:135px;">BS05M2913</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">hand held homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">9001272</td>
			<td style="width:260px;">TissueRuptor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">flow cytometer</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Fortessa SORP&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Flow cytometry software</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Diva</td>
		</tr>
	</tbody>
</table>

urinary tract infection in a small animal model: transurethral catheterization of male and female mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

In the development of this protocol, cohorts of female and male C57Bl/6 mice aged 6 to 8 weeks were instilled, and bacterial burden evaluated in bladders at time points ranging from 1 hr to 30 days. The results from these studies are detailed elsewhere (Zychlinsky Scharff et al., pending). Here, we present representative data from 24 hr infections. Notably, bacterial burden was equivalent between male and female mice at 24 hr post-infection (Figure 1). Little var...

Watch this Scientific Journal Video about Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice at JoVE.com

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

The overall goal of this experimental approach is to investigate the immune response to urinary tract infection, and to prevent direct comparison between male and female animals. This method can help answer key questions of mucosal immunology related to how males and females respond to urinary tract infection. The main advantage is that infection is established via the natural root.We first had the idea for this method after noting how often it is stated, that the installation of the bladder of the male mice is impossible. Though this method will provide insight into urinary tract infection, it can also be applied to other disease studies, such as bladder cancer. To begin, prepare one pediatric intravenous access canula for each group of mice to be infected.Using the inbuilt spring mechanism, divest each canula of its needle as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous canula. Sterilize the catheters in a laminar flow hood for one UV cycle of approximately 25 to 30 minutes.After going in overnight culture of uropathogenic e coli, or UPEC, according to the text protocol, spin the bacterial suspension in a table-top microcentrifuge at 17, 000 times g for one minute, and resuspend the resulting bacterial pellet at two times 10 to the eighth CFU per milliliter in PBS. Serially dilute an aliquot of suspension, and plate on LB agar with antibiotics if appropriate, to determine the exact inoculum for each infection. Draw the bacterial inoculum into a one milliliter syringe, and attach the catheter to the end of the syringe.Tap the syringe to remove any air, and depress the plunger to fill the dead air space in the catheter before beginning the instillation. After anesthetizing a female mouse according to an approved protocol, place the animal supine, and apply medium pressure to the lower abdomen to empty the bladder of urine. Full bladders feel like a pea under the skin, between the iliac crests.When the bladder is empty, using the non-dominant hand, place the thumb on the tail and a finger of the same hand on the abdomen of the mouse, and apply gentle pressure in opposing directions to hold the mouse firmly in place. Next, place the tip of the catheter perpendicular to the mouse at the urethral orifice, then, with gentle pressure, slide the catheter into the urethra until the hub meets the urethral orifice, while simultaneously lowering the syringe so that it is parallel to the working surface. Once the catheter is in place, use a finger from the non-dominant hand that's resting on the mouse abdomen to very gently pull the abdominal skin towards the head of the mouse.Note that the urethral orifice will not move. However, if the catheters is in the vagina, the tissue will move up and away from the catheter. With the hub of the catheter against the urethral orifice, slowly dispense 15 microliters of the bacterial inoculum.A slow installation rate minimizes vesicoureteral reflux into the kidney. Slowly remove the catheter to prevent leakage. Then place the animal in its cage in a supine position.To inoculate male mice, place two thumb forceps cranially and caudally to the mouses external genitalia. Retract the prepuce to fully expose the gland's penis. Then, once the penis is externally positioned, release the thumb forceps.Reposition the forceps to stabilize the protruding penis perpendicular to the animal, holding the organ gently but tautly. Then through the small opening in the tip of the urethral meatus, carefully introduce the catheter and gently guide it into the penis using forceps to gently maintain tension. Once the hub of the catheters meets the tip of the penis, very slowly dispense 50 microliters of the inoculum, while maintaining the position of the penis.Note the quality of the instillation according to a predetermined instillation quality score, such as the one shown here. Retract the catheter slowly over five seconds to prevent leakage of the inoculum. Place the mouse in its cage in a supine position.The animal should begin to recover 30 to 45 minutes following administration of the anesthetic. After sacrificing mice according to an approved protocol, use 70%Epinal to moisten the abdomen thoroughly to minimize contamination by the fur. Using scissors, make a two centimeter incision across the lower third of the mouses abdomen, and aseptically remove the bladder and any other desired organs.Transfer the organs into five milliliter polypropylene snap-cap tubes, containing one milliliter of sterile PBS and keep all samples on ice. With a hand-held homogenizer, homogenize the organs until almost no solid tissue remains. Discard any shiny, beige-white fat tissue that does not homogenize well.Then, use Epinal, followed by PBS to wash the homogenizer between each experimental or organ group. After preparing serial dilutions of homogenized organ suspension, plate dilutions on LB agar plates, with antibiotics when appropriate, and incubate at 37 degrees Celsius overnight. To carry out flow cytometry on bladder tissue, after isolating the bladder as demonstrated earlier in this video, use scissors to mince the tissue well.Add one minced bladder per tube containing digestion buffer. Then shake the tube to wash the minced tissue in the digestion buffer, and keep it on ice until all the bladders are dissected and minced. Incubate the minced bladders at 37 degrees Celsius, for 60 to 75 minutes with vigorous shaking by hand for five seconds, every 15 minutes.When the tissue has a glassy transparent appearance resembling wet tissue paper, digestion is complete. Inactivate the digestion enzymes by adding two to three milliliters of ice-cold flow cytometry buffer, and gently mix. Then place the tubes on ice.To ensure a single-cell suspension, and to remove any connective tissue, pass the contents of the tube through a 100 micrometer filter placed in a 15 milliliter conical tube. Then, with the end of a syringe plunger, gently press any remaining tissue through the filter. Wash the filter with an additional two milliliters of flow cytometry buffer, and keep the samples on ice.The wash the samples by centrifugation at 200 times g and four degrees Celsius, for seven minutes. Resuspend the pellets in 100 microliters of flow cytometry buffer containing Fc block, diluted to five micrograms per milliliter, and transfer to a 96 Well round bottom plate. After 10 to 15 minutes add 100 microliters of the desired antibody cocktail to each sample.Then incubate the samples on ice protected from light for 30 to 45 minutes. Wash the samples by centrifugation at 200 times g and four degrees Celsius for seven minutes. Finally, resuspend the cell pellets in 200 microliters of flow cytometry buffer, and pass the samples through a 40 micrometers cell strainer on a five milliliter polystyrene tube, just prior to acquisition on a flow cytometer.This figure shows representative data from mice infected for 24 hours. Bacterial burden was equivalent between male and female mice at 24 hours post infection. To determine whether loss of inoculum at the time of infection impacted the establishment of infection each instillation was scored on a scale of one to five, with one being the most optimal.Bacterial colonization was determined 24 hours post infection. No statistically significant differences were found among the CFU's obtained from animals with different instillation scores as assessed by a nonparametric Kruskal-Wallis test, comparing the mean rank of each column with the mean rank of every other column, and correcting for multiple comparisons using a Dunn's test. It can be concluded that suboptimal instillations, resulting in substantial leakage of bacterial inoculum during infection, do not significantly impact bacterial colonization at 24 hours.Importantly, the frequency of suboptimal instillation will decrease with practice. Finally, while the number of CD45 positive immune cells, present in naive animals was not different between female and male mice, there was a statistically-significant increase in infiltration into the bladders of infected female mice. While attempting catheterization, it is important to use slow, gentle movements.With its development, this technique has helped us to explore sex-based differences in immunity in the bladder of male and female animals.

urinary-tract-infection-small-animal-model-transurethral

Research

JoVE Journal

Immunology and Infection

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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

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

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

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

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 ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...