The authors would like to acknowledge Shelly Xie and Jennifer Payne for their expert technical assistance. This work was made possible through grants from the Society of Pediatric Urology and the Stanford Pediatric Research Fund.

I. Preparation of Urethral Catheters
<ol>
	<li>For each mouse bladder and kidney to be sampled, place 5-6 stainless steel beads (bladder, 1.6 mm size or 0.9-2.0 mm blend) or zirconium oxide beads (kidney, 0.5 mm size) in one cryovial tube, respectively. Autoclave the bead-containing tubes.</li>
	<li>After the tubes cool to ambient temperature, add 200 microliters of sterile PBS to each stainless steel bead-containing tube and 400 microliters of sterile PBS to each zirconium oxide bead-containing tube.</li>
	<li>We prepare urethral catheters as described by Hung and Hultgren13. Autoclave a clean pair of flat-head forceps and fine scissors (iris or similar type) and allow the instruments to cool to ambient temperature. Wipe down the surfaces of a laminar flow hood with 70% ethanol. In the hood, cut a 30 cm (approximate) segment of polyethylene tubing. Aseptically place a sterile 30 gauge needle (1/2 inch long needle) on a sterile 3 cc syringe. Pick up one end of the cut polyethylene tubing with the autoclaved forceps and slide the tubing onto the needle until it meets the hub. Cut the tubing so that approximately 2 cm extends beyond the tip of the needle. Remove the catheter from the syringe and place in a sterile petri dish.</li>
	<li>To reduce the likelihood of cross-contamination, we make one catheter for each mouse. After all catheters are made, sterilize them by exposure in an uncovered petri dish to UV irradiation for at least 30 minutes. Do not look directly at UV lamps or expose any part of the body in the hood while lamps are activated. After UV exposure, catheters can be stored long-term in the petri dish. We recommend sealing the dish with Parafilm to help maintain sterility.</li>
</ol>
II. Animal Inoculation
<ol>
	<li>Mice should arrive at least a week before experimental manipulation in order to avoid stress-induced confounding factors. Female mice are used because the male mouse urethra is extremely difficult to catheterize. CBA/J is a commonly utilized, UTI-susceptible inbred mouse strain. Mice should be used between 8 and 12 weeks of age, since this is the window of immunological maturity prior to senescence. On the first day of the experiment, dip the end of a sterile inoculating loop into a vial of frozen bacterial stock. Scrape up a small inoculum and streak out on an appropriate agar plate. 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 solid, yellowish-white translucent colonies on LB agar. Immediately return the bacterial stock to the freezer. Uropathogenic bacterial strains can become less virulent over time with repeated, serial cultures. Hence, we recommend streaking out a fresh agar plate for each experiment.</li>
	<li>On day 2 of the experiment, after confirming proper colony morphology, pick single colonies from agar plates and place in at least 5 cc of broth culture overnight. LB broth can be used for most uropathogenic bacterial strains (37&deg;C, 200 rpm in ambient air). Keep the caps loose on the broth culture tubes to allow aeration.</li>
	<li>On day 3 of the experiment, take 10% of the first broth culture and culture in broth again overnight (typically 37&deg;C and 200 rpm), to enhance type I pili expression. Serial broth cultures help to optimize bacterial uropathogenicity, since type I pili have been shown to be a critical virulence factor for uropathogens in vivo1. Keep the caps loose on the broth culture tubes to allow aeration.</li>
	<li>On day 4 of the experiment, measure the OD600 of the broth culture from day 3. If not already known for the bacterial strain being tested, perform ten-fold serial dilution plating over at least 7 logs (dilutions performed with PBS, cultures performed on LB agar, 37&deg;C incubation overnight) to determine the relationship between OD600 and cfu/ml. As a rule of thumb, an OD600 of 0.4-0.5 commonly corresponds to 1-2x107 cfu per 50 microliters. Once the relationship between OD600 and cfu/ml has been determined, one can adjust the broth culture to the OD600 corresponding to the desired cfu per mouse in 50 microliters of PBS. Some work has suggested that inoculation of 10 microliters, rather than 50, leads to less reflux into the kidneys6. However, most publications have reported use of 50 microliters per mouse. Generally, the desired cfu ranges between 10 6 and 108 cfu/mouse, but each bacterial/mouse strain combination needs to be empirically tested in vivo.</li>
	<li>To help ensure that inocula are not voided out immediately after instillation, mice should be deprived of water for at least 30 minutes prior to induction of general anesthesia. Another key maneuver is to induce voiding prior to anesthesia induction by scruffing mice and gently pressing the lower abdomen. Two to 2.5% isoflurane is a safe induction dose for 8-12 week old female CBA mice, followed by maintenance anesthesia with 1.8-2% isoflurane.</li>
	<li>Once anesthesia is achieved, mice are placed in the supine position with their head inserted in a nosecone connected to isoflurane-containing oxygen. The lower abdomen is massaged to evacuate urine from the bladder. If the bladder is relatively empty, this maneuver may not yield urine. The lower abdomen and perineum is soaked with 70% ethanol.</li>
	<li>A sterile 1 cc syringe without a needle is used to draw up the desired bacterial inoculum. Next, a sterile urethral catheter is aseptically placed on the syringe. The excess tubing is cut off the catheter with a pair of scissors so that only 1-2 millimeters of tubing remain distal to the tip of the needle. Then, the syringe is tapped and the plunger depressed with the syringe upright to remove all bubbles. A dollop of bacteriostatic lubricating jelly is squirted onto a sterile petri dish or the inside of sterile syringe wrapper. The tip of the catheter (with attached syringe) is dipped in the jelly. If multiple bacterial strains are being tested in the same experiment, be careful not to use the same drop of jelly to lubricate catheters containing different strains.</li>
	<li>Using a stereo microscope (0.8x to 5x zoom range) for magnification, the clitoral hood of the anesthetized mouse is gently grasped in the non-dominant hand with a pair of fine-toothed forceps and raised cephalad to expose the deep pink urethral meatus (Figure 1). The syringe (with attached catheter) is grasped in the dominant hand with a pencil grip and the tip of the catheter is engaged in the urethral meatus at a 45 degree angle. The clitoral hood is stretched along the long axis of the syringe, effectively "loading" the urethra onto the catheter. This maneuver is essential since the mouse urethra is redundant. The catheter should slide easily into the bladder (up to the hub, Figure 2), and can be gently twirled to help navigate the redundant folds of the urethra. Once the catheter is hubbed, the syringe can be lowered until it is parallel with the long axis of the mouse. The non-dominant hand can drop the forceps to help stabilize the position of the catheter and syringe as the dominant hand is used to slowly inject the inoculum (at least 5 seconds). If fluid is seen flowing around the catheter during injection, either the bladder is full or the catheter is in the vagina (just caudal to the urethra). Injection should be immediately stopped and the position of the catheter carefully examined. If the catheter was in the urethra, the given mouse should not be included in the experiment. On the other hand, a catheter misplaced in the vagina can be repositioned and the injection re-attempted. After injection, the catheter and syringe is slowly withdrawn from the mouse.</li>
	<li>To reduce the likelihood of early post-inoculation voiding, which may evacuate administered bacteria and thereby lead to variable infection levels, some investigators maintain mice under anesthesia for 30 minutes after transurethral inoculation10. After surgery, mice should recover on a clean warming blanket with continuous observation to confirm return of normal breathing patterns and activity. Once animals have fully recovered the ability to crawl, they may be returned to bedding-containing cages.</li>
</ol>
III. Measuring CFU in Infected Tissue
<ol>
	<li>Various mouse UTI publications have reported on culture results from 6 hours to one week or longer post-inoculation. The optimal time points need to be determined empirically for each bacterial/mouse strain combination. Voided urine is obtained for culture by scruffing mouse over a sterile Petri dish. Collected urine should be kept on ice at all times. Multiple attempts to obtain voided urine over many hours may be necessary, since mouse voiding is frequent, low volume (30-100 microliters per void), and stress-dependent.</li>
	<li>After obtaining voided urine specimens, we sacrifice mice using isoflurane and cervical dislocation. The abdomen is aseptically opened. If a voided urine specimen was not successfully obtained earlier, a small amount of urine can often be aspirated through the bladder wall using a 30 gauge needle and syringe.</li>
	<li>One or both kidneys are removed, minced into at least 4 pieces (each piece should be less than 50 micrograms) on a sterile petri dish, and placed into zirconium bead/PBS-containing cryovials on ice. The bladder is removed, minced into at least 4 pieces, and placed into stainless steel bead/PBS-containing cryovials on ice. The tissue/bead/PBS-containing cryovials are placed in a Bullet Blender homogenizer and the tissues are homogenized using the "8" speed setting for one minute. It is common for some tissue fragments to remain after homogenization. As long as 50-100 microliters of liquid homogenate can be pipetted, solid fragments are not necessarily a problem. If homogenization time is increased for some samples, it is critical to do so for all tissues in order to maintain consistency of homogenization. We utilize a Bullet Blender homogenizer because it avoids the possibility of cross-contamination, is more rapid than processing individual samples one by one using a standard homogenizer, and does not significantly heat the samples being homogenized.</li>
	<li>At least two or three ten-fold dilutions of collected urine and tissue homogenates should be made in a microtiter plate. For preliminary experiments it may be prudent to make at least seven ten-fold dilutions. Culture up to 100 microliters of urine, bladder, and/or kidney homogenates on appropriate agar. Since urine specimens may be volume-limited, it may be necessary to bring their volumes to 100 microliters prior to performing ten-fold dilutions. If this is required, the initial dilution (before ten-fold dilution) will need to be factored into final calculations of bacterial yields. We prefer culturing UPEC on eosin-methylene blue or MacConkey agar (overnight incubation at 37&deg;C), since these media are selective for Gram negative organisms. UPEC strains grow on MacConkey agar as centrally umbilicated, red-pink colonies due to their ability to ferment lactose.</li>
	<li>After overnight culture, count the cfu for each plate. For serially diluted samples, the plate dilution containing 50-100 colonies is considered the most accurate count. "Back" calculate cfu per tissue, per milligram of tissue, or per ml.</li>
</ol>
IV. Representative Results

<img src="/files/ftp_upload/2070/2070fig1.jpg" alt="Figure 1" /> 
Figure 1. Urethral meatus (dark pink tissue) exposed by lifting clitoral hood cephalad with forceps (beyond top of photo)

<img src="/files/ftp_upload/2070/2070fig2.jpg" alt="Figure 2" /> 
Figure 2. Catheterized urethra. The bifid clitoris can be seen just above the catheter.
<img src="/files/ftp_upload/2070/2070fig3.jpg" alt="Figure 3" /> 
Figure 3. Bladder cfu counts 2 days after transurethral infection of 8 week old female CBA/J mice with uropathogenic E. coli. Bladders were homogenized and cfu/ml determined by serial dilution plating on MacConkey agar. Diamonds indicate cfu/ml for individual mice, horizontal bar indicates median cfu/ml.

<ol>
	<li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect Immun. 55, 373-380 (1987).</li><li>Rouschop, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+CD44+facilitates+Escherichia+coli+infection+of+the+murine+urinary+tract.">Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract.</a> J Immunol. 177, 7225-7232 (2006).</li><li>Malaviya, R., Ikeda, T., Abraham, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mast+cells+to+bacterial+clearance+and+their+proliferation+during+experimental+cystitis+induced+by+type+1+fimbriated+E.+coli.">Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.</a> Immunol LettM. 91, 103-111 (2004).</li><li>Lane, M. C., Alteri, C. J., Smith, S. N., Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+flagella+is+coincident+with+uropathogenic+Escherichia+coli+ascension+to+the+upper+urinary+tract.">Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract.</a> Proc Natl Acad Sci U S A. 104, 16669-16674 (2007).</li><li>Hvidberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+long-term+ascending+urinary+tract+infection+mouse+model+for+antibiotic+treatment+studies.">Development of a long-term ascending urinary tract infection mouse model for antibiotic treatment studies.</a> Antimicrob Agents Chemother. 44, 156-163 (2000).</li><li>Hopkins, W. J., Hall, J. A., Conway, B. P., Uehling, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+urinary+tract+infection+by+intraurethral+inoculation+with+Escherichia+coli:+refining+the+murine+model.">Induction of urinary tract infection by intraurethral inoculation with Escherichia coli: refining the murine model.</a> J Infect Dis. 171, 462-465 (1995).</li><li>Hopkins, W. J., Gendron-Fitzpatrick, A., Balish, E., Uehling, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+host+responses+to+Escherichia+coli+urinary+tract+infection+in+genetically+distinct+mouse+strains.">Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains.</a> Infect Immun. 66, 2798-2802 (1998).</li><li>Gunther, N. W. t., Lockatell, V., Johnson, D. E., Mobley, H. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+responses+accelerate+bacterial+clearance+in+the+bladder.">Antigen-specific responses accelerate bacterial clearance in the bladder.</a> J Immunol. 176, 3080-3086 (2006).</li><li>Hagberg, L. <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, 273-283 (1983).</li><li>Hopkins, W. J., Hall, J. A., Conway, B. P., Uehling, D. 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No conflicts of interest declared.

Induction of mouse urinary tract infection by transurethral inoculation of bacteria is a long-established but technically demanding procedure11. Hung and Hultgren recently published an excellent review of transurethral techniques of mouse urinary tract infection13. In this paper, we attempt to further illustrate the finer points of this experimental approach.

Novices may practice their technique by performing a midline laparotomy to expose the bladder, injecting a dilu...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cryovial tubes, 2.0 ml polypropylene (no caps)</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>640201</td>
<td>&nbsp;</td>
<tr>
<td>Caps for cryovial tubes, with 'O' ring</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>440011-N</td>
<td>&nbsp;</td>
<tr>
<td>Bullet Blender</td>
<td>&nbsp;</td>
<td> Next Advance</td>
<td>BBX24</td>
<td>&nbsp;</td>
<tr>
<td>Zirconium Oxide Beads (0.5mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>ZROB05</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Beads (1.6mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB16</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Bead Blend (0.9-2.0mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB14B</td>
<td>&nbsp;</td>
<tr>
<td>CBA/J Mice, 8-12 week old female</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
<td>000656</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4)</td>
<td>&nbsp;</td>
<tr>
<td>LB agar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>1.10285.0500</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>NDC 10019-773-60</td>
<td>&nbsp;</td>
<tr>
<td>Inoculating loops</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-363-601</td>
<td>&nbsp;</td>
<tr>
<td>Eosin-methylene blue (EMB) agar powder</td>
<td>&nbsp;</td>
<td>Himedia Laboratories</td>
<td>MM022-500G</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>212123</td>
<td>&nbsp;</td>
<tr>
<td>LB agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244520</td>
<td>&nbsp;</td>
<tr>
<td>Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)]</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>427401</td>
<td>&nbsp;</td>
<tr>
<td>30 gauge needles, 1/2 inch</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>1 ml tuberculin Slip-Tip syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Luer-Lock syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Transurethral Induction of Mouse Urinary Tract Infection

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 ...

transurethral-induction-of-mouse-urinary-tract-infection

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Immunology and Infection

36.5K Views.  Stanford University. Questo video dimostra metodi per indurre transurethrally infezioni del tratto urinario del mouse e quantificare l'entità delle infezioni che ne derivano.

Video: Transurethral Induction of Mouse Urinary Tract Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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.

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, ...

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 ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

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 ...

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 ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...