eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 by the manufacturer. Discard the needles, preserving only the plastic intravenous cannula.</li>
	<li>Sterilize catheters in a laminar flow hood for one ultraviolet (U.V.) cycle, typically 25 - 30 min. 
	Note: Catheters can be used for more than one mouse, however, a new catheter should be used between experimental groups, sexes, or bacterial strains.</li>
</ol>
2. Preparation of Uropathogenic&#160;Escherichia coli for Infection
<ol>
	<li>At least two days prior to infection, using a sterile inoculation loop, streak uropathogenic Escherichia coli (UPEC) from a frozen bacterial stock onto a Luria Broth (LB) agar plate, containing antibiotics if appropriate. Incubate at 37 &#176;C overnight. Plates can be stored at 4 &#176;C for up to one week.</li>
	<li>In a 100 ml sterile Erlenmeyer flask, inoculate 10 ml LB, containing antibiotics if appropriate, with a single colony from the LB plate and incubate standing at 37 &#176;C overnight (approximately 16 - 18 hr). 
	Note: Standing cultures permit the expression of type 1 pili, which are necessary for infection. Cultures grown shaking will have less efficient pili expression, and therefore, inconsistent rates of infection.</li>
	<li>Measure the optical density (OD)600&#160;of the overnight culture and calculate the number of bacteria per ml using a pre-determined growth curve. As an example, for UPEC strain UTI89, OD600&#160;= 0.35 is equivalent to 2 x 108&#160;colony forming unit (CFU) per ml, thus, an overnight culture with an OD600&#160;= 2.4 would be equivalent to 13.7 x 108&#160;CFU per ml. Empirically determine the relationship between the OD600&#160;and viable bacteria with every new strain employed for infection.</li>
	<li>Determine the number of bacteria needed by considering the number of animals to be infected and that each mouse should receive approximately 107&#160;colony-forming units (CFU) in 50 &#956;l phosphate-buffered saline (PBS). Include in the calculation the ~130 &#181;l dead volume of the catheter and syringe nub and 100 &#181;l needed to determine the inoculum.
	<ol>
		<li>Spin the bacterial suspension in a tabletop microcentrifuge at 17,000 x g for 1 min and resuspend the resulting bacterial pellet at 2 x 108&#160;CFU per ml in PBS. Serially dilute an aliquot of this suspension and plate it on LB agar, with antibiotics if appropriate, to determine the exact inoculum for each infection.</li>
	</ol>
	</li>
	<li>Draw the bacterial inoculum into a 1 ml 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.</li>
</ol>
3. Preparation of Mice
<ol>
	<li>Anesthetize mice&#160;via&#160;intraperitoneal injection of 100 mg/kg ketamine and 5 mg/kg xylazine. Supplemental heat can be provided.&#160;</li>
	<li>Ensure that each mouse is fully sedated by squeezing the footpad with medium pressure. Mice are fully sedated when they do not react and require 3 - 5 min to reach this state.</li>
	<li>Place mice 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. 
	Note: Inhaled isoflurane has been used to catheterize female mice, however, it has not been tested whether male mice are sufficiently anesthetized by inhaled isoflurane for this procedure.</li>
</ol>
4. Transurethral Instillation of Female Mice
<ol>
	<li>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.</li>
	<li>Place the tip of the catheter perpendicular to the mouse at the urethral orifice. 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. The catheter requires no lubrication. Do not push or force the catheter into the urethra. The catheter should slide smoothly and easily into the urethra, with little to no resistance.
	<ol>
		<li>Before instilling the inoculum (step 4.3), once the catheter is in place, very gently pull the abdominal skin towards the head of the mouse with the finger from the non-dominant hand that is already on the abdomen of the mouse (step 4.1). If the catheter is in the urethra, the tissue composing the urethra orifice will not move, whereas if the catheter is incorrectly placed in the vagina, the tissue will move up and away from the catheter. This rapid test should be performed on each mouse.</li>
	</ol>
	</li>
	<li>When the hub of the catheter meets the urethral orifice, slowly dispense 50 &#181;l of the bacterial inoculum. A slow instillation rate minimizes vesicoureteral reflux into the kidney. Slowly remove the catheter to prevent leakage, over a count of 5. Place mice in their cages in a supine position.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Insyte Autoguard Shielded IV Catheters 24G, 0.7 mm external diameter, 14 mM long</td>
			<td>BD Medical</td>
			<td>381811 or 381411*</td>
			<td>* catalog number is country-dependent</td>
		</tr>
		<tr>
			<td>Inoculating loop</td>
			<td>Greiner Bio-One</td>
			<td>731171</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Miller broth</td>
			<td>Difco</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Miller agar</td>
			<td>Difco</td>
			<td>244520</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Bio-rad</td>
			<td>223-9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>B Braun</td>
			<td>9166017V</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Terumo</td>
			<td>BS05M2913</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a urinary tract infection model in female mice

Source: Zychlinsky Scharff, A. et al., <a href="https://app.jove.com/v/54432">Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

Establishment of a Urinary Tract Infection Model in Female Mice

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

Begin with an anesthetized female mouse in a supine position and empty its bladder.
Take a pre-assembled syringe with an attached catheter containing a suspension of uropathogenic&#160; Escherichia coli bacteria.
Insert the catheter through the urethral orifice into the bladder and slowly deliver the bacterial suspension.
Post-injection, recover the mouse in a supine position.
The fimbriae of bacteria bind to specific receptors on bladder epithelial cells, triggering their internalization within the underlying epithelial cells.
Inside bladder epithelial cells, bacteria multiply, with some adopting a filamentous morphology, and form dense intracellular bacterial communities or IBCs.
These IBCs exhibit biofilm-like properties, protecting the bacteria from the mouse&#39;s immune defense system and contributing to persistent infection.
Over time, IBCs rupture, releasing bacteria into the bladder lumen; the bacteria possessing the filamentous morphology adhere to neighboring epithelial cells.
This leads to the spreading of infection, establishing a urinary tract infection in the female mouse.

establishment-of-a-urinary-tract-infection-model-in-female-mice

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

173 vistas. Fuente: Zychlinsky Scharff, A. et al., Infección del tracto urinario en un modelo de animal pequeño: cateterismo transuretral de ratones machos y hembras.J. Vis. Exp. (2017) Este video demuestra el establecimiento de una infección uropatógena por Escherichia coli en una vejiga de ratón hembra a través de la instilación de catéter transuretral. Las bacterias inyectadas establecen con éxito una infección del tracto urinario dentro de la vejiga, proporcionando una valiosa plataforma para...

Video: Establecimiento de un modelo de infección del tracto urinario en ratones hembra - Concepto

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establecimiento y caracterización de IU y ITUAC en un modelo de ratón

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Investigación

JoVE Journal

Medicina

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.

Procedimiento transuretral instilación en ratón macho adulto

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

Inmunología e Infección

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Intrarrenal Inyección de<em&gt; Escherichia coli</em&gt; En un modelo de rata de pielonefritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Establishment of a Urinary Tract Infection Model in Female Mice

Transcripción

Establishment of a Urinary Tract Infection Model in Female Mice