This work was supported by R01HL124155 (CPH) and funding from the Research Institute at the University of Missouri to CPH.

The protocol for handling of mice and ultrasound imaging was approved by the University of Missouri Institutional Animal Care and Use Committee (animal protocol number 8799) and was conducted according to AAALAC International.
1. Equipment setup and preparation of mice
<ol>
	<li>Equipment setup
	<ol>
		<li>Turn on the ultrasound instrument, ultrasonic gel warmer and the heating pad.</li>
		<li>Open the ultrasound program and enter the study name and descriptive information for each mouse.</li>
		<li>Select the application as General Imaging.</li>
		<li>Choose the appropriate transducer for abdominal imaging (Figure 1B,C). In this experiment, MS400 transducer is used.</li>
		<li>Ensure anesthesia isoflurane and oxygen levels are adequate for each experimental session.</li>
		<li>Clean the ultrasound animal imaging platform.</li>
	</ol>
	</li>
	<li>Mouse preparation
	<ol>
		<li>Place the mouse cage on top of a heating pad (36.5 to 38.5 &#176;C).</li>
		<li>Gently hold the mouse by its tail base and place in the oxygen-filled isoflurane chamber.</li>
		<li>Direct the isoflurane and oxygen flow to the induction chamber.</li>
		<li>Turn on the isoflurane vaporizer and set the isoflurane level to 1-2% vol/vol. Turn on the oxygen tank pressure to 1-2 L/min.</li>
		<li>After ~2 min, confirm the adequate depth of anesthesia by the absence of withdrawal reflexes upon pinching the foot pad of the mouse.</li>
		<li>Next, turn off the induction chamber supply branch and turn on the branch directed to the anesthesia nose cone.</li>
		<li>Transfer the mouse from the induction chamber to the ultrasound imaging stage and position the anesthesia cone over the nose of the animal.</li>
		<li>Tilt the animal imaging platform around 10&#176; to the lower right corner for optimal scanning (Figure 1B).</li>
		<li>Put one drop of sterile ophthalmic solution in both eyes of mice to prevent drying under anesthesia.</li>
		<li>Position the mouse in the supine position with its nose inserted into the anesthesia cone.</li>
		<li>Apply the electrode gel to all four paws using a cotton swab and tape the paws to the copper leads on the animal imaging platform for electrocardiogram readings (Figure 1C).</li>
		<li>Use clippers to shave hair at the imaging site and then apply depilatory cream to remove remaining fur. Leave for less than 1 min.</li>
		<li>Gently wipe off the cream and hair with a damp paper towel.</li>
		<li>Monitor the breathing and ensure that heart rate is maintained between 450-550 beats/min. If below this level, reduce the isoflurane flow and wait until the heart rate recovers.</li>
		<li>Apply prewarmed ultrasonic gel (37 &#176;C) to the prepared skin site and attach the transducer to its holder and lower down until it touches the gel (Figure 1C).</li>
	</ol>
	</li>
</ol>
2. Ultrasound imaging of the abdominal aorta
<ol>
	<li>Position the transducer horizontally (i.e., perpendicular to the midline of the mouse).</li>
	<li>Smooth the ultrasonic gel and remove bubbles using the wood stick of a cotton swab.</li>
	<li>Lower the transducer and place 0.5 - 1 cm below the diaphragm after touching the gel. Now start to observe the images.</li>
	<li>Visualize the abdominal aorta in the short axis view (Figure 1C). 
	NOTE: B-mode is the default and most effective mode to anatomically locate the aorta and position the transducer. The abdominal aorta is identified by the presence of pulsatile flow using color Doppler and power Doppler modes in the short axis (i.e., the circumferential cross-section of the aorta). Adjust the micromanipulators on the animal stage and the transducer to bring the cross section of the aorta to the center of the image.</li>
	<li>Gently rotate the transducer 90&#176; clockwise, and slowly adjust the x-axis micromanipulator knob to visualize the aorta in long axis view (longitudinal section of the aorta). 
	NOTE: In many cases, gastro-intestinal gases may interfere with the image, or the aorta may not be at the optimal angle to allow a clear long axis view. Adjust the angle of the transducer slowly and horizontally until an acceptable long axis view is obtained. If problems persist, elevate the transducer, check for air bubbles under the transducer, slightly adjust the tilting angle of the animal stage, reapply gels, and repeat all the steps again.</li>
	<li>Set the focus zone and depth at the region of the aorta using the Focus Zone and Focus Depth toggles, respectively. Adjust the time gain compensation slider manually to darken the lumen of the aorta to achieve an optimal contrast of the aorta wall.</li>
	<li>Adjust the y-axis manipulator to visualize the branching points of the superior mesenteric and the right renal arteries. Use the right renal artery as a landmark to capture image of the suprarenal aorta (Figure 2A).</li>
	<li>Record at least 100 frames of B-mode images on the suprarenal aorta.</li>
	<li>Press cinestore to save the B-mode images.</li>
	<li>Press M-mode button on the instrument keyboard to enable M-mode recording. Roll the cursor ball to bring the yellow indicator line to a normal aorta sections with clear vessel wall image, or to the sections where maximal diameter of aneurysm is observed.</li>
	<li>Press the SV/gate toggle and adjust the cursor ball to ensure that vessel walls are included in the measurement bracket. Press update to record M-mode measurements and press cinestore to capture (Figure 2A,B). 
	NOTE: Maximal diameter of the aneurysm may not be in the same imaging plane as the optimal long axis view of the aorta. Adjust the x-axis manipulator knob slightly for each M-mode measurement to ensure that the MILD of each section is captured.</li>
	<li>To obtain ECG-gated Kilohertz Visualization (EKV) images, press the B-mode button to go back to B-mode recording. 
	NOTE: If the images are not sharp, adjust the x-axis manipulator to achieve the sharpest image of upper wall of the lumen over a section length (i.e., &#62; 6 mm).</li>
	<li>Press Physio Settings button on the keyboard and select Respiration Gating. Adjust the gating Delay and Window manually to record the data only during the flatter parts of the respiration wave. The recording sections will be shown as colored blocks on the tracing of the respiration wave. 
	NOTE: Without the adjustment of the respiration gating, the EKV images will be blurred due to the normal movement of animal during breathing.</li>
	<li>Press EKV button to enable the EKV mode. In the appropriate menu, select Standard Resolution and frame rate 3000 or higher. Select proceed to record EKV images. Press cinestore to save the images. Use EKV mode image to obtain measurements of pulse propagation velocity (PPV), distensibility and radial strain. 
	NOTE: EKV recording may fail if there are abnormal fluctuations in respiration, animal is respiring too rapidly, or frame rates settings are too high. In those case, set the frame rate lower and wait for the animal respiration to stabilize. Setting the frame rate at 3000 is usually appropriate for both mice and rats.</li>
</ol>
3. Post-imaging steps
<ol>
	<li>Gently wipe the ultrasonic gel from the abdominal area of the mouse with a paper towel moistened with warm water.</li>
	<li>Place the mouse back in its home cage on a heating pad.</li>
	<li>Turn off the isoflurane machine, clean the animal imaging platform and transducer with damp wipes.</li>
	<li>Transfer the image data collected during the ultrasound scan to the hard drive.</li>
	<li>Turn off the ultrasound instrument.</li>
	<li>After the mouse recovers from anesthesia and is alert, remove the heating pad and return the cage to the animal housing rack.</li>
</ol>
4. Analysis of abdominal aortic images
<ol>
	<li>Analysis of M-mode images to measure MILD
	<ol>
		<li>Open the ultrasound program and enter the study name and descriptive information for each mouse.</li>
		<li>Open the ultrasound data in the analysis software and open the M-mode image and pause the heartbeat.</li>
		<li>Click on Measurements.</li>
		<li>Select the vascular package from the drop-down options. Click on Depth and draw a line across the aortic lumen extending from inner wall to wall (Figure 2C,D). 
		NOTE: For consistency, the measurements should be taken at the systolic phase of the cardiac cycle when the aorta is maximally expanded. Draw three lines across three different heartbeats to obtain accurate and average measurements of MILD. In AAA, the measurements are taken at the maximal dilatation of the aorta. It is also advisable to fast the animals 4-6 h prior to collecting images to avoid interference from bowel motility and ensure image clarity.</li>
	</ol>
	</li>
	<li>Analysis for pulse propagation velocity (PPV)
	<ol>
		<li>Open the EKV image and pause the heartbeat.</li>
		<li>Open a new window on the analysis software (e.g., Vevo Vac) by clicking on the name icon.</li>
		<li>Click on the PPV option (arrow in Figure 3D). A small window will further appear with the image of the aorta.</li>
		<li>Draw a rectangular box by clicking on the upper vessel wall and dragging the pointer for about 4 mm covering both the walls of the suprarenal aorta. 
		NOTE: Keep the length of the box consistent (~4 mm) for all the images. The user can adjust the rectangular box by rotating to align the box and selecting the line then dragging to a new position on the vessel being analyzed to obtain the most appropriate and clear inflection of the pulse wave. The vertical lines of data from the rectangle will be displayed and identified as the Left (top image) and Right (bottom image) on the ROI. For a better visualization of the inflection of the pulse wave, it is sometimes useful to the draw box only on the upper wall as shown in Figure 3. The software will automatically calculate the PPV (m/s). However, it&#39;s always better to manually adjust the purple lines to set the exact inflection point on the pulse waves and PPV will change accordingly.</li>
		<li>Finally, select the Accept command to save the PPV values. Export the figures and the data to the data storage drive.</li>
	</ol>
	</li>
	<li>Analysis for distensibility and radial strain
	<ol>
		<li>Open the EKV image and pause the heartbeat.</li>
		<li>Click on the software icon. The software will open a new window.</li>
		<li>Click on the trace new ROI and draw a rectangular box on the both walls of the vessel. The software will automatically trace the upper and lower walls of the vessel. The user can adjust the trace to align on the wall by clicking on green points (Figure 4A,B).</li>
		<li>Now Accept the trace. The software will calculate the distensibility (1/Mpa) in the selected ROI.</li>
		<li>For the radial strain measurement, select the appropriate strain option from the menu bars on the top left. The images for radial strain and tangential strain will open.</li>
		<li>Obtain the value for radial strain (%) by moving the cursor on the peak of the curve. Export the data as images or in video format (Figure 4A,B).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Ultrasound imaging provides a powerful technique for determining functional properties of the aorta through measurements of PPV, distensibility and radial strain. These measurements are particularly instructive for studying mouse models of AAA and the in vivo approach allows for collection of longitudinal data that is potentially important to understanding temporal development of the aortic pathology. Specifically, measurements of in vivo aortic stiffness are determined locally in the abdominal aorta by PPV, distensibili...

An abdominal aortic aneurysm (AAA) represents a significant cardiovascular disease characterized by a permanent localized dilation of the aorta exceeding the original vessel diameter by 1.5 times1. AAA ranks among the top 13 causes of mortality in the United States2. The progression of AAA is attributed to the degeneration of the aortic wall and elastin fragmentation, ultimately leading to aortic rupture. These changes in the aortic wall may occur without a significant increase in the maximal intraluminal diameter (MILD), thus suggesting that MILD alone is not sufficient to predict the severity of the disease3. Therefore, additional factors need to be identified to detect initial changes in the aortic wall, which may guide early treatment options. The overall goal of this protocol is to provide a practical guide for assessing aortic functional properties using ultrasound imaging as characterized by measurements of pulse propagation velocity (PPV), distensibility and radial strain.
A well characterized experimental model to study AAA, first described by Daugherty and colleagues, involves subcutaneous infusion of angiotensin II (AngII) via osmotic pumps in Apoe-/- mice4. Precise measurement of MILD using ultrasound imaging has been instrumental in characterizing AAA in this mouse model5. Although histological changes during the development of AAA have been extensively studied, changes in the functional properties of the vessel wall such as aortic stiffness have not been well characterized. This protocol emphasizes the use of high-frequency ultrasound in combination with the sophisticated analyses as powerful tools for studying the temporal progression of AAA. Specifically, these approaches allow us to assess the functional properties of the vessel wall as measured by PPV, distensibility and radial strain.
Recent clinical studies in human subjects with AAA, as well as in the murine elastase-induced AAA model, suggest a positive correlation between aortic stiffness and aortic diameter6,7. PPV, an indicator of aortic stiffness, is accepted as an excellent measurement for quantifying changes in stiffness in vessel wall6,8. PPV is calculated by measuring the transit time of the pulse waveform at two sites along the vasculature, thus providing a regional assessment of aortic stiffness. We have recently demonstrated that increased aortic stiffness as measured by PPV, and at the cellular level as determined using atomic force microscopy, positively correlates with aneurysm development9. Further, the literature suggests that aortic stiffness may precede aneurysmal dilation and thus may provide useful information about regional intrinsic properties of the vessel wall during development of AAA10. Similarly, distensibility and strain measurements are the quantification tools to measure earlier changes of arterial fitness. Healthy arteries are flexible and elastic, whereas with increased stiffness and less elasticity, distensibility and strain is decreased. Here, we provide a practical guide and step by step protocol for the use of a high-frequency ultrasound system to measure MILD, PPV, distensibility and radial strain in mice. The protocol provides technical approaches that should be used in conjunction with the basic information provided by manuals for specific ultrasound imaging instruments and the accompanying video tutorial. Importantly, in our hands the described imaging protocol provides reproducible and accurate data that appear valuable in the study of the development and progression of experimental AAA.
To further demonstrate the utility of ultrasound imaging, we provide example images and measurements taken from our own studies aimed at using pharmacological approaches for preventing experimental AAA11. Specifically, notch signaling has been proposed to be involved in multiple aspects of vascular development and inflammation12. Using gene haploinsufficiency and pharmacologic approaches, we have previously demonstrated that Notch inhibition reduces the development of AAA in mice by preventing infiltration of macrophages at the site of vascular injury13,14,15. For the current article, using the pharmacological approach for Notch inhibition we focus on the relationship between aortic stiffness and factors relating to AAA. These studies illustrate that Notch inhibition reduces aortic stiffness, which is a measure of AAA progression11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma</td>
			<td>A9525</td>
			<td></td>
		</tr>
		<tr>
			<td>Apoe-/- mice</td>
			<td>The Jackon lab</td>
			<td><img src="/files/ftp_upload/60515/60515mateq.jpg"/></td>
			<td></td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>WAHL</td>
			<td>1854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Q-tips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td>LL9038</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode cream</td>
			<td>Sigma</td>
			<td>17-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel warmer</td>
			<td>Thermasonic (Parker)</td>
			<td>82-03 (LED)</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stryker</td>
			<td>T/pump professional</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>Fluriso TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Visualsonics</td>
			<td>VS4244</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricating ophthalmic ointment</td>
			<td>Lacri-lube</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic pumps</td>
			<td>Alzet</td>
			<td>Model 2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen tank</td>
			<td>Air gas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tranducer</td>
			<td>Visualsonics</td>
			<td>MS-400 or MS550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic gel</td>
			<td>Parker</td>
			<td>Aquasonic clear</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound Imaging System</td>
			<td>Visualsonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Vasc Software</td>
			<td>Visualsonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of pulse propagation velocity, distensibility and strain in an abdominal aortic aneurysm mouse model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

Representative M-mode images of the normal and aneurysmal abdominal aorta from mice are shown in Figure 2A and Figure 2B, respectively. The suprarenal abdominal aorta is identified by its location next to right renal artery and the superior mesenteric artery (Figure 2A). Representative images used for the calculation of MILD, at three different heartbeats of the systolic cardiac cyc...

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Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

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6.0K Views.  University of Missouri. This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

Video: Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

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

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

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

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

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

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

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

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

Single&#45;Copy Gene Expression for Characterizing Multidrug Efflux Systems

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...