Name: Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models (Video) | JoVE Journal
Uploaded: 2015-03-01T14:00:00.000+00:00
Duration: 8 min 32 s
Description: Learn videofluoroscopic swallow study methods adapted for murine disease models to detect and characterize dysphagia. This technique enables quantitative analysis of swallowing function in freely behaving rodents for preclinical research.

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5 minutes

We graciously thank additional members of the Lever Lab who contributed to data collection (Andries Ferreira, Danarae Aleman, Alexis Mok, Kaitlin Flynn, Elizabeth Bearce, and Matan Kadosh) and manuscript review (Andries Ferreira, Rebecca Schneider, and Kate Robbins). We also acknowledge Roderic Schlotzhauer and Edwin Honse from the MU Physics Machine Shop for their design input and fabrication of the rodent observation tubes used in this study. We are especially appreciative of Malea Jan Kunkel (Radiology Supervisor in the Veterinary Medicine and Surgery Department at the University of Missouri - College of Veterinary Medicine) and Jan Ivey (Manager of the Research Animal Cath Lab at the University of Missouri - School of Medicine) for demonstrating constant patience and motivation while operating the high energy fluoroscopes as we developed the murine VFSS protocol. Funding sources for this study included NIH/NIDCD (TE Lever), NIH/NINDS (GK Pavlath), Otolaryngology &#8211; Head and Neck Surgery start-up funds (TE Lever), MU PRIME Fund (TE Lever), Mizzou Advantage (TE Lever), and the MU Center on Aging (TE Lever).

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Open Access for this article is sponsored by Glenbrook.

Dysphagia (swallowing impairment) is a common symptom of numerous medical conditions affecting people of all ages. Examples include stroke, Parkinson&#8217;s disease, Alzheimer&#8217;s disease, cerebral palsy, muscular dystrophy, amyotrophic lateral sclerosis (ALS), Batten disease, head and neck cancer, premature birth, and advanced aging. Dysphagia is highly correlated with mortality, typically as a consequence of severe malnutrition or pneumonia that develops when bacterial-laden food/liquid/saliva is aspirated into the lungs1-4. This debilitating and life-threatening medical condition affects over 15 million people each year in the United States alone3. Despite the high prevalence and associated negative outcomes, current treatment options for dysphagia are limited to palliative (rather than curative) approaches, such as diet modification (e.g., avoiding specific food/liquid consistencies), postural changes (e.g., tucking the chin when swallowing), motor approaches (e.g., exercises targeting muscles in the oral cavity, pharynx, and larynx), sensory approaches (e.g., implementing flavor, temperature, and/or mechanical stimulation), and tube feeding (e.g., nutrition and hydration administered via nasogastric (NG) tube or percutaneous endoscopic gastrostomy (PEG) tube). These treatments merely serve as symptomatic therapy rather than targeting the underlying causes of the problem. Indeed, a major barrier to the discovery of novel, effective treatments for dysphagia is the limited scientific knowledge of the responsible pathological mechanisms, which are likely different for each disease.
Dysphagia diagnosis is predominantly made using a radiographic procedure called a videofluoroscopic swallowing study (VFSS), also known as a modified barium swallow study. Over the past 30-plus years, this diagnostic test has been considered the gold-standard for evaluating swallow function5-7. This test entails having the patient sit or stand within the path of the X-ray beam of a fluoroscopy machine while voluntarily ingesting food and liquid consistencies mixed with an oral contrast agent, typically barium sulfate8,9 or iohexol10. As the patient swallows, food and liquid containing contrast agent can be seen in real time via a computer monitor while traveling from the mouth to the stomach. Soft tissue structures also are visible and can be assessed relative to structure and function. Patients are asked to perform several swallows of each food and liquid consistency, all of which are video recorded for subsequent viewing and frame-by-frame analysis to quantify the presence and degree of dysphagia. Numerous physiological components of swallowing are typically analyzed, such as the anatomical trigger point of the pharyngeal swallow, bolus transit time through the pharynx and esophagus, extent and duration of laryngeal elevation, location and amount of post-swallow residue, and occurrence of and physiological reason for aspiration7,11.
Aspects of the human VFSS protocol were recently adapted for studying freely-behaving rats; however, results were limited because the rats did not remain in the videofluoroscopic field of view during testing12. VFSS has not previously been attempted with mice. Successful adaptation of the human VFSS protocol for use with mice and rats would provide a novel research method to investigate the hundreds of currently existing murine (mouse and rat) models of diseases that are known to cause dysphagia in humans. This new method (henceforth referred to as murine VFSS) would therefore hasten identification and validation of murine models of dysphagia that are suitable for investigating the underlying neurophysiological mechanisms within muscles, nerves, and brain tissue that are pathological and contributing to dysphagia in humans. Moreover, murine VFSS would permit identification of objective measures (biomarkers) of swallow function/dysfunction that could be directly compared with humans. These cross-species videofluoroscopic biomarkers could then serve as novel outcome measures to quantify treatment efficacy in preclinical trials with mice and rats, which would better translate to clinical trials with people.
To this end, the murine VFSS protocol was established using ~100 mice of either sex. All mice were either C57 or hybrid C57/SJL strains. The C57 mice were not genetically altered, whereas C57/SJL was the background strain for a colony of transgenic SOD1-G93A (or SOD1) mice, the most widely used animal model of ALS. The SOD1 colony was an approximate 50-50 mix of transgenic (i.e., ALS-affected) mice and nontransgenic (i.e., unaffected) littermates.
The murine VFSS protocol consists of three components:
<ol>
	<li>Custom-designed observation chambers that permit voluntary feeding and swallowing while standing unrestrained in a confined space within a fluoroscopy machine,</li>
	<li>Recipes that mask the aversive taste/odor of oral contrast agents and produce sufficient radiodensity to permit adequate visualization of swallowing,</li>
	<li>A step-by-step test protocol that maximizes animal compliance, minimizes total test time and radiation exposure, and permits quantification of several swallow parameters for each stage of swallowing (i.e., oral, pharyngeal, and esophageal).</li>
</ol>
The combined effect produces a comfortable, low stress, self-feeding examination environment that permits assessment of typical feeding and swallowing behaviors of mice.

<table><tbody><tr><td>Polycarbonate tubing for observation chambers</td><td>McMaster-Carr</td><td>3161T41</td><td>Body of observation tubes, 2&quot;X2&quot; diameter, 0.080&quot; thick wall</td></tr><tr><td>Polycarbonate sheet&amp;nbsp; for observation chambers</td><td>McMaster-Carr</td><td>9115K71</td><td>End-caps for observation tubes, 2&quot;x12&quot;x3/4&quot;</td></tr><tr><td>Polycarbonate sheet&amp;nbsp; for observation chambers</td><td>McMaster-Carr</td><td>8574K281</td><td>Peg-bowls for observation tubes</td></tr><tr><td>Silicone O-rings&amp;nbsp; for end-caps of observation chambers</td><td>McMaster-Carr</td><td>9396K108</td><td>S1138 AS568-029, pack of 25&lt;br/&gt; http://www.mcmaster.com/#o-rings/=t0wt5r&amp;nbsp;</td></tr><tr><td>Silicone stoppers for observation chambers</td><td>McMaster-Carr</td><td>2903K22</td><td>Package of 10 stoppers to plug the oval opening in the top of the observation chamber when using a peg-bowl&lt;br/&gt; http://www.mcmaster.com/#catalog/120/3803/=t0y5at</td></tr><tr><td>Centrifuge tubes for sipper tube bottles</td><td>Evergreen Scientific</td><td>222-3530-G80</td><td>30 ml freestanding centrifuge tubes, with caps, sterile&lt;br/&gt; https://www.evergreensci.com/labware-catalog/tubes-and-vials/30-and-50-ml-centrifuge-tubes/&amp;nbsp;</td></tr><tr><td>Silcone stoppers for sipper tube bottles</td><td>Saint-Gobain Performance Plastics</td><td>DX263031-10&amp;nbsp;</td><td>Number 31D, size: 26 mm bottom, 32 mm top, 30 mm high; 10 pack;&amp;nbsp;&lt;br/&gt; http://www.labpure.com/en/Products.asp?ID=179&amp;amp;PageBrand=STOPPERS</td></tr><tr><td>Stopper borers for sipper tube bottles</td><td>Thomas Scientific</td><td>3276G40</td><td>Cork Borer Set that ranges from 3/16-15/16 inch&amp;nbsp;&lt;br/&gt; http://www.thomassci.com/Supplies/Corks/_/CORK-BORER-SET-316-1516-IN?q=Humboldt</td></tr><tr><td>Drinking tubes for sipper tube bottles</td><td>Ancare</td><td>TD-100&amp;nbsp;</td><td>2 1/2&amp;rdquo; long drinking tubes with 5/16&amp;rdquo; opening, straight ball-spout&lt;br/&gt; http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point&amp;nbsp;</td></tr><tr><td>Iohexol for making oral contrast agent solution</td><td>GE Healthcare</td><td></td><td>350 mg iodine per ml&lt;br/&gt; http://www3.gehealthcare.com/en/products/categories/contrast_media/omnipaque&amp;nbsp;</td></tr><tr><td>Chocolate syrup for flavoring oral contrast agent</td><td>Herseys</td><td></td><td></td></tr><tr><td>10 ml syringe for syringe delivery system</td><td>Becton, Dickinson and Company</td><td>309604</td><td>Luer lock tip syringe without needle, 100 per box&lt;br/&gt; http://www.bd.com/hypodermic/products/syringeswithoutneedles.asp</td></tr><tr><td>Catheter tubing for syringe delivery system</td><td>Becton, Dickinson and Company</td><td>427451</td><td>Polyethylene Tubing (Non-Sterile) (PE 240) 100&#039;&lt;br/&gt; http://www.bd.com/ds/productCenter/427451.asp&amp;nbsp;</td></tr><tr><td>Needle for syringe delivery system</td><td>Becton, Dickinson and Company</td><td>427560</td><td>15-gauge needle, fits into PE 240 catheter tubing&lt;br/&gt; http://www.bd.com/ds/productCenter/427560.asp&amp;nbsp;</td></tr><tr><td>Delrin acetal resin rod for syringe delivery system</td><td>McMaster-Carr</td><td>8576K15</td><td>1/2 inch diameter, black&lt;br/&gt; http://www.mcmaster.com/#catalog/120/3609/=t0wvaf&amp;nbsp;</td></tr><tr><td>Acrylic sheeting for scissor lift</td><td>Ponoko</td><td></td><td>Laser cut&lt;br/&gt; http://www.ponoko.com&amp;nbsp;</td></tr><tr><td>3D printed ABS frame</td><td>Engineering Rapid Prototyping Facility, University of Missouri</td><td></td><td></td></tr><tr><td>Brass rods for scissor lift</td><td>Amazon</td><td>TTRB-03-12-03</td><td>made into axles&lt;br/&gt; http://www.amazon.com/Brass-Seamless-Round-Tubing-Length/dp/B000FN898M</td></tr><tr><td>Drawer slide for scissor lift</td><td>Richelieu</td><td>10292G116</td><td>Attaches to base of scissor lift&lt;br/&gt; http://www.lowes.com/pd_380986-93052-T35072G16_0__?productId=50041754</td></tr><tr><td>28BYJ-48 stepper motor for scissor lift</td><td></td><td></td><td>2 each</td></tr><tr><td>ULN2003 Darlington transistor array for scissor lift</td><td>Toshiba</td><td>ULN2003APG</td><td>Used as stepper drivers (2 each)</td></tr><tr><td>ATTINY85 microcontroller for scissor lift</td><td>Atmel</td><td>ATTINY85-20PU</td><td>2 each&lt;br/&gt; http://www.taydaelectronics.com/attiny85-attiny85-20pu-8-bit-20mhz-microcontroller-ic.html</td></tr><tr><td>Nylon spur gear</td><td>McMaster-Carr</td><td>57655K34</td><td>2 each&lt;br/&gt; http://www.mcmaster.com/#57655k34/=t0yaqz</td></tr><tr><td>Nylon spur gear rack</td><td>McMaster-Carr</td><td>57655K62</td><td>2 each&lt;br/&gt; http://www.mcmaster.com/#57655k62/=t0ybh9</td></tr><tr><td>4-40 nylon machine screws</td><td>McMaster-Carr</td><td>95133A315</td><td>Lift assembly&lt;br/&gt; http://www.mcmaster.com/#95133a315/=t0yd8q</td></tr><tr><td>4-40 nylon hex nuts</td><td>McMaster-Carr</td><td>94812A200</td><td>Lift assembly&lt;br/&gt; http://www.mcmaster.com/#94812a200/=t0ye29</td></tr><tr><td>Buna-N O-Ring AS568A Dash No. 104</td><td>McMaster-Carr</td><td>9452K318</td><td>Lift assembly&lt;br/&gt; http://www.mcmaster.com/#9452k318/=t0yem7</td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

adapting human videofluoroscopic swallow study methods to detect and characterize dysphagia in murine disease models

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

We have successfully designed a novel and replicable murine-specific VFSS protocol that includes test chambers that permit self-feeding, recipes for flavoring oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. The energy level capability of the fluoroscopy system determined which swallow parameters could be investigated in mice. We initially used high energy fluoroscopes designed for use with people and larger animals (e.g., GE Advantx, GE OEC 9600, and Omega Cardiac Cath CS-25, each at 30 frames per second). However, these systems had insufficient magnification capabilities for testing mice, which resulted in the animal filling only a small portion of the field of view (Figure 9). As a result, image quality was exceptionally poor, rendering it impossible to visualize most structures of the swallowing mechanism. Despite this limitation, we identified 7 objective VFSS swallow parameters that were consistently quantifiable for mice when using a conventional (i.e., high energy) fluoroscope in combination with the new murine VFSS protocol (Table 3). In addition, we identified the vallecular space as the anatomical trigger point for swallowing in healthy adult mice (3-17 months of age), as well as mice with conditions of advanced age (&gt;18 months) and end-stage ALS.
<img alt="Fluoroscopy of rodent swallowing; radiographic images, esophageal examination, medical study." src="/files/ftp_upload/52319/52319fig9highres.jpg" width="650" data-altupdatedat="1787630408000" data-alt="Figure 9"> 
Figure 9: High Energy Fluoroscopy Systems. Left: Representative image of a mouse obtained using high energy (i.e., conventional) fluoroscopy systems. Note that the mouse fills only a small portion of the fluoroscopy field of view, thereby demonstrating the insufficient magnification capability of conventional fluoroscopes for imaging rodents. Right: Same image magnified post-capture using a video editing software program. Black arrow: swallow trigger point (valleculae). White arrow: bolus in the distal esophagus, immediately prior to passing through the GE junction (white asterisk). <a href="https://www.jove.com/files/ftp_upload/52319/52319fig9highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SWALLOW PARAMETERS</td>
			<td style="width:140px">High Energy System</td>
			<td style="width:140px">Low Energy System</td>
		</tr>
		<tr>
			<td>Inter-Swallow Interval (ISI)</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Jaw Excursion Rate (Lick Rate Equivalent)</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Jaw Excursion Distance</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Lick-Swallow Ratio</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Swallow Rate </td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Pharyngeal Transit Time (PTT)</td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Bolus Speed through Pharynx</td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Esophageal Transit Time (ETT)</td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Bolus Speed through Esophagus</td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Bolus Speed through Pharynx and Esophagus</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>Bolus Area </td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Pharyngeal Residue Area</td>
			<td></td>
			<td>X</td>
		</tr>
		<tr>
			<td>Volume of Liquid Consumed</td>
			<td>X</td>
			<td>X</td>
		</tr>
	</tbody>
</table>
Table 3: Swallow Parameters Quantifiable Using High Versus Low Energy Fluoroscopy Systems.
We recently obtained a low energy magnification fluoroscopy system called The LabScope (Glenbrook Technologies, Randolph, NJ) that was specifically designed for our lab for use with mice and other small rodents (Figure 10). However, the markedly greater magnification levels of this system rendered it impossible to view the entire swallowing mechanism of a mouse in a single field of view. Instead, two test positions are required, as shown in Figure 11. Position 1 permits visualization of the entire head and proximal thoracic region. This position is necessary for assessing the oral and pharyngeal stages of swallowing. Position 2 permits visualization from the swallow trigger point (i.e., valleculae) to the gastroesophageal (GE) junction. This position is necessary for assessing the esophageal stage of swallowing. Preliminary work using The LabScope in combination with the new murine VFSS protocol has identified 13 objective swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using high energy (i.e., conventional) fluoroscopes (Table 3). This outcome is attributed to the advanced magnification capabilities of The LabScope, which allows for visualization of numerous anatomical structures (Figure 12) that were essentially invisible when using conventional systems: e.g., hyoid bone, trachea, and cervical vertebrae. As a result, we also were able to analyze the videos for evidence of laryngeal penetration and aspiration. Neither penetration nor aspiration was observed for any mouse in this study, regardless of health or disease conditions.
<img alt="X-ray imaging setup with camera, source on scissor lift for observation." src="/files/ftp_upload/52319/52319fig10highres.jpg" width="650" data-altupdatedat="1787630408000" data-alt="Figure 10"> 
Figure 10: The LabScope. Left: The LabScope performs as a desktop fluoroscope for small animals. Right: Close-up view of The LabScope with labeled components. The scissor lift table is positioning an observation chamber within the fluoroscope field of view. <a href="https://www.jove.com/files/ftp_upload/52319/52319fig10highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="X-ray comparison, animal jaw positions; radiographic analysis, skeletal structure, veterinary study." src="/files/ftp_upload/52319/52319fig11highres.jpg" width="650" data-altupdatedat="1787630408000" data-alt="Figure 11"> 
Figure 11: Low Energy Fluoroscopy System. Images of a mouse obtained using a low energy fluoroscopy system. Note that the high magnification capability prevents visualization of the entire swallow mechanism within the fluoroscopy field of view. Left: Position 1 – permits visualization of the entire head and proximal thoracic region. The swallow trigger point (black arrow) is essentially centered within the field of view. Right: Position 2 – permits visualization from the swallow trigger point (black arrow) to the GE junction (white asterisk). Note the bolus passing through the distal esophagus (white arrow). <a href="https://www.jove.com/files/ftp_upload/52319/52319fig11highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="X-ray diagrams of avian skull anatomy, highlighting mandible and hyoid orientation." src="/files/ftp_upload/52319/52319fig12highres.jpg" width="650" data-altupdatedat="1787630408000" data-alt="Figure 12"> 
Figure 12: Anatomical Structures Visible Using a Low Energy Fluoroscopy System. Even at the lowest magnification setting (left), boney structures of the head and neck of a mouse are readily visible using our low energy fluoroscopy system (i.e., The LabScope). Anatomical structures within the black square are shown (and labeled) at higher magnification to the right. Improved visualization of boney structures permits quantification of several additional swallow parameters that were impossible to analyze using high energy fluoroscopes. <a href="https://www.jove.com/files/ftp_upload/52319/52319fig12highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Swallow rate and inter-swallow interval are representative VFSS parameters that can be quantified using either low or high energy fluoroscopy systems in combination with the new murine VFSS protocol. These two swallow parameters were quantified for three groups of mice: SOD1-G93A (SOD1) transgenic mice (i.e., a model of ALS) at disease end-stage between 4-5 months of age, aged C57 mice (18-24 months of age), and a control group of healthy young (4-8 months of age) C57 mice and nontransgenic littermates from the SOD1 colony. All data pertain to spout drinking only, using either a low or high energy fluoroscopy system. No significant differences were found between young C57 mice and young nontransgenic (control) mice from the SOD1 colony relative to these two swallow parameters; therefore, data were combined into a general “control” group of young healthy mice for comparison with aged C57 mice and end-stage SOD1 mice. Swallow rate (i.e., the number of swallows during 2 consecutive seconds of uninterrupted drinking) was significantly slower for SOD1 mice compared to aged C57 mice and controls. Inter-swallow interval (i.e., the time between two successive swallows) was not significantly different between groups. These findings support the notion that dysphagia profiles are likely to be distinctly different for each disease condition (Figure 13).
<img alt="Bar charts comparing swallow rate and inter-swallow interval; control, SOD1, aged C57 groups." src="/files/ftp_upload/52319/52319fig13highres.jpg" width="650" data-altupdatedat="1787630408000" data-alt="Figure 13"> 
Figure 13: Preliminary Findings. This figure shows representative preliminary findings for two VFSS swallow parameters quantified using the murine VFSS protocol: swallow rate (left) and inter-swallow interval (right). Swallow rate was significantly slower for SOD1 mice compared to aged C57 mice and controls. No significant group differences were identified for inter-swallow interval. Lines at the top of the bars indicate statistically significant differences (p &lt; 0.05) between groups, identified using Bonferroni pairwise comparisons. Error bars represent ± 1 SEM. <a href="https://www.jove.com/files/ftp_upload/52319/52319fig13highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

The overall goal of this procedure is to study swallowing function in freely behaving rodents, referred to as a video fluoroscopic swallow study or VFSS first. A feeding chamber is constructed to confine rodents in a small space during testing. The second step is to perform behavioral conditioning with a test solution to ensure maximal participation.The next step is to conduct VFSS testing with the preconditioned mice. Finally, videos are subjected to frame by frame analysis to quantify swallow parameters. Ultimately, this novel assessment tool can be used to detect and characterize dysphagia in mirroring disease models and provide functional biomarkers for use in preclinical trials.We first had the idea for using observation chambers because VFSS testing of humans is voluntary and unrestrained, and we wanted to closely mimic the human clinical procedure. The main advantage of this technique over similar existing methods is that it quickly elicits voluntary feeding behaviors in rodents to permit rapid testing of all stages of swallowing. Generally, individuals new to this method will struggle with video analysis because the small and rapid movements of swallowing and mice are difficult to discern.Begin by assembling the observation chambers next, assemble a syringe delivery system to deliver solution to peg bowls during testing. Once assembled, place the observation chamber onto a motorized scissor lift for remote positioning. The main advantage of the remote controlled scissor lift table is improved radiation safety for researchers while maintaining freely behaving rodents in the fluoroscopy field of view.Prepare the mice by habituating them to the observation chamber and subjecting them to an overnight water regulation period. When ready for testing, remove the chamber from the cage and clean it thoroughly. Use a dry erase marker to label each cleaned tube before placing it back in the home cage.Next, prepare the chocolate flavored iohexol solution to set up the fluoroscopy environment. Use a spare observation chamber and peg bowl to determine the optimal height and position within the fluoroscope beam that permits visualization of drinking in the lateral plane. Set the fluoroscopy frame rate to 30 frames per second.Higher frame rates can be used if available. Ensure that a radiopaque calibration marker is appropriately placed on the fluoroscope camera so that it is visible on the display monitor during the entire test. This step is necessary to permit calibration of length measurements to reduce stress due to handling.Observe the cage until a mouse freely enters the observation chamber. Once entered, lift the chamber out of the cage and gently attach the second end cap. With repeated testing, mice can be easily coaxed to enter the chamber when it is placed in front of them inside the cage, or when suspended by the tail over the chamber opening.Position the observation chamber within the fluoroscopy machine to begin VFSS testing in the lateral plane. Next, use a syringe delivery system to fill the peg bowl with chocolate flavored iohexol solution. Start the video fluoroscopy recording.When the mouse starts drinking during testing, use the remote controlled lift table to adjust the position of the observation chamber so that the swallowing mechanism is visible in the field of view. Pause recording each time the mouse turns away from the peg bowl to minimize the duration of radiation exposure. Resume recording when the mouse returns to the peg bowl, refill the peg bowl as needed.The goal is to record several long bouts of continuous drinking, which is typical for most mice. Within the first two minutes of testing, stop testing if the mouse does not drink within five minutes, return non-compliant mice to the home cage for retesting at a later time the same day. Do not exceed a 24 hour water regulation.When testing multiple mice from the same home cage. Clean the peg bowl and tubing with a dry paper towel between mice. Trim the tip of the PE tubing as needed.If chewed during testing, clean the observation chamber as needed to remove any splattered iohexol on the chamber walls. Rinse the chamber with tap water and dry with a paper towel. Use a video editing software program that permits frame by frame analysis of the video fluoroscopy recordings.To quantify the swallow parameters of interest, at least two trained reviewers should analyze each video in a blinded fashion. The primary reviewer views each video to identify and analyze three to five long drinking bouts. This criterion is based on published non-radiographic swallow studies showing that this parameter is sufficient for statistical analysis.The secondary reviewer then independently analyzes the three to five measures per swallow parameter for each mouse that were initially identified and analyzed by the primary reviewer. Reviewer discrepancies are then identified for each mouse and reanalyzed until 100%consensus is reached. The undisputed values for each swallow parameter are then averaged to obtain the mean value for each mouse.When fewer than three measures are obtained for a swallow parameter, enter a missing value in the statistical database for that measure. These examples were obtained using a low energy fluoroscopy system position. One permits visualization of the entire head and proximal thoracic region.The swallow trigger point is centered within the field of view. Position two permits visualization from the swallow trigger point to the gastroesophageal junction. Note, the bolus passing through the distal esophagus even at the lowest magnification setting.Bony structures of the head and neck are readily visible using the low energy fluoroscopy system. Anatomical structures are also examined at higher magnification to permit quantification of several additional swallow parameters. This figure shows representative preliminary findings for two VFSS swallow parameters, swallow rate, and inter swallow interval swallow rate was significantly slower for SOD one mice compared to aged C 57.Mice and controls inters swallow interval was not significantly different between groups. Once mastered video capture can be completed in just under two minutes per mass if it is performed properly. While attempting this procedure, it's important to remember to utilize the chocolate flavored test solution within a few hours of preparation to prevent changes in viscosity and palatability that can result in avoidance behaviors.This new technique is paving the way for researchers in the field of neuroscience to explore natural feeding and swallowing behaviors in healthy rodents and disease models. After watching this video, you should have a good understanding of how to successfully perform VFSS testing of freely behaving rodents To quantify typical feeding behaviors and swallowing function, Don't forget that working with radiation emitting equipment can be extremely hazardous, and radiation precautions for time, distance, and shielding should always be followed while performing this procedure.

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Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Live Imaging of Mouse Secondary Palate Fusion

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

The article introduces the µTongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

This protocol describes a serial transoral laryngoscopy approach for mice and rats that permits close-up, unobstructed video imaging of the larynx during breathing and swallowing using an optimized anesthetic regimen and finely tuned endoscopic manipulation techniques.

Minimally Invasive Murine Laryngoscopy for Close&#45;Up Imaging of Laryngeal Motion During Breathing and Swallowing

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Learn videofluoroscopic swallow study methods adapted for murine disease models to detect and characterize dysphagia. This technique enables quantitative analysis of swallowing function in freely behaving rodents for preclinical research.

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

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