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).

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 border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >Polycarbonate tubing for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >3161T41</td>
			<td>Body of observation tubes, 2&#34;X2&#34; diameter, 0.080&#34; thick wall</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9115K71</td>
			<td>End-caps for observation tubes, 2&#34;x12&#34;x3/4&#34;</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >8574K281</td>
			<td>Peg-bowls for observation tubes</td>
		</tr>
		<tr >
			<td >Silicone O-rings&#160; for end-caps of observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9396K108</td>
			<td>S1138 AS568-029, pack of 25 
			http://www.mcmaster.com/#o-rings/=t0wt5r&#160;</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 
			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 
			https://www.evergreensci.com/labware-catalog/tubes-and-vials/30-and-50-ml-centrifuge-tubes/&#160;</td>
		</tr>
		<tr >
			<td >Silcone stoppers for sipper tube bottles</td>
			<td >Saint-Gobain Performance Plastics</td>
			<td >DX263031-10&#160;</td>
			<td>Number 31D, size: 26 mm bottom, 32 mm top, 30 mm high; 10 pack;&#160; 
			http://www.labpure.com/en/Products.asp?ID=179&#38;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&#160; 
			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&#160;</td>
			<td>2 1/2&#8221; long drinking tubes with 5/16&#8221; opening, straight ball-spout 
			http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point&#160;</td>
		</tr>
		<tr >
			<td >Iohexol for making oral contrast agent solution</td>
			<td >GE Healthcare</td>
			<td ></td>
			<td>350 mg iodine per ml 
			http://www3.gehealthcare.com/en/products/categories/contrast_media/omnipaque&#160;</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 
			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&#39; 
			http://www.bd.com/ds/productCenter/427451.asp&#160;</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 
			http://www.bd.com/ds/productCenter/427560.asp&#160;</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 
			http://www.mcmaster.com/#catalog/120/3609/=t0wvaf&#160;</td>
		</tr>
		<tr >
			<td >Acrylic sheeting for scissor lift</td>
			<td >Ponoko</td>
			<td ></td>
			<td>Laser cut 
			http://www.ponoko.com&#160;</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 
			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 
			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 
			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 
			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 
			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 
			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 
			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 
			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

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 (&#62;18 months) and end-stage ALS.
<img alt="Figure 9" src="/files/ftp_upload/52319/52319fig9highres.jpg" width="650" /> 
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&#160;</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&#160;</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="Figure 10" src="/files/ftp_upload/52319/52319fig10highres.jpg" width="650" /> 
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="Figure 11" src="/files/ftp_upload/52319/52319fig11highres.jpg" width="650" /> 
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 &#8211; 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 &#8211; 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="Figure 12" src="/files/ftp_upload/52319/52319fig12highres.jpg" width="650" /> 
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 &#8220;control&#8221; 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="Figure 13" src="/files/ftp_upload/52319/52319fig13highres.jpg" width="650" /> 
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 &#60; 0.05) between groups, identified using Bonferroni pairwise comparisons. Error bars represent &#177; 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>

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

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 ...

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20.9K Views.  University of Missouri. This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.