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><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

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>

Watch this Scientific Journal Video about Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models at JoVE.com

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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21.2K Views.  University of Missouri.  Deze studie succesvol menselijk Videofluoroscopisch slikken studie (VFSS) methoden voor gebruik met murine ziektemodellen behoeve van translationeel dysfagie onderzoek vergemakkelijken aangepast. 

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

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

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

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

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

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

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

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

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.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

Cancer Research

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

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

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

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

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

Neuroscience

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

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

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...

Endoscopic Approach for Studying Laryngeal Dysfunction in Rodents

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

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty&#160;breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions.
To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification).
Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

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.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to ...

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

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