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.

בליעה (בליעת ירידת ערך) היא סימפטום נפוץ של מצבים רפואיים רבים המשפיעים על אנשים בכל הגילים. דוגמאות כוללות שבץ, מחלת פרקינסון, מחלת אלצהיימר, שיתוק מוחין, ניוון שרירים, טרשת לרוחב amyotrophic (ALS), מחלה באטן, סרטן ראש והצוואר, לידה מוקדמת, והזדקנות מתקדמת. בליעה מתאם גבוהה עם תמותה, בדרך כלל כתוצאה של תת תזונה חמורה או דלקת ריאות שמתפתחת כאשר מזון / נוזל / רוק חיידקים-לאדן הוא להישאף לריאות 1-4. מצבו הרפואי מתיש ומסכנות חיים זה משפיע על יותר מ -15 מיליון בני אדם בכל שנה בארצות הברית לבדה 3. למרות השכיחות הגבוהה ותוצאות שליליות נלוות, אפשרויות טיפול הנוכחיות לבליעה מוגבלות לפליאטיבי (ולא מרפא) מתקרב, כגון שינוי תזונה (למשל, הימנעות consistencies מזון / נוזל הספציפי), שינויים ביציבה (למשל, tucking הסנטר כאשר בליעה), גישות מוטוריות (למשל, תרגילים לחיזוק שרירי בחלל הפה, הלוע, גרון ו), גישות חושיות (למשל, טעם יישום, טמפרטורה, ו / או גירוי מכאני), וצינור האכלה (לדוגמא, תזונה ולחות מנוהלת באמצעות צינור nasogastric (NG) או צינור percutaneous גסטרונומית אנדוסקופית (PEG)). טיפולים אלה משמשים רק טיפול סימפטומטי ולא כמיקוד הגורמים הבסיסיים של הבעיה. ואכן, מכשול עיקרי לגילוי רומן, טיפולים יעילים לבליעה הוא מהמנגנונים פתולוגיים האחראים, אשר צפוי שונים לכל מחלה הידע המדעי המוגבל. אבחון הפרעת בליעה הוא בעיקר נעשה באמצעות הליך רדיוגרפי נקרא מחקר videofluoroscopic בליעה (VFSS), הידוע גם במחקר סנונית בריום שונה. במהלך שנות 30 פלוס העבר, בדיקת אבחון זה כבר נחשבה זהב סטנדרטי עבור evaluating פונקצית סנונית 5-7. בדיקה זו כרוכה בצורך בסבלנות לשבת או לעמוד בדרכה של קרן רנטגן של מכונה שיקוף בזמן בליעת מזון וסדירויות נוזל מעורבבת עם חומר ניגוד אוראלי מרצון, בדרך כלל 8,9 בריום סולפט או iohexol 10. כמטופל בולע, ניתן לראות מזון ונוזלים המכילים חומר ניגוד בזמן אמת באמצעות צג מחשב בעת נסיעה מן הפה אל הקיבה. מבני רקמות רכים הם גם נראים לעין וניתן להעריך ביחס למבנה ותפקוד. חולים מתבקשים לבצע כמה סנוניות של כל מזון ועקביות נוזלית, אשר כולם וידאו המוקלט לצפייה שלאחר מכן וניתוח מסגרת לפי מסגרת לכמת את הנוכחות ואת מידת הבליעה. רכיבים פיסיולוגיים רבים של בליעה הם בדרך כלל מנותחים, כגון נקודה אנטומיים ההדק של סנונית בלוע, זמן מעבר בולוס דרך הלוע והוושט, במידה ומשך larynהעלאה גאל, מיקום וכמות משקעי פוסט-סנונית, ומופע של וסיבה פיזיולוגית לשאיפה 7,11. היבטים של פרוטוקול VFSS אדם הותאמו לאחרונה ללימוד חולדות באופן חופשי-מתנהגים; עם זאת, תוצאות היו מוגבלות בגלל החולדות לא נשארו בתחום videofluoroscopic מבט במהלך בדיקת 12. VFSS שעד עתה לא ניסה עם עכברים. הסתגלות מוצלחת של פרוטוקול VFSS האנושי לשימוש עם עכברים וחולדות הייתה לספק שיטת מחקר חדשנית לחקור מאות עכברי קיימים כיום (עכבר וחולדה) מודלים של מחלות שידועות כגורמים לבליעה בבני אדם. שיטה זו חדשה (המכונה מעתה כVFSS עכברי) הייתה לכן לזרז זיהוי ואימות של מודלים עכבריים של בליעה המתאימים לחוקר את המנגנונים הנוירו-פיסיולוגיים הבסיסיים בתוך רקמת שרירים, עצבים, והמוח שפתולוגי ותורם לבליעה iבני אדם n. יתר על כן, VFSS העכברי יאפשר זיהוי של מדדים אובייקטיביים (סמנים ביולוגיים) של פונקצית סנונית / תפקוד לקוי שניתן להשוות באופן ישיר עם בני אדם. אז סמנים ביולוגיים videofluoroscopic בין מינים אלה יכולים לשמש כמדדי תוצאת רומן לכמת את יעילות טיפול בניסויים פרה-קליניים בעכברים וחולדות, שהיה טובים יותר לתרגם לניסויים קליניים עם אנשים. לשם כך, פרוטוקול VFSS העכברי הוקם באמצעות ~ משני מינים 100 עכברים. כל העכברים היו או C57 או זני C57 / SJL היברידי. עכברי C57 לא עברו שינוי גנטי, ואילו C57 / SJL היה מתח הרקע למושבה של SOD1-G93A המהונדס (או SOD1) עכברים, מודל החיה הנפוצה ביותר של ALS. המושבה SOD1 הייתה 50-50 תמהיל משוער של מהונדס (כלומר, מושפע-ALS) עכברים והמלטת nontransgenic (כלומר, לא נפגע). פרוטוקול VFSS העכברי מורכב משלושה מרכיבים: <ol style=";text-align:right;direction:rtl"><li&gt; תאי תצפית מעוצבים מותאמים אישית המאפשרים האכלה ובליעה מרצון בעת ​​שעמדה חסר מעצורים בחלל מצומצם בתוך מכונה שיקוף, </li><li style=";text-align:right;direction:rtl"> מתכונים שלהסוות את הטעם / הריח מרתיע של סוכנים בניגוד אוראליים ולייצר radiodensity מספיק כדי לאפשר הדמיה מספקת של בליעה, </li><li style=";text-align:right;direction:rtl"> פרוטוקול בדיקת צעד-אחר-צעד הממקסם עמידת בעלי חיים, ממזער את זמן בדיקה כולל וחשיפה לקרינה, ומאפשר כימות של מספר פרמטרים סנונית לכל שלב בבליעה (כלומר, אוראלי, בלוע, וושט). </li></ol> ההשפעה המשולבת מייצרת מתח נמוך, סביבה נוחה, האכלה עצמי בדיקה המאפשרת הערכה של האכלה טיפוסית והתנהגויות בליעה של עכברים.

<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

מחקר זה הותאם videofluoroscopic אדם בליעת מחקר שיטות (VFSS) לשימוש עם דגמי מחלה עכבריים לצורך קידום מחקר הבליעה translational. תוצאות מוצלחות תלויות בשלושת מרכיבים קריטיים: חדרי בדיקה המאפשרים האכלה עצמית בעמידה בלתי מרוסן בחלל מצומצם, מתכונים שלהסוות את הטעם / ריח מרתיע של סוכנים בניגוד אוראליים, זמינים מסחרי, ופרוטוקול בדיקת צעד-אחר-צעד ש מאפשר כימות של פיזיולוגיה סנונית. חיסולו של אחד או יותר ממרכיבים אלה יהיה השפעה לרעה על תוצאות המחקר. יתר על כן, יכולת רמת האנרגיה של מערכת השיקוף תקבע איזה לבלוע פרמטרים יכולים להיחקר. רוב מרכזי המחקר fluoroscopes אנרגיה גבוהה מיועד לשימוש עם אנשים ובעלי חיים גדולים יותר, וכתוצאה מכך איכות תמונה ירודה במיוחד כאשר בודקים עכברים ומכרסמים קטנים אחרים. למרות מגבלה זו, זיהינו שבעה VFSפרמטרים S שהם לכימות באופן עקבי בעכברים בעת שימוש פלואורוסקופ אנרגיה גבוה בשילוב עם פרוטוקול VFSS העכברי החדש. לאחרונה הושגו מערכת שיקוף אנרגיה נמוכה עם יכולות רזולוציה הדמיה וגדלה גבוהות במיוחד, שנועדה לשימוש עם עכברים ומכרסמים קטנים אחרים. עבודה ראשונית באמצעות מערכת חדשה זו, בשילוב עם פרוטוקול VFSS העכברי החדש, זיהתה 13 פרמטרים סנונית שהם כימות באופן עקבי בעכברים, שהוא כמעט כפול המספר שהושג באמצעות (כלומר, אנרגיה גבוהה) קונבנציונלית fluoroscopes. זיהוי של פרמטרים סנונית נוספים צפוי כפי שאנו לייעל את היכולות של מערכת חדשה זו. תוצאות עד כה להדגים את התועלת של שימוש במערכת שיקוף אנרגיה נמוכה כדי לזהות ולכמת שינויים עדינים בפיזיולוגיה סנונית שאחרת עשויה להתעלם בעת השימוש בfluoroscopes אנרגיה הגבוהה כדי לחקור מודלים עכבריים מחלה.

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.

התאמת שיטות סנונית Videofluoroscopic אדם מחקר כדי לזהות ולאפיין בליעה במודלי Murine מחלות

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

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21.0K 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.

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).

שיטות לכמת שינויים מושרה פרמקולוגית בתפקוד המוטורי ב SCI הושלמה האדם

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.

בידוד של oligomers PRP המסיס ובלתי מסיס במוח האנושי הרגיל

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.

Karyotyping רפאים ללמוד חריגות כרומוזום בבני אדם ועכברים עם מחלת כליות פוליציסטיות

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

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

מודלים לאינטראקציה בין סביבת גנים כדי לחשוף מנגנוני רגישות במחלת פרקינסון

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

דגם Murine הדמיה בלתי פולשנית לזיהוי ומעקב סרטן השחלות הישנות

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

בידוד וכריתה של אב העורקים Murine; טכניקה רב-תכליתית בחקר לב וכלי דם מחלות

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

שימוש בנגיף Adeno הקשורים ככלי לחקר חסמי רשתית במחלה

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Sacs Ex Vivo המעיים להעריך חדירות ריריות מודלים של מחלות במערכת העיכול

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

שיטות חריפה subacute Murine Hindlimb איסכמיה

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

מי כלוריד-induced Ferric מודלים פקקת Murine

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...