Name: a new ex vivo model for the evaluation of endoscopic submucosal injection material performance
Uploaded: 2018-10-19T13:30:00
Description: Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

עבודה זו נתמכה על ידי קיוטו הטכנולוגיה החדשנית למחקר רפואי & פיתוח מערכת תמיכה, ועל ידי התוכנית המחקר Translational; קידום אסטרטגי עבור היישום המעשי של טכנולוגיה רפואית חדשנית (TR-ספרינט) מהסוכנות יפן למחקר רפואי, פיתוח (AMED).

להלן כללי התנהגות מנחים את טיפול בבעלי חיים של אוניברסיטת קיוטו הפאכטאראל מראש של התרופה.
1. הכנת דגימות באמצעות בטן חזירי
הערה: הצעד הראשון הוא להכין דגימות כדי לשמש הדגם ex-vivo (איור 1). עובי הקיר קיבה חזירי משתנה באזורים שונים של הקיבה. השתמ?...

<ol>
	<li>Ono, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection+for+treatment+of+early+gastric+cancer.">Endoscopic mucosal resection for treatment of early gastric cancer.</a> Gut. 48 (2), 225-229 (2001).</li><li>Conio, M., Ponchon, T., Blanchi, S., Filiberti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection.">Endoscopic mucosal resection.</a> The American journal of gastroenterology. 101 (3), 653-663 (2006).</li><li>Soetikno, R. M., Gotoda, T., Nakanishi, Y., Soehendra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection.">Endoscopic mucosal resection.</a> Gastrointestinal endoscopy. 57 (4), 567-579 (2003).</li><li>Iishi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+resection+of+large+sessile+colorectal+polyps+using+a+submucosal+saline+injection+technique.">Endoscopic resection of large sessile colorectal polyps using a submucosal saline injection technique.</a> Hepato-gastroenterology. 44 (15), 698-702 (1997).</li><li>Katsinelos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+50%+dextrose+and+normal+saline+solution+on+their+ability+to+create+submucosal+fluid+cushions+for+endoscopic+resection+of+sessile+rectosigmoid+polyps.">A comparative study of 50% dextrose and normal saline solution on their ability to create submucosal fluid cushions for endoscopic resection of sessile rectosigmoid polyps.</a> Gastrointestinal endoscopy. 68 (4), 692-698 (2008).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+endoscopic+mucosal+resection+using+sodium+hyaluronate.">A novel method of endoscopic mucosal resection using sodium hyaluronate.</a> Gastrointestinal endoscopy. 50 (2), 251-256 (1999).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+successful+single-step+endoscopic+resection+of+a+40+millimeter+flat-elevated+tumor+in+the+rectum:+endoscopic+mucosal+resection+using+sodium+hyaluronate.">A successful single-step endoscopic resection of a 40 millimeter flat-elevated tumor in the rectum: endoscopic mucosal resection using sodium hyaluronate.</a> Gastrointestinal endoscopy. 50 (5), 701-704 (1999).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+and+safety+of+0.4%+sodium+hyaluronate+solution+as+a+submucosal+fluid+&amp;#34;cushion&amp;#34;+in+endoscopic+resection+for+gastric+neoplasms:+a+prospective+multicenter+trial.">Usefulness and safety of 0.4% sodium hyaluronate solution as a submucosal fluid &amp;#34;cushion&amp;#34; in endoscopic resection for gastric neoplasms: a prospective multicenter trial.</a> Gastrointestinal endoscopy. 67 (6), 830-839 (2008).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+en-bloc+resection+of+large+superficial+tumors+in+the+stomach+and+colon+using+sodium+hyaluronate+and+small-caliber-tip+transparent+hood.">Successful en-bloc resection of large superficial tumors in the stomach and colon using sodium hyaluronate and small-caliber-tip transparent hood.</a> Endoscopy. 35 (8), 690-694 (2003).</li><li>Kishihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+sodium+hyaluronate+solution+in+colorectal+endoscopic+mucosal+resection.">Usefulness of sodium hyaluronate solution in colorectal endoscopic mucosal resection.</a> Digestive endoscopy. 24 (5), 348-352 (2012).</li><li>Yoshida, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection+with+0.13%+hyaluronic+acid+solution+for+colorectal+polyps+less+than+20+mm:+a+randomized+controlled+trial.">Endoscopic mucosal resection with 0.13% hyaluronic acid solution for colorectal polyps less than 20 mm: a randomized controlled trial.</a> Journal of gastroenterology and hepatology. 27 (8), 1377-1383 (2012).</li><li>Uraoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+glycerol+as+a+submucosal+injection+for+EMR.">Effectiveness of glycerol as a submucosal injection for EMR.</a> Gastrointestinal endoscopy. 61 (6), 736-740 (2005).</li><li>Conio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+performance+in+the+porcine+esophagus+of+different+solutions+used+for+submucosal+injection.">Comparative performance in the porcine esophagus of different solutions used for submucosal injection.</a> Gastrointestinal endoscopy. 56 (4), 513-516 (2002).</li><li>Moss, A., Bourke, M. J., Metz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind+trial+of+succinylated+gelatin+submucosal+injection+for+endoscopic+resection+of+large+sessile+polyps+of+the+colon.">A randomized, double-blind trial of succinylated gelatin submucosal injection for endoscopic resection of large sessile polyps of the colon.</a> The American journal of gastroenterology. 105 (11), 2375-2382 (2010).</li><li>Lee, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+EMR:+submucosal+injection+of+a+fibrinogen+mixture.">A new method of EMR: submucosal injection of a fibrinogen mixture.</a> Gastrointestinal endoscopy. 59 (2), 220-224 (2004).</li><li>Hurlstone, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMR+using+dextrose+solution+versus+sodium+hyaluronate+for+colorectal+Paris+type+I+and+0-II+lesions:+a+randomized+endoscopist-blinded+study.">EMR using dextrose solution versus sodium hyaluronate for colorectal Paris type I and 0-II lesions: a randomized endoscopist-blinded study.</a> Endoscopy. 40 (2), 110-114 (2008).</li><li>Huai, Z. Y., Feng Xian, W., Chang Jiang, L., Xi Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submucosal+injection+solution+for+endoscopic+resection+in+gastrointestinal+tract:+a+traditional+and+network+meta-analysis.">Submucosal injection solution for endoscopic resection in gastrointestinal tract: a traditional and network meta-analysis.</a> Gastroenterology research and practice. 2015, 702768 (2015).</li><li>Yandrapu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+saline+solution+versus+other+viscous+solutions+for+submucosal+injection+during+endoscopic+mucosal+resection:+a+systematic+review+and+meta-analysis.">Normal saline solution versus other viscous solutions for submucosal injection during endoscopic mucosal resection: a systematic review and meta-analysis.</a> Gastrointestinal endoscopy. , (2016).</li><li>Fernandez-Esparrach, G., Shaikh, S. N., Cohen, A., Ryan, M. B., Thompson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+reverse-phase+polymer+as+a+submucosal+injection+solution+for+EMR:+a+comparative+study+(with+video).">Efficacy of a reverse-phase polymer as a submucosal injection solution for EMR: a comparative study (with video).</a> Gastrointestinal endoscopy. 69 (6), 1135-1139 (2009).</li><li>Tran, R. T., Palmer, M., Tang, S. J., Abell, T. L., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+drug-eluting+elastomeric+polymer:+a+novel+submucosal+injection+material.">Injectable drug-eluting elastomeric polymer: a novel submucosal injection material.</a> Gastrointestinal endoscopy. 75 (5), 1092-1097 (2012).</li><li>Akagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+alginate+as+an+ideal+submucosal+injection+material+for+endoscopic+submucosal+resection:+preliminary+experimental+and+clinical+study.">Sodium alginate as an ideal submucosal injection material for endoscopic submucosal resection: preliminary experimental and clinical study.</a> Gastrointestinal endoscopy. 74 (5), 1026-1032 (2011).</li><li>Eun, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+sodium+alginate+as+a+submucosal+injection+material+for+endoscopic+mucosal+resection+in+animal.">Effectiveness of sodium alginate as a submucosal injection material for endoscopic mucosal resection in animal.</a> Gut and Liver. 1 (1), 27-32 (2007).</li><li>Hirose, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+ex+vivo+model+for+evaluation+of+endoscopic+submucosal+injection+materials+performance.">Development of a new ex vivo model for evaluation of endoscopic submucosal injection materials performance.</a> Journal of the Mechanical Behavior of Biomedical Materials. 79, 219-225 (2018).</li></ol>

הקיבה חזירי המשמש עבור הדגם החדש לאחסן במקפיא מיד לאחר כריתה, לשמש בתוך כמה חודשים אחרי קפוא, מאז הרעננות של הקיבה החזירים חיונית למדידה SEH. (נמדד SEH באמצעות דגימות קיבה מוקפאים ולא מוקפאים, ואנו אישר כי לא היה הבדל בתוצאה מידה סה.)
האיכות של דגימות קיבה מושפעת במידה רבה השונות...

ניתוח אנדוסקופי submucosal (ESD) והן כריתה אנדוסקופית הרירית (אלקטרומגנטית) הם הטיפולים הנפוצים כיום בשלב מוקדם סרטן מערכת העיכול1,2. הזרקת חומר הזרקה submucosal (SIM) לתוך submucosa הוא אחד הצעדים החשובים ביותר עבור EMR והן ESD הליכים2,3. העלאת רמת הרשאות submucosal גבוהה תחזוקה של העלאת רמת הרשאות submucosal קריטריונים קריטיים עבור בבטחה ניצוח EMR/ESD בקרב אנשי עסקים ותיירים כאחד.
למרות תמיסת מלח (NS) שימש בתור SIM מאז המצאת4,טיפול אנדוסקופי5, נתרן היאלורונית (HA) הונהג כטיפול בבשנים האחרונות6,7. חה הפך להיות בשימוש נרחב טיפולים אנדוסקופיים SIM סופריור עקב שלו ביצועים גבוהים8,9,10,11. כיום, נערכה השוואה ביצועים בין סימס הקיים, ביצועים גבוהים סימס פותחו כדי לזהות עוד מעולה SIM5,12,13,14, 15,16,17,18.
המודל שמחוץ באמצעות טיפוס הבטן חזירי שימש כדי להעריך את ביצועי ה-SIM, כי ההערכה של ה-SIM ביצועים במערכת העיכול האנושית היא קשה מאוד19,20,21 , 22. עם זאת, הדגם הזה קונבנציונלי שמחוץ היא פשוטה מאוד, ויש לו ההיקף לשיפור. שכפול סביבה קרובה יותר אל רירית מערכת העיכול האנושית תאפשר הערכה מדויקת של ביצועי ה-SIM.
במחקר הקודם שלנו, פיתחנו שמחוץ מודל חדש ניתן להשתמש כדי להעריך את הביצועים של סימס שונות בפירוט על-ידי החלת מתח מתמיד לקצוות של הדגימה. אנחנו גם אישר כי מודל לשעבר vivo החדש המוצע, מאפשר מדידה מדויקת היא תחת תנאים אחידים, השוואה מפורטת בין ההופעות של סוגים שונים של סימס23.
במחקר זה, אנו מציגים מראה שלם של הדגם ex-vivo החדש, המתודולוגיה הגדרה מפורטים של הדגם ex-vivo החדש מוסבר בפירוט באמצעות קטעי וידאו ודמויות. הדגם ex-vivo החדש כולל חלקים זמינים בקלות, ניתן להגדיר במהירות. תיאורים של הגדרה מפורטים מתודולוגיה יתרום להפצת המודל החדש.

<table>
	<tbody>
		<tr>
			<td>weight (153.1 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fixed type pulley</td>
			<td>H.H.H. MANUFACTURING</td>
			<td>VS25</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel wire with a diameter of 0.45 mm</td>
			<td>Nissa Chain</td>
			<td>Cut wire Y-5</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel clip of width 147 mm</td>
			<td>KOKUYO</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel key wire with a length of 12 cm</td>
			<td>Nissa Chain</td>
			<td>P-702</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel S shaped hook</td>
			<td>TRUSCO NAKAYAMA</td>
			<td>TCS1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>lockable stainless steel S-shaped hook</td>
			<td>Mizumoto Machine Mfg</td>
			<td>B2054</td>
			<td></td>
		</tr>
		<tr>
			<td>rectangular wooden base (45 x 60 cm)</td>
			<td>none</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber plate (5 x 5 cm)</td>
			<td>none</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>digital height gage</td>
			<td>Mitutoyo</td>
			<td>HDS-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5-mL syringe</td>
			<td>Terumo</td>
			<td>SS-02SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>23-gauge needle</td>
			<td>Terumo</td>
			<td>NN-2332R</td>
			<td></td>
		</tr>
		<tr>
			<td>MucoUp</td>
			<td>Boston Scientific</td>
			<td>none</td>
			<td>0.4% sodium hyaluronate (HA)</td>
		</tr>
		<tr>
			<td>saline (20 mL)</td>
			<td>Otsuka Pharmaceutical</td>
			<td>none</td>
			<td>normal saline (NS)</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7 software</td>
			<td>GraphPad Inc</td>
			<td>none</td>
			<td></td>
		</tr>
	</tbody>
</table>

a new ex vivo model for the evaluation of endoscopic submucosal injection material performance

הגברת הביצועים של חומרי הזרקה submucosal (סימס) חשוב עבור טיפול אנדוסקופי של מוקדם סרטן במערכת העיכול. זה חיוני להקים דגם ex-vivo שיכול להעריך ביצועי ה-SIM במדויק, לפיתוח סימס ביצועים גבוהים. במחקר הקודם שלנו, פיתחנו שמחוץ מודל חדש ניתן להשתמש כדי להעריך את הביצועים של סימס שונות בפירוט על-ידי החלת מתח מתמיד לקצוות של הדגימה. אנחנו גם אישר כי המודל לשעבר vivo החדש המוצע מאפשר העלאת רמת הרשאות submucosal מדויק מדידת גובה (סה) תחת תנאים אחידים והשוואות מפורט של המופעים של סוגים שונים של סימס. כאן, אנו מתארים את החדשה ex-vivo דגם ולהסביר את המתודולוגיה הגדרה מפורטים של מודל זה. מאז כל החלקים של המודל החדש היה קל להשיג, ההתקנה של הדגם החדש תסתיים במהירות. סה של סימס שונות ניתן למדוד בצורה מדויקת יותר באמצעות המודל החדש. ניתן לזהות הגורם הקריטי הקובע את ביצועי ה-SIM בעזרת המודל החדש. SIM פיתוח מהירות יעלה אחרי הגורם זוהתה.

סה נמדדה לאורך זמן דגם ex-vivo חדש או קונבנציונלי שמחוץ מודל. הערכים של SEH (NS) נמדד באמצעות מודל המקובלת [NS שהוזרק למחזור submucosa של הדגימה קבוע עם סיכות (0.0 N)] היו 5.7 mm (0 דקות), 3.6 מ מ (5 דקות), 3.0 מ מ (10 דקות), ו- 2.2 מ"מ (30 דקות). בדרך זו, הערכים של SEH ירד עם הגדלת הקפצות הזרקה. ניתוח ?...

Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

פיתחנו שמחוץ מודל חדש חל מתח מתמיד הדגימה קיבה חזירי. התפתחות זו אפשרו להעריך את הביצועים (את הגובה ואת משך העלאת רמת הרשאות submucosal) של סימס שונות באופן מדויק.  המתודולוגיה הגדרה מפורטים של המודל החדש הוא הסביר.

מודל לשעבר Vivo חדש על ההערכה של ביצועי חומרים הזרקה Submucosal אנדוסקופי

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

שיטה זו יכולה לעזור לענות על שאלות מפתח בתחום הביו-רפואי על עניין רפואי. היתרון העיקרי של טכניקה זו הוא כי הביצועים של נושאים שונים הזרקת submucosal ניתן להתחמק בצורה מדויקת יותר מאשר להיות שיטות קונבנציונליות. התחל על ידי חיתוך שישה על שישה ריבועים סנטימטר מהחלק השלישי העליון של קיבה פורצ'ין ומיד אחסון הדגימות במינוס 30 מעלות צלזיוס.כדי להגדיר את התקן המתיחה, חבר קליפ נירוסטה לחוט מפתח וו בצורת S, וחבר את חוט המפתח ואת הוו למשקל. חבר את הקרס לקצה השני של החוט ותקן גלגלות בשני קצות הבסיס. לאחר מכן מניחים צלחת גומי של שישה על שישה סנטימטרים, במרכז הבסיס וממקמים דגימת קיבה מופשרת על צלחת הגומי.לאחר מכן, לצבוט את הדגימה מסתיים עם קליפ של התקן המתיחה, לתלות את המשקל דרך שתי גלגלות להחיל מתח מתמיד על הדגימה, ולהשתמש במד דיגיטלי כדי למדוד את גובה גובה submucosal הזרקה מראש. לאחר מכן תקן את הסופר בגובה משטח הריחוף ודחוף מראש, כדי לבצע התאמת נקודת אפס של מד הגובה. כדי לבצע הליך העלאת תת-מדור, השתמש במזרק 2.5 מיליליטר המצויד במחט 23 מד, כדי להזריק שני מיליליטר של 0.4% נתרן היאלורנט, אופקית לתוך תת-המזרק משולי הדגימה.לאחר מכן השתמש במד הדיגיטלי כדי למדוד את גובה הגובה submucosal מיד, ובנקודות הזמן שצוינו לאחר ההזרקה. הקלטת הגובה המוצג במד הגובה, בעת תיקון הסופר לראש גובה תת-התת-טקסט. בניסויים מייצגים אלה נמדד גובה הגובה התת-מומוסל לאחר הזרקת חומצת מלח רגילה, או חומצת היאלורונט, במודלים הקונבנציונליים והחדשים של ניתוח אקס ויוו.למרות שגובה הגובה התת-מומוסל ירד עם הזמן לאחר הזרקת כל אחד מהתמיסות, בשני הדגמים, גובה הגובה התת-מומוסל שנוצר, לאחר הזרקת חומצת היאלורונט, היה גבוה באופן עקבי מאלה שנמדדו לאחר הזרקת מלח רגילה, ללא קשר לזמן שלאחר ההזרקה. יש לציין כי יישום של מתח מתמיד הביא לשונות כוללת נמוכה יותר בתוך המדידות, בהשוואה למודל המדידה הקונבנציונלי ללא מתח. ואכן, גובה הגובה התת-מומוסל שהושג במתח של 3.0 ניוטון, היה נמוך משמעותית מגבהי הגובה התת-מומוסל שהושגו ב-1.5 ניוטון.בעוד, אין הבדלים משמעותיים בגבהים תת-מומוסלים נצפו באמצעות המודל הקונבנציונלי, בהשוואה למודל המתח הקבוע. לאחר התפתחותה, טכניקה זו סוללת את הדרך לחוקרים בתחום העניינים הביו-רפואיים, לבטא את השימוש בחומרי הזרקת תת-mucosal בעלי ביצועים גבוהים לטיפול אנדוסקופי.

a-new-ex-vivo-model-for-evaluation-endoscopic-submucosal-injection

Research

JoVE Journal

Bioengineering

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

עיצוב של bioreactor לחץ על מחזורי Ex Vivo מחקר של מסתמי הלב אבי העורקים

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

בדיקת Microtensile לסביבה מבוקרת של nanocomposites הפולימר מכאני-מסתגל ל<em&gt; לשעבר vivo</em&gt; אפיון

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

קביעת תעריף הובלת xenobiotics ו ננו מעבר לשליה באמצעות<em&gt; לשעבר vivo</em&gt; דגם זלוף שליה אנושי

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Assay וMicroinjection פצע<em&gt; בvivo</em&gt; לוקליזציה של עוריות פצע הכתבים תגובה ב<em&gt; דרוזופילה</em&gt; עוברים.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin&#160;O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

הקמת דגם בניתוח מושרה בעכברים כדי לחקור את תפקיד מגן של progranulin בדלקת מפרקים ניוונית

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

הדמיה גידים קולגן המפוגל<em&gt; בvivo</em&gt; ו<em&gt; Ex vivo</em&gt; באמצעות הכלאה-מופעל על תמונה של Caged קולגן כוזב פפטידים

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

טכניקה כירורגית להשתלה של רקמות שתלי כלי דם מהונדסים ולאחר<em&gt; In vivo</em&gt; ניטור

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Athymic עכברוש מודל להערכת הנדסי קדמי צולב גידים שתלי

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

ראפיד סריקת אלקטרונים פאראמגנטיים תהודה פותחת אפיקים חדשים עבור פרמטרים חשובים מבחינת פיזיולוגית ההדמיה<em&gt; In vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.