The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

הערה: כל השיטות שמוצגים בפרוטוקול זה אושרו על ידי ועדת הטיפול ושימוש בבעלי חיים המוסדיים. 1. הכנת שולחן ניתוחים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדר את כרית החימום עם הסירקולציה מחודשת מים חמים על שולחן הניתוחים, תחת מיקרוסקופ לנתיחה. מניחים את צלחת בסיס נירוסטה על כרית החימום. הנח כרית סופגת סטרילית על צלחת הבסיס וכרית חימום. </li><li style=";text-align:right;direction:rtl"> לארגן כלים כירורגיים סטרילי על משטח סופג סטרילית סמוכה. לאסוף שני זוגות מלקחיים דקים שקצהו בזווית, שני זוגות מלקחיים זווית, שלושה מלקחיים עוצר דמום, שני חוטי מפשקים, מספריים כירורגיות קטנים, מיקרו מספריים, חוט אנגיופלסטיקה אחד (בקוטר 0.15 אינץ &#39;) ושלושה מגזרי 6 אינץ&#39; של תפר 6.0 משי . </li><li style=";text-align:right;direction:rtl"> תכופף את הקצה העגול של חוט אנגיופלסטיקה כדי להתאים את העקמומיות של עורק הירך. זה יעשה את זה קל יותר לקדם את החוט במהלך הניתוח. </li></ol> 2. הכנתעכבר לכירורגיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדימי עכבר על ידי שאיפה מתמדת של isoflurane 2.5%. הקפד לעקוב אחר מצבו של בעל החיים לאורך כל ההליך. לבצע בדיקת קמצוץ של כף הרגל של העכבר כדי לוודא שהוא הוא בהרדמה מלאה. ודא בעלי החיים אינו זז בעת מבחן קמצוץ מנוהל. </li><li style=";text-align:right;direction:rtl"> החל משחה סיכה לעיניו של בעל החיים כדי למנוע התייבשות. אבטח את החיה במצב שכיבה לכרית סופגת באמצעות קלטת כירורגית. המשך מתן isoflurane 2.5% באמצעות חרטומו. </li><li style=";text-align:right;direction:rtl"> שימוש בקרם מקריח, להסיר פרווה מהרגל ובבטן לקו אמצע. יש לשטוף את העור ביסודיות עם מים. אפילציה יכולה להתבצע יום אחד מראש של ניתוח. שים לב שטיפול מוגזם עם הקרם עלול להוביל לגירוי בעור. </li><li style=";text-align:right;direction:rtl"> מייד לפני הניתוח, חל povidone- יוד לאזור המקולף עם המוליך כותנה שקצה לעקר את העור. יש לשטוף את העור המקולף עם אתנול 70% ויבשיםעם המוליך כותנה שקצה סטרילי. חזור 3 פעמים. </li><li style=";text-align:right;direction:rtl"> שלו מנה טרום ניתוחית של 5 מ&quot;ג / קילוגרם של carprofen באמצעות הזרקה תת עורית. </li></ol> 3. בידוד של עורק הירך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעזרת מספריים כירורגיות קטנים, לעשות חתך מעוגל בעור מעל עורק הירך. בבוטות לנתח ולאבטח את רקמות סובבות באמצעות תופסנים מפשק מגנטי מפשקים כדי לאתר את עורק הירך. להרטיב את הרקמות מעת לעת באמצעות מי מלח להשקיה החל מלוח באמצעות המוליך כותנה שקצה סטרילי. </li><li style=";text-align:right;direction:rtl"> לבודד את עורק הירך באמצעות מלקחיים. בעדינות להפריד את העצב מחבילת כלי הדם באמצעות מלקחיים קנס שקצו. למנוע ניקוב הווריד, ולא לגרום נזק לעצב. לדחוף את העצב מהחבילה, כדי למנוע גירוי זה. </li><li style=";text-align:right;direction:rtl"> בעדינות להפריד את וריד הירך מעורק הירך, איתור הסתעפות הירך. האזור של הסתעפות קשה במיוחד לנתח. </li><li style=";text-align:right;direction:rtl"> Posterior להסתעפות, לולאת תפר 6.0 משי תחת עורק הירך ומאובטח עם hemostat. תפר הפרוקסימלי זה ישמש ללהגביל את זרימת דם בעורק. הערה: קיימת שונות קלות בקשרים בעת ביצוע שיטת קשירה לעומת שיטת הצריבה (ראה איור 1 ואיור 2). </li><li style=";text-align:right;direction:rtl"> דיסטלי להסתעפות, לולאת 6.0 תפר משי תחת עורק הירך ומאובטח עם hemostat. עזרים זה דיסטלי תפר במיצוב של העורק. </li><li style=";text-align:right;direction:rtl"> שני תפרי הלולאה תחת הענף השרירי של עורק הירך, מראש לקשור אותם ובטוחים עם hemostat. זכור כדי ללחלח את הרקמות עם מי מלח. אם ביצוע שיטת כויה של פגיעת חוט, רק אחד תפר כרך הוא הכרחי על הענף שרירי. </li></ol> 4. ביצועים של עורק הירך חוט פגיעה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להגביל את זרימת דם לעורק הירך על ידי משיכת התפר הפרוקסימלי. מעט למשוך את hemost דיסטליובhemostat הבטחת הסניף לחשוף את האתר לarteriotomy. ולקשור את סניף השרירים על ידי קשירת התפר סביבו. הערה: הרמת העורק כלפי מעלה יהיה יעילה יותר להגביל את זרימת דם מאשר מושך אופקי לבד. </li><li style=";text-align:right;direction:rtl"> לנתק את הסניף הקטן עם כויה בין שני התפרים. באמצעות מיקרו מספריים, לבצע arteriotomy בענף הצד השני של הסתעפות. שימוש במלקחיים מאוזנת בגליל של סרט כירורגית כדי לייצב את מיקרו המספריים. </li><li style=";text-align:right;direction:rtl"> לאשר את קיומו של arteriotomy באמצעות מלקחיים קנס שקצו. רם בעדינות את פתיחת arteriotomy עם המלקחיים. להציג את הסוף המעוגל של החוט לתוך arteriotomy באמצעות מלקחיים. כדי להקל על קידום התיל, להוסיף טיפה אחת או שתיים של לידוקאין לאזור באמצעות מזרק. </li><li style=";text-align:right;direction:rtl"> כאשר החוט מגיע התפר הפרוקסימלי, לשחרר את התפר ולהתאים אותו כך שהוא לא יכול לעכב את קידומו של החוט. הכנס את החוט עד שהוא לא יכול להתקדם עוד יותר. קצה החוט דואר צריך להפסיק באזור הרצועה מפשעתי. </li><li style=";text-align:right;direction:rtl"> לאפשר לחוט להישאר בעורק הירך לרגע אחד. אחרי דקה אחת, לחזור בו ולקדם את החוט בתנועת ניסור עשר פעמים לפגוע ולערטל האנדותל של עורק הירך. לפציעתם של חומרה פחותה, להקטין את מספר הפעמים שהחוט נמשך ויוצא מהעורק או להשאיר את החוט בעורק דקות 1 בלבד. </li><li style=";text-align:right;direction:rtl"> לחזור בו מהחוט לאט. כאשר בסופו של סיבוב החוט עבר את התפר הפרוקסימלי, להגביל את הזרימה לעורק על ידי משיכת התפר הפרוקסימלי. לחזור בו מהתיל לחלוטין. </li></ol> 5. ligating סניף השרירים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הדק את התפר שנותר על הענף שרירי. זה ימנע דימום מarteriotomy. חזור זרימה לסניף שרירי, ולאשר דם שלא דולף ממנו. חתוך את הקצוות מהתפרים בסניף שרירי. </li></ol> 6. אלטרנטיבי שיטה: המותאם למקום Cauterization של Arteriotomy הערה: ניתן לנקוט גישה חלופית כדי למנוע קשירה של סניף השרירים ולאפשר גישה של כלי דם דרך עורק הירך העיקרי הגדול יותר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החל מ 4.1, להגביל את הזרימה לעורק הירך על ידי משיכת התפר הפרוקסימלי. משוך את תפר דיסטלי ותפר הבטחת סניף השרירים מעט כדי לחשוף את האתר לarteriotomy (איור 1 ואיור 2). </li><li style=";text-align:right;direction:rtl"> באמצעות מיקרו מספריים, לבצע arteriotomy בנקודת עורק הירך ושרירי הסניף הסניף. אם מבצע החתך בדופן העורק, זה יכול להיות קל יותר לצרוב. שימוש במלקחיים מאוזנת בגליל של סרט כירורגית כדי לייצב את מיקרו המספריים. </li><li style=";text-align:right;direction:rtl"> כדי לבדוק את ההצלחה של צריבה, לשחזר את הזרימה לעורק הירך על ידי שחרור התפר הפרוקסימלי. אם דימום מתרחש מarteriotomy, לחזור על כויה. אם כויה היא מוצלחת, זרימת דם תהיה מחדשמאוחסן דיסטלי arteriotomy. </li><li style=";text-align:right;direction:rtl"> להציג את החוט ולבצע פגיעת החוט כאמור בסעיף 4. </li><li style=";text-align:right;direction:rtl"> המשך שמגביל את זרימת דם לעורק הירך. מחממים כויה דקה שקצהו לפחות 6 סנטימטרים מהעכבר. כעצת כויה מתקררת, להחיל אותו באורח קל בצד של arteriotomy כדי לסגור את החתך. </li><li style=";text-align:right;direction:rtl"> כדי לבדוק את ההצלחה של צריבה, לשחזר את הזרימה לעורק הירך על ידי שחרור התפר הפרוקסימלי. אם דימום מתרחש מarteriotomy, לחזור על כויה. אם כויה היא מוצלחת, זרימת דם תשוחזר דיסטלי arteriotomy. הסר את הפרוקסימלי הזמנית וקשרי דיסטלי. </li></ol> 7. השרשה של perivascular החדרת תרופות תיקון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור תיקון תרופת perivascular ניתן ליצור כפי שתואר במחקרים קודמים 11 או בשיטות דומות. להקהות את הפינות של אזור המחסום של תיקון משלוח סמים באמצעות מספריים סטרילי </li><li style=";text-align:right;direction:rtl"> הנח את טפיחת משלוח הסמיםch על עורק הירך הפצוע עם הצד משחרר תרופה מול העורק. במידת צורך, להשתמש במלקחיים כדי לשפר את מעמדו של התיקון. </li></ol> 8. פצע סגר ושחזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לסגור את הפצע עם תפר שהופסק פשוט המורכב של קשרים מרובעים. תפר רציף מאריך סגירת פצעים במקרה של בעלי החיים מנסים להסיר את התפר. </li><li style=";text-align:right;direction:rtl"> כבה את ההרדמה ולהסיר את החיה מלהגדיר. לאפשר לבעלי החיים להתאושש על כרית החימום. </li><li style=";text-align:right;direction:rtl"> תמשיך לעקוב אחר ההתאוששות של העכבר. בדוק את אתר חתך בכל יום כדי לוודא שהוא נותר סגור. לטיפול בכאב לאחר ניתוח, לנהל הזרקה תת עורית של 5 מ&quot;ג / קילוגרם של carprofen כל 12 שעות לאחר ניתוח ולאחר מכן כל 12 שעות למשך 2 ימים. אם כאב ממשיך מעבר 2 ימים הראשונים, להתייעץ עם וטרינר לכיוונים במשככי כאבים נוספים. </li></ol> 9. עורקי קציר עצם הירך להיסטולוגיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בגיל 28 ימים שלאחר ניתוח, לבצע המתת חסד פחמן דו חמצני בעכבר. הערה: קצב הזרימה המתאים של פחמן דו חמצני צריך לעקור 10-30% של החדר לדקה וישתנה בהתאם לגודלו של התא בשימוש. חיתוך של הסרעפת ולנקב לב צריכה להתבצע כשיטת משנית של המתת חסד. </li><li style=";text-align:right;direction:rtl"> לאבטח את העכבר במצב שכיבה באמצעות קלטת כירורגית. עושה חתך לאורך עורק הירך, שבו החתך הניתוחי הראשוני נעשה. </li><li style=";text-align:right;direction:rtl"> לשניהם את העורק נפצע ועורק וללא כל הפגע מהרגל הנגדית, בבוטות לנתח ולהבטיח שמסביב רקמות באמצעות כלי כתיבה ותופסנים מפשק מגנטיים כדי לאתר את עורק הירך. להרטיב את הרקמות מעת לעת באמצעות מי מלח להשקיה. החל מלוח באמצעות המוליך כותנה שקצה סטרילי. הקפד שלא לפגוע בעורק. </li><li style=";text-align:right;direction:rtl"> לאחר עורק הירך כבר מבודד מהאתר של arteriotomy המקורי לאב העורקים בבטן, לקשור SEGment של תפר משי בסמוך לאתר המקורי של arteriotomy. תפר זה יעזור לזהות את הקצה הדיסטלי ביותר של עורק הירך ולהקל טיפול במדגם. </li><li style=";text-align:right;direction:rtl"> בעזרת המספריים מיקרו לנתיחה שיחתוך את עורק הירך. לעשות חתך אחד דיסטלי לתפר. הפוך את החתך האחר בקצה השני של עורק הירך, ליד אב העורקים בבטן. </li><li style=";text-align:right;direction:rtl"> העבר את העורק נכרת לצלחת פטרי זכוכית המכילה תמיסת מלח. לנתח את העורק נוסף כדי להסיר את רקמת חיבור עודפת או שומן. להסיר בעדינות את דם מלומן על ידי שטיפה עם מי מלח. </li><li style=";text-align:right;direction:rtl"> העבר את העורק לבקבוקון של 10% שנאגרו פורמלין. אחסן את הבקבוקון על 4 מעלות צלזיוס עם הנדנדה עדינה במשך 48 שעות. </li><li style=";text-align:right;direction:rtl"> העבר את העורק הקבוע עד 70% אתנול, לאחסן עד שהוא מעובד להיסטולוגיה. </li><li style=";text-align:right;direction:rtl"> להטביע את העורק בלוקים וסעיף בלוקים עבור מכתים פרפין. </li><li style=";text-align:right;direction:rtl"> לבצע צביעת histochemical וimmunochemical להעריך את מידת הפגיעה HYP וintimalerplasia. הערה: לקבלת תוצאות הנציג, השתמשנו Hematoxylin ו Eosin לדמיין את הגרעינים ומורפולוגיה כללית או כתם Pentachrome של Movat לדמיין lamellae אלסטי ורכיבי עורקים אחרים. </li></ol>

<ol>
	<li>Hoffmann, R., Mintz, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+in-stent+restenosis+-+predictors,+treatment+and+prevention.">Coronary in-stent restenosis - predictors, treatment and prevention.</a> Eur Heart J. 21 (21), 1739-1749 (2000).</li><li>Erbel, 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=Coronary-artery+stenting+compared+with+balloon+angioplasty+for+restenosis+after+initial+balloon+angioplasty.+Restenosis+Stent+Study+Group.">Coronary-artery stenting compared with balloon angioplasty for restenosis after initial balloon angioplasty. Restenosis Stent Study Group.</a> N Engl J Med. 339 (23), 1672-1678 (1998).</li><li>Farooq, V., Gogas, B. D., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restenosis:+delineating+the+numerous+causes+of+drug-eluting+stent+restenosis.">Restenosis: delineating the numerous causes of drug-eluting stent restenosis.</a> Circ Cardiovasc Interv. 4 (2), 195-205 (2011).</li><li>Iqbal, J., Gunn, J., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stents:+historical+development,+current+status+and+future+directions.">Coronary stents: historical development, current status and future directions.</a> Br Med Bull. 106, 193-211 (2013).</li><li>Garg, S., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stents:+current+status.">Coronary stents: current status.</a> J Am Coll Cardiol. 56 (10 Suppl), S1-S42 (2010).</li><li>Park, S. J., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+percutaneous+coronary+intervention+with+drug-eluting+stents+in+Asia.">Current status of percutaneous coronary intervention with drug-eluting stents in Asia.</a> Circulation. 118 (25), 2730-2737 (2008).</li><li>Alfonso, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+drug-eluting+stent+restenosis+the+new+pilgrimage:+quo+vadis.">Treatment of drug-eluting stent restenosis the new pilgrimage: quo vadis.</a> J Am Coll Cardiol. 55 (24), 2717-2720 (2010).</li><li>Dake, M. D., 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=Paclitaxel-eluting+stents+show+superiority+to+balloon+angioplasty+and+bare+metal+stents+in+femoropopliteal+disease:+twelve-month+Zilver+PTX+randomized+study+results.">Paclitaxel-eluting stents show superiority to balloon angioplasty and bare metal stents in femoropopliteal disease: twelve-month Zilver PTX randomized study results.</a> Circ Cardiovasc Interv. 4 (5), 495-504 (2011).</li><li>Bosiers, 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=Results+of+the+Protege+EverFlex+200-mm-long+nitinol+stent+(ev3)+in+TASC+C+and+D+femoropopliteal+lesions.">Results of the Protege EverFlex 200-mm-long nitinol stent (ev3) in TASC C and D femoropopliteal lesions.</a> J Vasc Surg. 54 (4), 1042-1050 (2011).</li><li>Duda, 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=Drug-eluting+and+bare+nitinol+stents+for+the+treatment+of+atherosclerotic+lesions+in+the+superficial+femoral+artery:+long-term+results+from+the+SIROCCO+trial.">Drug-eluting and bare nitinol stents for the treatment of atherosclerotic lesions in the superficial femoral artery: long-term results from the SIROCCO trial.</a> J Endovasc Ther. 13 (6), 701-710 (2006).</li><li>Kumar, A., Lindner, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+with+neointima+formation+in+the+mouse+carotid+artery+after+cessation+of+blood+flow.">Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow.</a> Arterioscler Thromb Vasc Biol. 17 (10), 2238-2244 (1997).</li><li>Sata, 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Roque, 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=Mouse+model+of+femoral+artery+denudation+injury+associated+with+the+rapid+accumulation+of+adhesion+molecules+on+the+luminal+surface+and+recruitment+of+neutrophils.">Mouse model of femoral artery denudation injury associated with the rapid accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils.</a> Arterioscler Thromb Vasc Biol. 20 (2), 335-342 (2000).</li><li>Lindner, V., Fingerle, J., Reidy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+arterial+injury.">Mouse model of arterial injury.</a> Circ Res. 73 (5), 792-796 (1993).</li><li>Edelman, E. R., Adams, D. H., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+adventitial+heparin+delivery+on+smooth+muscle+cell+proliferation+following+endothelial+injury.">Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury.</a> Proc Natl Acad Sci U S A. 87 (10), 3773-3777 (1990).</li><li>Stemerman, M. B., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+arteriosclerosis.+I.+Fibrous+plaque+formation+in+primates,+an+electron+microscope+study.">Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study.</a> J Exp Med. 136 (4), 769-789 (1972).</li><li>Brouchet, L., 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=Estradiol+accelerates+reendothelialization+in+mouse+carotid+artery+through+estrogen+receptor-alpha+but+not+estrogen+receptor-beta.">Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta.</a> Circulation. 103 (3), 423-428 (2001).</li><li>Filipe, C., 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=Estradiol+accelerates+endothelial+healing+through+the+retrograde+commitment+of+uninjured+endothelium.">Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2822-H2830 (2008).</li><li>Feuls, 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=Microvascular+denudation+of+the+femoral+artery+of+the+mouse+as+a+model+for+restenosis.">Microvascular denudation of the femoral artery of the mouse as a model for restenosis.</a> Rofo. 175 (7), 952-957 (2003).</li><li>Holt, A. W., Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Rat+and+Mouse+Carotid+Artery+Surgery:+Injury+&amp;+Remodeling+Studies.">Experimental Rat and Mouse Carotid Artery Surgery: Injury &amp; Remodeling Studies.</a> ISRN Minim Invasive Surg. 2013, (2013).</li><li>Iafrati, M. D., 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=Estrogen+inhibits+the+vascular+injury+response+in+estrogen+receptor+alpha-deficient+mice.">Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.</a> Nat Med. 3 (5), 545-548 (1997).</li><li>Sullivan, T. 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=Estrogen+inhibits+the+response-to-injury+in+a+mouse+carotid+artery+model.">Estrogen inhibits the response-to-injury in a mouse carotid artery model.</a> J Clin Invest. 96 (5), 2482-2488 (1995).</li><li>Yin, Y., Zhao, X., Fang, Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+wire+injury+mouse+model+with+a+nonmicrosurgical+procedure.">Carotid artery wire injury mouse model with a nonmicrosurgical procedure.</a> Vascular. 18 (4), 221-226 (2010).</li><li>Nam, D., 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=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li><li>Nam, D., 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+model+of+disturbed+flow-induced+atherosclerosis+in+mouse+carotid+artery+by+partial+ligation+and+a+simple+method+of+RNA+isolation+from+carotid+endothelium.">A model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of RNA isolation from carotid endothelium.</a> J Vis Exp. (40), (2010).</li><li>Kuhlmann, M. 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=Implantation+of+a+carotid+cuff+for+triggering+shear-stress+induced+atherosclerosis+in+mice.">Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.</a> J Vis Exp. (59), (2012).</li><li>Carmeliet, 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=Vascular+wound+healing+and+neointima+formation+induced+by+perivascular+electric+injury+in+mice.">Vascular wound healing and neointima formation induced by perivascular electric injury in mice.</a> Am J Pathol. 150 (2), 761-776 (1997).</li></ol>

We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire inju...

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table><tbody><tr><td>Straight spring wire, 0.15&amp;rdquo; diameter</td><td>Cook</td><td>G02426</td><td></td></tr><tr><td>High Temperature cautery</td><td>Bovie Medical Corp.</td><td>HIT1</td><td></td></tr><tr><td>High-temperature fine tip for cautery</td><td>Bovie Medical Corp.</td><td>H101</td><td></td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15000-13</td><td>For performance of arteriotomy</td></tr><tr><td>Angled fine-tipped forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>For blunt dissection of vascular bundle</td></tr><tr><td>Angled forceps</td><td>Roboz</td><td>RS-5069</td><td>For clearing tissues</td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td>RS-5840</td><td>For cutting skin</td></tr><tr><td>Retractor</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Retractor wire</td><td>Fine Science Tools</td><td>18200-05</td><td>Attached to retractor</td></tr><tr><td>Base plate</td><td>Fine Science Tools</td><td>18200-03</td><td>For use with retractor</td></tr><tr><td>Magnetic retractor fixator</td><td>Fine Science Tools</td><td>18200-01</td><td></td></tr><tr><td>Needle Holder</td><td>Roboz</td><td>RS-7822</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Biomedical Research Instruments, Inc.</td><td>34-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Meiji Techno</td><td>EMZ-5TR</td><td></td></tr><tr><td>Microscope light source</td><td>Meiji Techno</td><td>FT191</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-500</td><td>For maintaining mouse body temperature</td></tr><tr><td>Reusable heating pad</td><td>Gaymar</td><td>TP-R 22G</td><td>For maintaining mouse body temperature</td></tr><tr><td>Lidocaine</td><td>Various</td><td></td><td></td></tr><tr><td>4.0 Vicryl suture with half circle needle</td><td>Ethicon</td><td>J494G</td><td>For post-surgical wound closure</td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Puritan</td><td>25-806-2WC</td><td>For application of depilatory cream and absorbing fluids</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Various</td><td></td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>6.0 braided silk suture</td><td>Teleflex Medical</td><td>4-S</td><td>For isolation of femoral artery during surgery</td></tr><tr><td>0.9% sodium chloride</td><td>Various</td><td></td><td>For irrigating tissues</td></tr><tr><td>Gel eye lubricant</td><td>Various</td><td></td><td></td></tr><tr><td>Glass Petri dish</td><td>Pyrex</td><td>3160-60</td><td>For femoral artery harvest</td></tr><tr><td>10% buffered formalin</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>Bouin&#039;s fluid</td><td>Electron Microscopy Sciences</td><td></td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcian blue, 1%</td><td>Electron Microscopy Sciences</td><td>26385-01</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alkaline alcohol</td><td>Electron Microscopy Sciences</td><td>26385-02</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Orcein, 0.2%</td><td>Electron Microscopy Sciences</td><td>26385-03</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Hematoxylin alcoholic, 5%</td><td>Electron Microscopy Sciences</td><td>26385-04</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Ferric chloride, 10%</td><td>Electron Microscopy Sciences</td><td>26385-05</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Lugol&#039;s Iodine</td><td>Electron Microscopy Sciences</td><td>26385-06</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Woodstain scarlet-acid fuchsin working solution</td><td>Electron Microscopy Sciences</td><td>26385-07</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Acetic acid, 0.5%</td><td>Electron Microscopy Sciences</td><td>Various</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Phosphotungstic acid, 5%</td><td>Electron Microscopy Sciences</td><td>26385-09</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcoholic saffron, 6%</td><td>Electron Microscopy Sciences</td><td>26385-10</td><td>For Movat&#039;s Pentachrome staining</td></tr></tbody></table>

murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

בעקבות פציעת חוט, היפרפלזיה neointimal מתפתחת לאורך זמן ונבחנה בדרך כלל לאחר 14 עד 28 ימים. הטכניקות מתוארות בעבודה זו תוביל לדור חזק של היפרפלזיה intimal בעכברים, כפי שמוצגים תוצאות היסטולוגית באיור 3. עורק הירך וללא כל פגע ידגימו lamellae שלם אלסטי ועובי רגיל והיקף. עורק ?...

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

דגם Murine של עורק הירך חוט פגיעה בהשרשה של perivascular החדרת תרופות תיקון

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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17.9K Views.  University of Texas at Austin. We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. 

Video: Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

דגם Murine של השתלת תומכן בעורק הראשי לחקר restenosis

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

משלוח סמים האוסמוטי לhindlimbs איסכמי וטפטוף של כלי דם עם Microfil למייקרו-טומוגרפיה ממוחשבת הדמיה

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

מערכת רדיו-telemetric לפקח פונקצית לב וכלי דם אצל חולדות עם חוט השדרה חיתוך רוחב ועוברי גזע עצבי שתלי סלולארי

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

דגם Murine של עורקי היצרות: היבטים טכניים של עצם הירך חוט פגיעה

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Hydrogel לבנות והפיברין מבוסס גישת דבק כדי לספק Therapeutics באוטם שריר הלב דגם Murine.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

אינדוקציה של טרשת עורקים מואצת בעכברים: מודל "פגיעת חוט"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

תיקון אנגיופלסטיקה בחולדת אב העורקים או נחות Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

השתלה של תיקון ביו-מודפס בתלת מיתד במודל מורין של אוטם שריר הלב

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Bioengineering

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

השתלת שתל כלי דם מהונדס רקמות בעורק הצוואר של העכבר בטכניקת שרוול

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

הדמיה תוך-חיונית מולטיפוטון לניטור גיוס לויקוציטים במהלך דלקת עורקים במודל מורין גפיים אחוריות

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...