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>

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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 injury model with a perivascular patch is that it allows the use of genetically modified mice or murine models of disease. The endovascular manipulation of small arteries is technically demanding and this aspect has motivated the creation of models that create injury through a number of extravascular methods including ligation,11,24,25 perivascular cuffing26 and electric shock.27 In our procedures, a contralateral sham surgery can be used as a control by performing the protocol without the wire injury or, alternatively, non-operated control arteries can be used. The critical steps within the protocol are the following: (1) the surgical cut down and dissection of the femoral artery from surrounding structures, (2) the formation of the arteriotomy, (3) performance of the wire injury, (4) closure of the arteriotomy and (5) implantation of the perivascular cuff. Below we will review the major steps and address potential pitfalls in their performance. 
For the surgical cut down, the major challenge is the separation of the femoral artery from the femoral vein. Care should be taken at this stage, as it is easy to cause bleeding during the separation and the vein tears easily in comparison to the artery. Using forceps to blunt dissect and remove adventitia surround the artery and vein can help this process (see techniques shown in the video). Also, the artery may have branches underneath that can be torn if an overly aggressive technique is used. 
For the formation of the arteriotomy and wire injury, it is critical to use high quality microsurgical scissors. It is often helpful to cut at a 45-degree angle to the axis of the artery and to stabilize the cut with forceps as shown in the video. Too much or too little tension on the artery can make cutting difficult and it is sometimes helpful pull the distal tie before the proximal tie to fill the artery with blood to aid in the performing the arteriotomy. After the cut, opening the hole with the closed point of the scissors or forceps can aid in allowing entry of the wire. The size of the arteriotomy should cut approximately half way through the artery. Again, too much tension on the artery will make insertion of the wire difficult and may tear the vessel. A low to moderate level of tension should be used with the ties. When inserting the wire a slight twisting motion can be helpful but too much twisting will cause the artery to seize, preventing advancement of the wire.
A key step in performing our alternative method is the controlled local cautery that seals the vessel without blocking flow. The cautery should be heated away from the tissue and then touched to the side of the artery. Heating with the cautery while touching the vessel will burn surrounding tissues including the vein and femoral artery branches. One potential limitation of this technique is that if the arteries are not consistently cauterized there may be variations in the flow following the surgery or in the amount of inflammation caused by the burn. Analysis of the artery away from this region and consistent technique can minimize the potential impact of these limitations. We hope this alternative method will make the wire injury surgery more accessible and reduce the need for older mice for vascular injury studies.

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 border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:239px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Straight spring wire, 0.15&#8221; diameter</td>
			<td style="width:239px;">Cook</td>
			<td style="width:239px;">G02426</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High Temperature cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">HIT1</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High-temperature fine tip for cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">H101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Micro-scissors</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">15000-13</td>
			<td style="width:239px;">For performance of arteriotomy</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Angled fine-tipped forceps</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">11251-35</td>
			<td style="width:239px;">For blunt dissection of vascular bundle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Angled forceps</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5069</td>
			<td style="width:239px;">For clearing tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Surgical scissors</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5840</td>
			<td style="width:239px;">For cutting skin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-10</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor wire</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-05</td>
			<td style="width:239px;">Attached to retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Base plate</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-03</td>
			<td style="width:239px;">For use with retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Magnetic retractor fixator</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Needle Holder</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-7822</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Hemostatic forceps</td>
			<td style="width:239px;">Biomedical Research Instruments, Inc.</td>
			<td style="width:239px;">34-1000</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Dissecting microscope</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">EMZ-5TR</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Microscope light source</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">FT191</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Warm water recirculator</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-500</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Reusable heating pad</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-R 22G</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lidocaine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">4.0 Vicryl suture with half circle needle</td>
			<td style="width:239px;">Ethicon</td>
			<td style="width:239px;">J494G</td>
			<td style="width:239px;">For post-surgical wound closure</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Sterile cotton-tipped applicators</td>
			<td style="width:239px;">Puritan</td>
			<td style="width:239px;">25-806-2WC</td>
			<td style="width:239px;">For application of depilatory cream and absorbing fluids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Depilatory cream</td>
			<td style="width:239px;">Nair</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Isoflurane</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Betadine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">6.0 braided silk suture</td>
			<td style="width:239px;">Teleflex Medical</td>
			<td style="width:239px;">4-S</td>
			<td style="width:239px;">For isolation of femoral artery during surgery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">0.9% sodium chloride</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;">For irrigating tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Gel eye lubricant</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Glass petri dish</td>
			<td style="width:239px;">Pyrex</td>
			<td>3160-60</td>
			<td>For femoral artery harvest</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">10% buffered formalin</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Bouin&#39;s fluid</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td></td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcian blue, 1%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-01</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alkaline alcohol</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-02</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Orcein, 0.2%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-03</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Hematoxylin alcoholic, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-04</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Ferric chloride, 10%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-05</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lugol&#39;s Iodine</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-06</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Woodstain scarlet-acid fuchsin working solution</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-07</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Acetic acid, 0.5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>Various</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Phosphotungstic acid, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-09</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcoholic saffron, 6%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-10</td>
			<td style="width:239px;">For Movat&#39;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 הירך נפצע יציג היפרפלזיה intimal, מושפל lamellae אלסטי ולהפגין מחדש endothelialization בנקודות זמן מאוחר יותר. Re-endothelialization הוא בדרך כלל מלא בסביבות 21 ימים, אבל זה תלוי בזן הרקע של העכברים המשמשים וניתן להעריך באמצעות immunostaining עבור סמנים האנדותל בסעיפים או, עדיף, שנמדדו בen הכנות פנים באמצעות הצבע הכחול של אוון, במיקרוסקופ אלקטרונים סורק או immunostaining עם מיקרוסקופיה confocal בen הכנות פנים של העורק. 17,18 לכימות של היפרפלזיה intimal זההדרך טובה ביותר הוא למדוד את שטח intimal או יחס intimal לתקשורת בשלושה אתרים לאורך העורק הפגוע כדי לפצות על סטיות פוטנציאליות לאורכו של האזור שנפגע בעורק. <img alt="איור 1" src="/files/ftp_upload/52403/52403fig1highres.jpg" /> איור 1: תמונות של השלבים הקריטיים של שתי שיטות לפגיעה חוט בעורק הירך השוואה של הצעדים לשיטת קשירה (למעלה) לשיטת כויה (למטה) לביצוע פציעת חוט.. בשיטת קשירה, שני תפרי הבטחת סניף השרירים הם מראש קשורים כך שניתן יהיה להדק ולקשור את הסניף שרירי. יותר הדיסטלי של שני תפרי סניף השרירים מתהדק. הכנסת arteriotomy והחוט מבוצעת בסניף שרירי. אחרי הפציעה מתבצעת, התפר שנותר בסניף השרירים מתהדק וגזוז. בשיטת כויה, אחד של רקuture סובב תחת ענף השרירים. Arteriotomy והחדרת תיל מבוצעים בנקודת הסניף של סניף השרירים מעורק הירך. אחרי הפציעה מתבצעת, חתך חטא ללא ligating או עורק הירך או סניף שרירי. <a href="https://www.jove.com/files/ftp_upload/52403/52403fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/52403/52403fig2highres.jpg" /> איור 2: תמונות בהגדלה גבוהות יותר של השלבים הקריטיים של שתי טכניקות לפגיעה חוט השוואת תקריב של צעדים לשיטת קשירה (למעלה) לשיטת כויה (למטה).. המיקום של arteriotomy הוא נתחם עם הקו המקווקו. <a href="https://www.jove.com/files/ftp_upload/52403/52403fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של זה</a>דמות. <img alt="איור 3" src="/files/ftp_upload/52403/52403fig3highres.jpg" /> איור 3:. תוצאות היסטולוגית נציג של פגיעת חוט 14 ימים לאחר ההליך היסטולוגית מסעיפי העורקים הוכתמו Hematoxylin ו Eosin (למעלה) והצביעה של Movat pentachrome (למטה). כותרת בתמונה היא לומן (L), אינטימה (I) וlamina אלסטי (EL).

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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17.8K 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

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

קשירת עורקים כלילית לבין הזרקת Intramyocardial ב מודל Murine של אוטם

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

התכווצות העורקים mesenteric ללימודי הרפיה באמצעות Myography בנקאית אוטומטית

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

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 ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

דגם של שריר הלב איסכמיה reperfusion הפגיעה Murine באמצעות קשירת של העורק השמאלי קדמי יורד

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

דגם Murine של פגיעת המעיים איסכמיה reperfusion

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

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. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Contractility איזומטרי מדידה של עורק מצע המעי העליון העכבר באמצעות חוט Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

עכברוש עורק הראש מבוקר לחץ על פגיעה בבלון מגזרי עם יישום טיפולי Periadventitial

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

אספקה תוך-תוך-קרדיאלית מושהית של תאי גזע לאחר פציעת איסכמיה רפרציה במודל מורין

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...