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.

ملاحظة: وقد تمت الموافقة على جميع الطرق هو مبين في هذا البروتوكول من قبل لجنة رعاية واستخدام الحيوان المؤسسي. 1. إعداد جدول الجراحي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد سادة التدفئة مع إعادة تدوير المياه الدافئة على طاولة الجراحة، تحت المجهر تشريح. وضع غير القابل للصدأ لوحة الصلب قاعدة على لوحة التدفئة. وضع وسادة ماصة معقمة على لوحة قاعدة وسادة التدفئة. </li><li style=";text-align:right;direction:rtl"> ترتيب الأدوات الجراحية المعقمة على وسادة ماصة معقمة المجاورة. جمع اثنين من أزواج من الملقط ذات الرؤوس غرامة الزاوية، واثنين من أزواج من ملقط الزاوية، ثلاثة ملقط مرقئ، وهما الكامشات الأسلاك، مقص جراحي صغير، مقص الدقيقة، والأسلاك الاوعية الدموية واحد (0.15 بوصة القطر) وثلاث شرائح 6 بوصة من خياطة 6.0 الحرير . </li><li style=";text-align:right;direction:rtl"> ثني نهاية إيابا للسلك قسطرة لتتناسب مع انحناء الشريان الفخذي. وهذا سيجعل من الاسهل للمضي قدما في السلك أثناء الجراحة. </li></ol> 2. إعدادالماوس لجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الماوس عن طريق الاستنشاق المستمر من 2.5٪ الأيزوفلورين. تأكد من مراقبة حالة الحيوان في جميع أنحاء الداخلي. إجراء اختبار قرصة القدم الماوس، لتأكيد أن يتم تخدير بشكل كامل. ضمان الحيوان لا يتحرك عندما يخضع للامتحان قرصة. </li><li style=";text-align:right;direction:rtl"> تطبيق مرهم التشحيم لعيون الحيوان لمنع جفاف. تأمين الحيوان في موقف ضعيف للسادة ماصة باستخدام الشريط الجراحية. تواصل إدارة 2.5٪ الأيزوفلورين عبر مخروط الأنف. </li><li style=";text-align:right;direction:rtl"> باستخدام كريم مزيل الشعر، إزالة الفراء من الساق والبطن إلى خط منتصف. شطف الجلد جيدا بالماء. إزالة الشعر لا يمكن أن يؤديها قبل يوم واحد من عملية جراحية. لاحظ أن العلاج المفرطة مع كريم قد يؤدي إلى تهيج الجلد. </li><li style=";text-align:right;direction:rtl"> على الفور قبل الجراحة، وتطبيق بوفيدون اليود إلى منطقة نزعه مع قضيب من القطن ذات الرؤوس لتعقيم الجلد. شطف الجلد نزعه مع 70٪ من الإيثانول والجافةمع القطن ذات الرؤوس المعقم قضيب. كرر 3 مرات. </li><li style=";text-align:right;direction:rtl"> إدارة جرعة ما قبل المنطوق من 5 ملغم / كغم من كاربروفين عن طريق الحقن تحت الجلد. </li></ol> 3. عزل الشريان الفخذي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام مقص جراحي صغيرة، وجعل شق المنحني في الجلد فوق الشريان الفخذي. تشريح بصراحة وتأمين الأنسجة المحيطة باستخدام الكامشات التبعي ضام المغناطيسي لتحديد موقع الشريان الفخذي. ترطيب الأنسجة بشكل دوري باستخدام المياه المالحة للري تطبيق المالحة باستخدام القطن ذات الرؤوس المعقم قضيب. </li><li style=";text-align:right;direction:rtl"> عزل الشريان الفخذي باستخدام ملقط. فصل بلطف العصب من حزمة الأوعية الدموية باستخدام ملقط غرامة ذات الرؤوس. تجنب ثقب الوريد، ولا يتلف الأعصاب. دفع العصبية بعيدا عن حزمة لتجنب تحفيز ذلك. </li><li style=";text-align:right;direction:rtl"> فصل بلطف الوريد الفخذي من الشريان الفخذي، وتحديد مكان والتشعب الفخذ. منطقة التشعب هي صعبة للغاية لتشريح. </li><li style=";text-align:right;direction:rtl"> Posterior إلى التشعب، حلقة خياطة الحرير 6.0 تحت الشريان الفخذي وآمنة مع مرقئ. وسوف تستخدم هذه خياطة الداني لتقييد تدفق الدم في الشريان. ملاحظة: هناك اختلاف طفيف في العلاقات عند تنفيذ طريقة ربط مقابل طريقة الكي (انظر الشكل 1 والشكل 2). </li><li style=";text-align:right;direction:rtl"> القاصي إلى التشعب، حلقة 6.0 خياطة الحرير تحت الشريان الفخذي وآمنة مع مرقئ. هذه المساعدات خياطة البعيدة في المواقع من الشريان. </li><li style=";text-align:right;direction:rtl"> حلقة اثنين من الغرز ضمن الفرع العضلي للشريان الفخذ، قبل التعادل لهم وتأمين مع مرقئ. تذكر لترطيب الأنسجة بمحلول ملحي. إذا كان أداء طريقة الكي الإصابة الأسلاك، واحد فقط خياطة يحلق ضرورية على الفرع العضلي. </li></ol> 4. أداء فخذي الشريان إصابة سلك <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تقييد تدفق الدم في الشريان الفخذي عن طريق سحب خياطة الداني. سحب قليلا من hemost القاصيفي ومرقئ تأمين فرع لفضح الموقع لبضع الشريان. Ligate فرع العضلات عن طريق ربط الخيط حوله. ملاحظة: رفع الشريان أعلى تقييد تدفق الدم بشكل أكثر فعالية من سحب أفقيا وحدها. </li><li style=";text-align:right;direction:rtl"> قطع فرع صغير مع الكي بين الغرز اثنين. باستخدام مقص الصغرى، إجراء بضع الشريان في فرع جانب من التشعب. استخدام ملقط متوازنة على لفافة من الشريط الجراحية لتحقيق الاستقرار في المقص الصغير. </li><li style=";text-align:right;direction:rtl"> تأكيد وجود بضع الشريان باستخدام ملقط غرامة ذات الرؤوس. رفع بلطف افتتاح بضع الشريان مع ملقط. إدخال نهاية مدورة من السلك في بضع الشريان باستخدام ملقط. لتخفيف النهوض السلك، إضافة واحد أو اثنين من قطرات من يدوكائين في المنطقة باستخدام حقنة. </li><li style=";text-align:right;direction:rtl"> عندما السلك يصل خياطة الداني، والإفراج عن خياطة وتعديله بحيث لا يمكن أن تحول دون تطور من السلك. أدخل السلك حتى أنه لا يمكن دفع أكثر من ذلك. غيض من عشريجب أن يتوقف الأسلاك الإلكترونية في المنطقة في الرباط الإربي. </li><li style=";text-align:right;direction:rtl"> السماح للسلك بالبقاء في الشريان الفخذي لمدة دقيقة واحدة. بعد دقيقة واحدة، ودفع التراجع السلك في حركة نشر عشر مرات تجرح وتعرية البطانة من الشريان الفخذي. لإصابة من شدة أقل، وانخفاض عدد المرات التي يتم سحبها السلك الدخول والخروج من الشريان أو ترك السلك في الشريان لمدة 1 دقيقة فقط. </li><li style=";text-align:right;direction:rtl"> التراجع السلك ببطء. عندما نهاية الجولة من السلك اجتاز خياطة الداني، وتقييد تدفق في الشريان عن طريق سحب خياطة الداني. التراجع السلك تماما. </li></ol> 5. Ligating فرع العضلات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تشديد خياطة المتبقية على فرع العضلي. هذا سوف يمنع نزيف من بضع الشريان. عودة تدفق إلى فرع العضلات، وتؤكد أن الدم لا يتسرب منه. تقليم نهايات من الغرز على الفرع العضلي. </li></ol> 6. الأسلوب البديل: المترجمة كاوterization من بضع الشريان ملاحظة: يمكن اتخاذ نهج بديل لتجنب ربط فرع العضلات وتسمح الوصول الى الاوعية الدموية من خلال شريان الفخذ الرئيسي أكبر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدءا من 4.1، وتقييد تدفق إلى الشريان الفخذي من خلال سحب على خياطة الداني. سحب الخيط القاصي وخياطة تأمين فرع العضلات قليلا لفضح الموقع لبضع الشريان (الشكل 1) والشكل (2). </li><li style=";text-align:right;direction:rtl"> باستخدام مقص الصغرى، إجراء بضع الشريان عند نقطة فرع من الشريان الفخذي وفرع العضلي. إذا تم إجراء شق في جانب الشريان، قد يكون من الأسهل ليكوي. استخدام ملقط متوازنة على لفافة من الشريط الجراحية لتحقيق الاستقرار في المقص الصغير. </li><li style=";text-align:right;direction:rtl"> لاختبار نجاح الكي، واستعادة تدفق إلى الشريان الفخذي من خلال تخفيف خياطة القريبة. في حالة حدوث نزيف من بضع الشريان، كرر الكي. إذا كان الكي ناجحا، سيتم إعادة تدفق الدمالمخزنة البعيدة إلى بضع الشريان. </li><li style=";text-align:right;direction:rtl"> إدخال السلك وأداء إصابة الأسلاك كما هو موضح في قسم 4. </li><li style=";text-align:right;direction:rtl"> تواصل تقييد تدفق الدم في الشريان الفخذي. تسخين مكواة ذات الرؤوس غرامة لا تقل عن 6 بوصات بعيدا عن الماوس. كما يبرد طرف الكي، وتطبيق ذلك على محمل الجد جانب بضع الشريان لإغلاق الجرح. </li><li style=";text-align:right;direction:rtl"> لاختبار نجاح الكي، واستعادة تدفق إلى الشريان الفخذي من خلال تخفيف خياطة القريبة. في حالة حدوث نزيف من بضع الشريان، كرر الكي. إذا كان الكي ناجحا، سوف يتم استعادة تدفق الدم البعيدة لبضع الشريان. إزالة القريبة مؤقت والعلاقات البعيدة. </li></ol> 7. زرع المحيطة بالأوعية تسليم المخدرات التصحيح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء التصحيح المخدرات ماحول الأوعية يمكن أن تنشأ كما هو موضح في الدراسات السابقة 11 أو باستخدام أساليب مماثلة. بلانت زوايا المنطقة حاجز التصحيح تسليم المخدرات باستخدام مقص العقيمة </li><li style=";text-align:right;direction:rtl"> وضع بات تسليم المخدراتالفصل على الشريان الفخذي بجروح مع الجانب بين الافراج عن المخدرات التي تواجه الشريان. عند الضرورة، واستخدام ملقط لتحسين وضع التصحيح. </li></ol> 8. الجرح إغلاق والاسترداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إغلاق الجرح مع خياطة انقطاع بسيطة تتكون من عقدة مربع. A خياطة متقطعة يطيل إغلاق الجرح في حالة يحاول الحيوان لإزالة خياطة. </li><li style=";text-align:right;direction:rtl"> إيقاف التخدير وإزالة الحيوان من مجموعة لأعلى. السماح للحيوان لاسترداد على لوحة ظاهرة الاحتباس. </li><li style=";text-align:right;direction:rtl"> تواصل رصد الانتعاش الفأر. التحقق من موقع شق كل يوم لضمان بقائه مغلقا. لعلاج الألم بعد العمليات الجراحية، وإدارة الحقن تحت الجلد من 5 ملغم / كغم من كاربروفين كل 12 ساعة بعد الجراحة وبعد ذلك كل 12 ساعة لمدة 2 أيام. إذا استمر الألم ما بعد الأيام الأولى 2، يجب استشارة طبيب بيطري للاتجاهات على مزيد من الدواء الألم. </li></ol> 9. الشرايين حصاد الفخذ لعلم الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في 28 يوما بعد الجراحة، نفذ ثاني أكسيد الكربون القتل الرحيم على الماوس. ملاحظة: معدل تدفق مناسب من ثاني أكسيد الكربون يجب أن تحل محل 10-30٪ من الغرفة في الدقيقة الواحدة، وسوف تختلف تبعا لحجم الغرفة المستخدمة. يجب أن يتم تنفيذ قطع الحجاب الحاجز وثقب في القلب كوسيلة من وسائل الثانوي القتل الرحيم. </li><li style=";text-align:right;direction:rtl"> تأمين الماوس في موقف ضعيف باستخدام الشريط الجراحية. إجراء شق خلال شريان الفخذ، حيث تم إجراء شق جراحي الأولي. </li><li style=";text-align:right;direction:rtl"> لكل من الشريان المصاب والشريان لم يصب من الساق المقابل، بصراحة تشريح وتأمين الأنسجة باستخدام الكامشات والتبعي ضام المغناطيسي لتحديد موقع الشريان الفخذي المحيطة بها. ترطيب الأنسجة بشكل دوري باستخدام المياه المالحة للري. تطبيق المالحة باستخدام القطن ذات الرؤوس المعقم قضيب. الحرص على عدم تلف الشريان. </li><li style=";text-align:right;direction:rtl"> بعد أن تم عزل الشريان الفخذي من موقع لبضع الشريان الأصلي إلى الشريان الأبهر في البطن، وربط SEGمنة للخياطة الحرير بالقرب من الموقع الأصلي للبضع الشريان. هذا وسوف خياطة تساعد في تحديد نهاية معظم البعيدة للشريان الفخذ وسهولة التعامل مع العينة. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص تشريح الصغيرة لاستئصال الشريان الفخذي. جعل شق واحد القاصي إلى خياطة. جعل شق آخر في الطرف المقابل من الشريان الفخذي، بجانب الشريان الأورطي البطني. </li><li style=";text-align:right;direction:rtl"> نقل الشريان رفعه إلى طبق بتري الزجاج تحتوي على محلول ملحي. تشريح الشريان أيضا على إزالة النسيج الضام الزائدة أو الدهون. بلطف إزالة الدم من تجويف قبل الشطف بمحلول ملحي. </li><li style=";text-align:right;direction:rtl"> نقل الشريان إلى قارورة من 10٪ من الفورمالين مخزنة. تخزين القارورة في 4 درجات مئوية مع هزاز لطيف لمدة 48 ساعة. </li><li style=";text-align:right;direction:rtl"> نقل الشريان الثابتة إلى 70٪ من الإيثانول، لتخزين حتى تتم معالجتها لالأنسجة. </li><li style=";text-align:right;direction:rtl"> تضمين الشريان في كتل البارافين وقسم الكتل لتلطيخ. </li><li style=";text-align:right;direction:rtl"> أداء تلطيخ النسيجية وimmunochemical لتقييم مدى الإصابة وباطنة HYPerplasia. ملاحظة: للحصول على نتائج تمثيلية، استخدمنا الهيماتوكسيلين ويوزين لتصور النوى والتشكل العام أو Movat في Pentachrome وصمة عار لتصور صفائح مرنة ومكونات الشرايين الأخرى. </li></ol>

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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>
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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.

وبعد إصابة الأسلاك، وتضخم neointimal يتطور مع مرور الوقت وعادة ما يتم فحص بعد 14 إلى 28 يوما. التقنيات الموضحة في هذا الاعلان من العمل لجيل قوي من باطنة تضخم في الفئران كما أظهرت نتائج النسيجية في الشكل (3). ومن شأن الشريان الفخذي لم يصب تثبت صفائح مرنة سليمة وسمك طبيعي ومحيط. سوف شريان الفخذ المصاب تظهر باطنة تضخم، تدهور صفائح مرنة والتظاهر إعادة تبطنن-في نقطة زمنية لاحقة. إعادة تبطنن اكتمال عادة في حوالي 21 يوما ولكن هذا يعتمد على سلالة خلفية الفئران المستخدمة ويمكن تقييمها باستخدام المناعية للعلامات البطانية على أقسام أو ويفضل أن يكون، ويقاس على أون الاستعدادات الوجه باستخدام صبغة زرقاء وإيفان، والمجهر الإلكتروني أو المناعية مع المجهر متحد البؤر على أون استعدادات مواجهة الشريان. 17،18 لتقدير حجم باطنة تضخم عليههو الأفضل لقياس منطقة باطنة أو نسبة باطنة إلى وسائل الإعلام في ثلاثة مواقع على طول الشريان المصاب لحساب التغيرات المحتملة على طول المنطقة الجرحى من الشريان. <img alt="الشكل (1)" src="/files/ftp_upload/52403/52403fig1highres.jpg" /> الشكل 1: صور من الخطوات الحاسمة من طريقتين للإصابة سلك في الشريان الفخذي مقارنة بين الخطوات لطريقة ربط (أعلى) إلى أسلوب الكي (القاع) لأداء إصابة الأسلاك. في طريقة ربط، واثنين من الغرز تأمين فرع العضلات هي ما قبل ربط بحيث يمكن شددت على ligate فرع العضلي. وشددت على المزيد من القاصي اثنين من الغرز فرع العضلات. يتم تنفيذ بضع الشريان والأسلاك الإدراج في فرع العضلي. بعد يتم تنفيذ الإصابة، وتشديد خياطة المتبقية على فرع العضلات وقلص. في طريقة الكي، ق واحد فقطويحلق uture ضمن الفرع العضلي. يتم تنفيذ بضع الشريان والأسلاك الإدراج عند نقطة فرع من فرع العضلات من الشريان الفخذي. بعد يتم تنفيذ الإصابة، وأكتوي شق دون ligating إما الشريان الفخذي أو فرع العضلي. <a href="https://www.jove.com/files/ftp_upload/52403/52403fig1large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/52403/52403fig2highres.jpg" /> الشكل 2: صور التكبير أعلى من الخطوات الحاسمة من التقنيات اثنين للإصابة سلك المقارنة عن قرب من الخطوات لطريقة ربط (أعلى) إلى أسلوب الكي (القاع). وترسيمها الموقع من بضع الشريان مع خط متقطع. <a href="https://www.jove.com/files/ftp_upload/52403/52403fig2large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا</a>الرقم. <img alt="الشكل (3)" src="/files/ftp_upload/52403/52403fig3highres.jpg" /> الرقم 3: النتائج النسيجية التمثيلية للإصابة سلك بعد 14 يوما من إجراء نسيجية من أقسام الشرايين وملطخة الهيماتوكسيلين ويوزين (أعلى) وpentachrome تلطيخ Movat في (القاع). وصفت في الصورة هي لمعة (L)، البطانية (I) والصفيحة المرنة (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.

نموذج الفئران من فخذي الشريان إصابة سلك مع غرس المحيطة بالأوعية تسليم المخدرات التصحيح

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.

الشريان التاجي ربط Intramyocardial وحقن الفئران في نموذج من احتشاء

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.

الشريان المساريقي تقلص والاسترخاء عن طريق الدراسات الآلي Myography أسلاك

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.

نموذج الفئران من زرع دعامات في الشريان السباتي لدراسة عودة التضيق

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.

A الفئران نموذج من عضلة القلب إصابة نقص التروية، ضخه من خلال ربط الشريان الأمامي اليسار تنازليا

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.

والفئران نموذج من عودة التضيق الشرياني: الجوانب الفنية من الفخذ إصابة سلك

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.

نموذج الفئران من المعوية الإسكيمية-ضخه إصابة

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.

تحريض تصلب الشرايين المتسارع في الفئران: نموذج "الأسلاك- إصابة"

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.

متساوي القياس كونتراكتيليتي القياس الشريان المساريقي العلوي الماوس باستخدام أسلاك ميوجرافي

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.

إصابة بالون فئلي من ضغط الشريان السباتي الجرذ مع تطبيق علاجي شامل

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.

تأخر التسليم داخل القلب للخلايا الجذعية بعد إصابة إعادة ضخ الإقفاري في نموذج مورين

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