Name: a model of long-term ventricular fibrillation in isolated rat hearts
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تم تنفيذ هذا العمل بدعم من جراحة القلب والأوعية الدموية ، المركز الطبي الأول ، مستشفى PLA الصيني العام ومركز المختبر ، مستشفى PLA الصيني العام.

تمت مراجعة جميع الإجراءات والبروتوكولات التجريبية المستخدمة في هذا التحقيق والموافقة عليها من قبل لجنة رعاية واستخدام الحيوان في مستشفى PLA العام.
1. تحضير جهاز لانجيندورف
<ol><li>قم بإعداد المخزن المؤقت Krebs-Henseleit (K-H). لتحضير المخزن المؤقت K-H ، أضف ما يلي إلى الماء المق?...

<ol>
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(174), e62335 (2021).</li><li>Jungen, 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=Impact+of+intracardiac+neurons+on+cardiac+electrophysiology+and+arrhythmogenesis+in+an+ex+vivo+Langendorff+system.">Impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo Langendorff system.</a> Journal of Visualized Experiments. 135, e57617 (2018).</li><li>Koretsune, Y., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+calcium+in+the+pathophysiology+of+ventricular+fibrillation+and+in+the+pathogenesis+of+postarrhythmic+contractile+dysfunction.">Cell calcium in the pathophysiology of ventricular fibrillation and in the pathogenesis of postarrhythmic contractile dysfunction.</a> Circulation. 80 (2), 369-379 (1989).</li><li>Brazier, J. 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ينشئ هذا البروتوكول نموذجا حيوانيا ل VF طويل الأجل في قلوب الفئران المعزولة التي لم يتم الإبلاغ عنها من قبل. بالإضافة إلى ذلك ، تمت مقارنة ظروف التحفيز الكهربائي المختلفة في هذه الدراسة. تقدم هذه الدراسة نموذجا للدراسات المتعلقة بتوقف الرجفان البطيني أثناء جراحة القلب.
يعد ?...

عادة ما يتم إجراء جراحة القلب عن طريق بضع الصدر ، مع انسداد الشريان الأورطي والتروية بمحلول شلل القلب لإيقاف القلب. يمكن أن تكون جراحة القلب المتكررة أكثر صعوبة من الجراحة الأولية ، مع ارتفاع المضاعفات ومعدلات الوفيات1،2،3. علاوة على ذلك ، قد يتسبب نهج بضع القص المتوسط التقليدي في تلف أوعية الجسر خلف القص والشريان الأورطي الصاعد والبطين الأيمن والهياكل المهمة الأخرى. النزيف الشديد بسبب انفصال النسيج الضام ، وعدوى الجرح القصي ، والتهاب العظم والنقي القصي بسبب بضع القص كلها مضاعفات محتملة. يزيد التشريح الشامل من خطر الإصابة بالآفات والنزيف في الهياكل القلبية الحيوية.
مع تطور جراحة القلب طفيفة التوغل ، أصبحت الشقوق أصغر ، ويصعب أحيانا تحقيق السكتة القلبية. تكرار جراحة القلب تحت الرجفان البطيني (VF)4,5 آمن وممكن وقد يوفر حماية أفضل لعضلة القلب. لذلك ، يقدم هذا البروتوكول طريقة السكتة القلبية VF في الجراحة مع الدورة الدموية خارج الجسم طفيفة التوغل. يفقد القلب تقلصا فعالا أثناء VF ، وبالتالي ، ليست هناك حاجة لخياطة ومنع الشريان الأورطي الصاعد أثناء الجراحة ، مما يبسط الإجراء. ومع ذلك ، حتى لو كان القلب معطرا باستمرار ، فقد يظل VF طويل المدى ضارا بالقلب.
نظرا لاستخدام هذه الطريقة على نطاق واسع ، تصبح مسألة كيفية حماية القلب أثناء VF ذات أهمية متزايدة. سيتطلب ذلك دراسات مكثفة ومتعمقة باستخدام نماذج حيوانية من VF طويل الأجل. في الماضي ، استخدمت الأبحاث في هذا المجال في الغالب الحيوانات الكبيرة 6,7 وتطلبت التعاون بين الجراحين وأطباء التخدير وأخصائيي الإرواء وغيرهم من الباحثين. استغرقت هذه الدراسات وقتا طويلا ، وكانت أحجام العينات صغيرة في كثير من الأحيان ، وركزت الدراسات بشكل عام على وظيفة القلب وأقل على التقييمات الميكانيكية والجزيئية. حتى الآن ، لم تبلغ أي دراسة عن بروتوكول مفصل لإنشاء نموذج VF طويل الأجل.
وبالتالي ، يوفر هذا البروتوكول التفاصيل اللازمة لتطوير نموذج فأر VF طويل الأجل باستخدام جهاز Langendorff. البروتوكول بسيط واقتصادي وقابل للتكرار ومستقر.

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	<tbody>
		<tr>
			<td>0 Non-absorbable suture</td>
			<td>Ethicon, Inc.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>95% O2 + 5% CO2</td>
			<td>Beijing BeiYang United Gas Co., Ltd.&#160;</td>
			<td></td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>AcqKnowledge software</td>
			<td>BIOPAC Systems Inc.</td>
			<td>Version 4.2.1</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Automatic biochemistry analyzer</td>
			<td>Rayto Life and Analytical Sciences Co., Ltd.</td>
			<td>Chemray 800</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>BIOPAC research systems</td>
			<td>BIOPAC Systems Inc.</td>
			<td>MP150</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Blunt needle (20 G, TWLB)</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10005861</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>CK-MB assay kits&#160;</td>
			<td>Changchun Huili Biotech Co., Ltd.</td>
			<td>C060</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>Curved forcep</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009717</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>BIOPAC Systems Inc.</td>
			<td>STEMISOC</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>63005518</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Tianjin Biochem Pharmaceutical Co., Ltd.</td>
			<td>H120200505</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>21082201</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20025118</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Needle electrodes</td>
			<td>BIOPAC Systems Inc.</td>
			<td>EL452</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Ophthalmic clamp</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Perfusion tube</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10016318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10018960</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10019318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20040718</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sprague-Dawley (SD) rats</td>
			<td>SPF (Beijing) biotechnology Co., Ltd.</td>
			<td>Male, 300-350g</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Jiangsu Jingchuang Electronics Co., Ltd.</td>
			<td>GSP-6</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Tissueforceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Toothed forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Chengdu Instrument Factory</td>
			<td>DKX-150</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Water bath1</td>
			<td>Ningbo Scientz Biotechnology Co.,Ltd.</td>
			<td>SC-15</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Water bath2</td>
			<td>Shanghai Yiheng Technology Instrument Co., Ltd.</td>
			<td>DK-8D</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
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</table>

a model of long-term ventricular fibrillation in isolated rat hearts

الرجفان البطيني (VF) هو عدم انتظام ضربات القلب القاتل مع ارتفاع معدل الإصابة في مرضى القلب ، ولكن توقف VF تحت التروية هو طريقة مهملة للتوقف أثناء العملية في مجال جراحة القلب. مع التطورات الحديثة في جراحة القلب ، زاد الطلب على دراسات VF المطولة تحت التروية. ومع ذلك ، يفتقر الحقل إلى نماذج حيوانية بسيطة وموثوقة وقابلة للتكرار من الرجفان البطيني المزمن. يحفز هذا البروتوكول VF على المدى الطويل من خلال التحفيز الكهربائي للتيار المتردد (AC) للنخاب. تم استخدام ظروف مختلفة للحث على VF ، بما في ذلك التحفيز المستمر بجهد منخفض أو مرتفع للحث على VF طويل المدى والتحفيز لمدة 5 دقائق بجهد منخفض أو مرتفع للحث على VF التلقائي طويل المدى. تمت مقارنة معدلات نجاح الحالات المختلفة ، وكذلك معدلات إصابة عضلة القلب واستعادة وظائف القلب. أظهرت النتائج أن التحفيز المستمر للجهد المنخفض يحفز VF على المدى الطويل وأن 5 دقائق من التحفيز منخفض الجهد تسبب في VF التلقائي طويل الأجل مع إصابة عضلة القلب الخفيفة وارتفاع معدل استعادة وظيفة القلب. ومع ذلك ، فإن نموذج VF طويل الأجل منخفض الجهد والمحفز باستمرار كان له معدل نجاح أعلى. قدم التحفيز عالي الجهد معدلا أعلى من تحريض VF ولكنه أظهر معدل نجاح منخفض لإزالة الرجفان ، وضعف استعادة وظائف القلب ، وإصابة عضلة القلب الشديدة. على أساس هذه النتائج ، يوصى بالتحفيز المستمر لتيار متردد النخاب منخفض الجهد لمعدل نجاحه العالي ، والاستقرار ، والموثوقية ، والتكاثر ، والتأثير المنخفض على وظائف القلب ، وإصابة عضلة القلب الخفيفة.

تم استخدام ما مجموعه 57 فأرا في التجارب ، استوفى 30 منها معايير التضمين. تم تقسيم الحيوانات المدرجة إلى خمس مجموعات ، مع ستة في كل مجموعة: المجموعة الضابطة (المجموعة C) ، ومجموعة VF طويلة المدى ذات الجهد المنخفض المحفزة باستمرار (Group LC) ، ومجموعة VF طويلة المدى ذات الجهد العالي المحفزة باستمرار (Gr...

Watch this Scientific Journal Video about A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts at JoVE.com

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

يقدم هذا البروتوكول نموذجا للرجفان البطيني طويل الأمد في قلوب الفئران الناجم عن التحفيز المستمر بتيار متناوب منخفض الجهد. يتمتع هذا النموذج بمعدل نجاح مرتفع ، وهو مستقر وموثوق وقابل للتكرار ، وله تأثير منخفض على وظيفة القلب ، ولا يسبب سوى إصابة خفيفة في عضلة القلب.

نموذج للرجفان البطيني طويل الأمد في قلوب الفئران المعزولة

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

يوفر هذا البروتوكول حاجة مفصلة لتطوير نموذج فأر الرجفان البطيني طويل الأمد باستخدام جهاز Langendorff. يملأ فجوة الرجفان البطيني على المدى الطويل في النموذج الحيواني الصغير. التقنية بسيطة واقتصادية وقابلة للتكرار ومستقرة.يمكن استخدام هذه التقنية في دراسة توقف الرجفان البطيني تحت التروية والذي يتم إجراؤه في حالات سريرية معينة في مجال جلسات وعمليات جراحة القلب. للبدء ، قم بإعداد المخزن المؤقت Krebs-Henseleit أو KH بإضافة 118 مليمول كلوريد الصوديوم ، 4.7 مليمول كلوريد البوتاسيوم ، 1.2 مليمولار كلوريد المغنيسيوم ، 1.2 مليمولار فوسفات هيدروجين الصوديوم ، 1.8 مليمولار كلوريد الكالسيوم ، 25 مليمول بيكربونات الصوديوم ، 11.1 مليمولار الجلوكوز ، و 0.5 مليمولار EDTA في الماء المقطر. ثم قم بإعداد نظام نضح Langendorff المعدل.الغاز المستمر القارورة التي تحتوي على عازلة KH مع 95 ٪ الأكسجين و 5 ٪ ثاني أكسيد الكربون عند ضغط حوالي 80 ملليمتر من الزئبق. ضع أحد طرفي أنبوب التروية في المخزن المؤقت KH ، وقم بتمرير منتصف أنبوب التروية عبر الحمام المائي ، وقم بتوصيل إبرة قياس 20 حادة بالطرف الآخر من أنبوب التروية. بعد تعليق الإبرة على حامل سلكي ، اضبط درجة حرارة الحمام المائي بحيث تكون درجة حرارة المخزن المؤقت KH من نهاية نظام التروية 37 درجة مئوية.استخدم مسجل إشارة فسيولوجية لرقمنة وتسجيل جميع الإشارات التناظرية. استخدم قطبين كهربائيين بإبرة من الفولاذ المقاوم للصدأ لتسجيل مخطط كهربية القلب ثنائي القطب أو ECG وقطبين كهربائيين بإبرة من الفولاذ المقاوم للصدأ للتحفيز الكهربائي. قم بتوصيل أحد طرفي الأقطاب الكهربائية الأربعة بمسجل الإشارات الفسيولوجية والطرف الآخر بالقرب من المنطقة التي سيتم وضع القلب فيها بعد توصيله بالجهاز.بعد ذلك ، استخدم برنامج الكمبيوتر المحمول للتعرف تلقائيا على معلمات تخطيط القلب ثنائي القطب ومعلمات الدورة الدموية وضبطها وتسجيلها. اضبط معلمات المحفز الكهربائي على تيار متناوب 30 هرتز مع مجموعة الجهد المنخفض التي تستقبل 2 فولت ومجموعة الجهد العالي التي تتلقى 6 فولت. مع توصيل الفئران المخدرة بجهاز التنفس الصناعي بعد تشريح عنق الرحم والتنبيب الرغامي ، ارفع الجلد عن عملية الخنجري بالملقط المسنن وقم بعمل شق عرضي بطول ثلاثة سنتيمترات في الجلد بمقص الأنسجة.قم بتمديد شقوق الجلد والأضلاع إلى الإبطين على كلا الجانبين على شكل حرف V. استخدم تشريحا حادا لفصل الجذع العضدي الرأسي عن الأنسجة المحيطة. ثم قم بتثبيت الجذع العضدي الرأسي بالملقط المنحني لتسهيل إزالة القلب.اغمر القلب على الفور في طبق بتري مع مخزن مؤقت KH عند 0 إلى 4 درجات مئوية لغسل وضخ الدم المتبقي. نقل القلب إلى طبق بتري الثاني. قطع الأنسجة الزائدة عن الحاجة وتحديد الشريان الأورطي.استخدم ملقطين للعين لرفع الشريان الأورطي وإدخال الشريان الأورطي في الإبرة الحادة لجهاز Langendorff. ثم بمساعدة مساعد ، اربط عقدة بخيط خياطة 0. قم بتشغيل منظم تدفق التروية.ادفع البالون عبر الصمام التاجي إلى البطين الأيسر. املأ البالون بالماء المقطر لتحقيق ضغط انبساطي نهائي من 5 إلى 10 ملليمترات من الزئبق. قم بتوصيل مخطط كهربية القلب وأقطاب التحفيز الكهربائي بالقلب.ضع القلب في وسط الحجرة الزجاجية المغلفة عند 37 درجة مئوية. اسمح للقلب بالتوازن لمدة 20 دقيقة داخل الغرفة. ثم اضبط درجة حرارة حمام الماء للحفاظ على درجة الحرارة داخل الغرفة الزجاجية المغلفة عند 30 درجة مئوية.بعد أن تصل درجة الحرارة إلى المستوى المطلوب ، قم بتنشيط مفتاح التحفيز الكهربائي على برنامج الكمبيوتر المحمول. إذا كان الحيوان جزءا من الرجفان البطيني طويل الأمد أو مجموعة VF التي يتم تحفيزها باستمرار ، فاترك 90 دقيقة من التحفيز الكهربائي. بعد 90 دقيقة من VF ، استخدم الأقطاب الكهربائية لإعطاء 0.1 جول من إزالة الرجفان بالتيار المباشر.في نفس الوقت ، قم بتنظيم درجة حرارة حمام الماء للسماح لدرجة الحرارة بالارتفاع ببطء داخل الغرفة الزجاجية المغلفة إلى 37 درجة مئوية لمدة 10 دقائق. بعد إزالة الرجفان ، اترك القلب ينبض لمدة 60 دقيقة ثم أوقف النبض عن طريق التروية البطيئة مع 10٪ كلوريد البوتاسيوم عند حوالي 37 درجة مئوية. إزالة القلب لكيناز الكرياتين أو مقايسة CK-MB والتحليل النسيجي.يتم عرض معدلات VF ومعدل نجاح إزالة الرجفان ومعدل نجاح نموذج VF هنا. تلقت المجموعة LC والمجموعة HC تحفيزا كهربائيا مستمرا ، وبالتالي حدث VF بمعدل نجاح 100٪. لكن المجموعة HC أظهرت معدلات نجاح أقل لإزالة الرجفان.المجموعة LI والمجموعة HI التي تم فيها إيقاف التحفيز الكهربائي بعد خمس دقائق كان لها معدلات مختلفة من VF ، لكن معدل VF كان أبطأ في كلتا المجموعتين مقارنة بالمجموعة LC والمجموعة HC. كان لدى كل من Group LC و Group LI معدلات نجاح أفضل في إزالة الرجفان. ولكن بشكل عام ، حققت Group LC أعلى معدل نجاح للنموذج ، في حين كان لدى Group LI معدل نجاح نموذج أقل. يتم عرض معدل ضربات القلب أو HR ، وتدفق الشريان التاجي أو CF ، وفرق ضغط البطين الأيسر أو معدلات استرداد LVPD هنا.ظلت ديناميكا الدم في المجموعة C مستقرة أثناء التجربة وأظهرت انخفاضا طفيفا في HR و CF و LVPD. كان للمجموعتين ذات الجهد المنخفض الناجم عن VF أداء مماثل ومعدل استرداد جيد. لم يكن معدل HR و LVPD مختلفين بشكل كبير في تلك المجموعات مقارنة بالمجموعة C.لكن معدل استرداد CF كان أفضل بكثير مما كان عليه في المجموعة C.In النقيض ، كان معدل استرداد الدورة الدموية للمجموعتين ذوات الجهد العالي الناجم عن VF طويل الأجل ضعيفا وأظهرت مجموعة VF طويلة الأجل ذات الجهد العالي المحفز باستمرار أسوأ معدل استرداد.أظهر تحليل سوائل الانصباب التاجي أن مستويات CK-MB كانت أعلى في كلتا مجموعتي الجهد العالي. لم يتم العثور على فروق بين مجموعتي الجهد المنخفض وأظهر تلطيخ المجموعة C.Hematoxylin و eosin منطقة حرق القطب في المجموعة HC. هذا البروتوكول مناسب للدراسة المتعلقة بتوقف الرجفان البطيني والتروية في جراحة القلب والأوعية الدموية.

a-model-of-long-term-ventricular-fibrillation-in-isolated-rat-hearts

Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

تقييم وظيفة القلب وعلم الطاقة في قلوب معزولة عن طريق الماوس 31 P الرنين المغناطيسي الطيفي

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH الإسفار التصوير من البطينين متفرقة العامل قلوب الأرنب

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

تخفيض في البطين الأيسر الإجهاد الحائط والتحسين في وظيفة في عدم قلوب باستخدام Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

طريقة معزولة<em&gt; فيفو السابقين</em&gt; الإرواء الرئة في الفئران نموذج: الدروس المستفادة من تطوير برنامج EVLP الجرذ

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

نموذج فأر من الرجفان البطيني والإنعاش التي كتبها التقليدية مغلقة الصدر تقنية

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

الاستقراء وتقييم الإصابة الإسكيمية-ضخه في Langendorff perfused لالجرذ القلوب

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

الموضع الجراحي القسطرة على المدى الطويل القلب والأوعية الدموية ممارسة اختبار في الخنازير

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

على المدى الطويل قياس ضغط الدم في الانتقال بحرية الفئران عن طريق القياس

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

نموذج قلب مقلوب لجمع الخلالي الخلالي من القلب المعزول الجرذ

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

نموذج موحد من الرجفان البطيني ودعم الحياة القلبية المتقدمة في الخنازير

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...