注:所有程序都经寿命机构动物护理和使用委员会，并根据国家研究委员会指南护理和使用实验动物10的所有动物和程序。 1.气球制作和左心室压力监测电路<ol><li>根据Miller 等人构建LV的气球。11在装有搅拌棒的烧杯中，混合9.5毫升蒸馏水，14.2毫升轻玉米糖浆33.8克蔗糖和热的热板上，混合直至糖被溶解。继续加热溶液直至它达到约150ºC。 注:许多方法可用来构建LV的气球包括安全套的技巧和保鲜膜结构9,12修改。我们发现上面的方法比较容易构建无泄漏的气球，但对保鲜膜的气球优势包括以前的特性进行适当的frequenCY -反应关系9。 </li><li>打破干面条股成片约5厘米的长度和浸每一块的一侧约1厘米深成糖溶液和慢慢除去。 </li><li>将面食链到聚苯乙烯泡沫块的干端，并暂停所以糖混合物滴落并形成泪滴形状的模具。离开O / N在干燥器因此模具变硬。 </li><li>翌日，浸模具成硅氧烷分散体凝胶。将面条股回聚苯乙烯泡沫块和地方AA 37ºC烤箱2小时，或直至干燥。重复此步骤一次，以便硅氧烷两层被应用。 </li><li>一旦干燥，在水中发生数小时，以协助去除气囊从该模具。商店气球在0.02％叠氮化钠溶液中。 </li><li>用23克血收集组通过削减针来创建一个钝头，并放置在凹槽针产生一个定制的气球导管。 </li><li>油管连接到反压力能器，并在保持管和插管冲洗用水完全淹没，以防止空气进入系统。将已充满水到套管一个气球，用2-0丝线绑到套管。通过能力保持压力测试气球（约100毫米汞柱至少1小时） </li></ol> 2.准备离体心脏灌注系统<ol><li>最初，洗暖系统。打开一个37ºC温水浴循环器已连接到Langendorff装置，和填充缓冲储水用蒸馏水。连接新的玻璃纤维过滤器管道流入设备，并开启泵冲洗系统。在准备时间，监测水利用温度探针从所述的Langendorff块排出的温度，以确保水浴设定准确。另外，打开冷冻循环泵，并设置为20ºC将在心脏的心脏停搏使用。 </li><li>一世n个同时准备了以下解决方案。制备1升的心麻痹液（110毫摩尔NaCl，16毫米氯化钾，16毫米氯化镁2，1.5mM的氯化钙 ，10mM的碳酸氢钠 ）和过滤。另外，准备2升KHB（118 mM氯化钠，4.8 mM的氯化钾，1.2mM的磷酸二氢钾，1.7mM的MgSO 4干燥，2mM的丙酮酸钠，6mM的葡萄糖，24.9毫碳酸氢钠 （预充有CO 2）的 ，1.4毫米氯化钙2（最后加入），一旦溶解后，通过过滤到烧瓶中使用一个5微米的玻璃烧结的漏斗除去沉淀物。将一些成在冰上小碟手术和分离过程中使用。 </li><li>充氧的灌注液（KHB）用95％O 2/5％CO 2下在使用前至少30分钟。灌注的Langendorff系统KHB从系统中删除任何残留的水和缓冲水库。对于小鼠，设置初始泵速度来〜2.0毫升/分。附近放置插管灌注2-0和4-0丝线缝合。运行泵直至管道和设备中充满机智ħ灌洗液并确保气泡strup填充灌注液。 注:对于灌注和性能稳定的长期强烈建议在灌注回路安装一个在线的玻璃纤维过滤器（约1微米），收集可能形成血栓，在心脏沉淀物的任何</li></ol> 3.鼠标手术 3.1）鼠标麻醉和处理<ol><li>制备注射器用80毫克/千克氯胺酮和5mg / ml的甲苯噻嗪混合物麻醉剂的剂量，并加入无菌的0.9％生理盐水以调体积至0.2ml。注射肝素IP（50微升的1000单位/毫升溶液）。 </li><li>将鼠标放回运输容器（〜10-20分钟），并等待它失去了知觉肝素生效。定期做一个脚趾捏监控疼痛反射。 </li></ol> 3.2）拆下心<ol><li>一旦鼠标是完全无意识的，不响应脚趾捏，用针或25摹其固定到一个合适的手术台<html>通过四肢注射器提示。进行开胸暴露心脏。切一小口正下方的胸骨和切口延伸到鼠标避免膜片的两侧。 </li><li>迅速切断膜片，然后迅速切开胸廓的侧面。翻转回肋骨像蛤壳，露出胸腔。轻轻地抓住了心脏的，将剪刀下，取出心脏。 注:关键是要快速清除心脏一旦在胸腔暴露和肺部非功能。 </li></ol> 3.3）清洁的心<ol><li>将心脏入菜含有冰冷KHB和剪掉任何附加大块肺组织。使用镊子拿起心脏和定位主动脉。轻轻挤压心脏和寻找看来，因为这应该是主动脉的开口端的任何血液。用细尖镊子在主动脉的开口端保持心脏。 </li></ol>E"&gt; 4。安装了心脏，开始灌注，并放置气球 4.1）安装和Cannulating心<ol><li>前运送心脏的Langendorff装置，转向泵，确保灌注压很低（〜20毫米汞柱）。按住心脏的主动脉刚下套管。使用另一套精尖镊子，小心地打开主动脉和​​心脏滑动到了插管。用一组镊子举行的地方，然后用软边夹将其暂时固定到套管。 注:心脏快速安装到灌注导管是一个很好的准备是至关重要的。随着经验的积累，从打开胸腔，直至安装心脏的程序应采取1至2分钟。 </li><li>领带，并开始灌注如下，使用4-0丝线缝合安全主动脉正下方的夹子套管。确保领带也是围绕金属套管主动脉，所以主动脉没有得到时收紧打结结。一旦一条领带牢牢地放置，取出夹子。 </li><li>使用额外的丝线牢靠固定主动脉并确保打结低于任何血管分支可能脱落主动脉插管。这些通常可以通过灌注液泄漏或主动脉拍摄出被发现。 注:在灌注的最初阶段的血应该从心脏洗涤和整个心脏应该出现一个软的粉红色。不洗出暗的变色表明缺血区可能是由于空气栓子或血栓和心脏不应使用。 </li></ol> 4.2）建立和灌注测量LVP <ol><li>慢慢增加灌注压，增加泵速，直到灌注压力达到70毫米汞柱。 注:标准灌注压的小鼠灌注可以是70-90毫米汞柱，但必须保持恒定，从动物到动物的实验中。 </li><li>清洁剩余组织（肺，甲状腺等）可能仍然附着到心脏。用剪刀切断左心房，以便创造一个开口进入左心室。 </li><li>将气球上的保持器和放气。定位气球导管近主动脉插管，直接在开口进入左心室。小心向下插入气球进入左心室，而持有的心脏到位，使主动脉不撕裂。 </li><li>一旦到位慢慢开始充气气球，直到达到LVEDP 8〜毫米汞柱。将温度探头对心脏的底部，所以它测量出水的温度。 </li></ol> 4.3）测量基础<ol><li>在水夹套灌注腔密封的心脏。在此期间，不断灌注心脏与KHB，并确保温度继续上升。监控温度并相应地调整水浴中，直到它达到大约〜37ºC。 注:监测Temperat .. [温度URE灌注的初始阶段是至关重要的，因为冠状动脉流量，随后温度可以从心脏到心脏。出水灌流的温度为所需温度的心肌通过温度探头放置在心脏的顶点监控。此外，相较于其他的Langendorff灌注协议，心脏没有灌注或逮捕时淹没在CP或KHB，这主要是做快速调节温度，以及提供有效的CP输送无扩散。 </li><li>开始使用连接到合适的传感器数据采集系统，包括灌注压，左心室压力（附连到LVP气球压力传感器），温度和电生理参数（心电图，MAP如果配备）连续记录功能测量（附件将根据制造商和特定于各个传感器和数据采集系统）。一旦出水达到KHB〜37ºC至少15<html>分和心脏功能的参数是稳定的，记下时间为基线测量。 注:纳入/排除标准适用于每一个心脏。对&lt;60毫米汞柱基线时LVDP表示心脏，应该从分析中删除。此外，冠状动脉血流&gt;4.5毫升/分钟，或无法维持在基线灌注压可能表示一个泄漏或撕裂主动脉。也有心脏功能（ 即，增加左室舒张末压，心律失常极），它可以直观地确认清楚缺血区（ 即血块），或表现出局部缺血有关的任何削减都心从分析中删除。 </li><li>测量通过收集冠状动脉流出物的冠脉流量量筒1分钟。关闭该室，并允许温度和功能以返回到基线值。备选地测量经由流量探针置于泄压电路后冠状动脉连续流中的灌注管线。 </li></ol>"&gt; 5。起爆心脏停搏<ol><li>将100毫升冷水停跳缓冲到另一个水库。的KHB储插管转移到心麻痹水库。 </li><li>分离水套住房和灌注升温电路由温水浴，并连接到冷冻循环利用油管快速接头。使用单独的加热和冷却循环，使心脏和灌注液的温度急剧变化。 </li><li>按照气泡的开关过程中被引入到管路，并一旦它到达靠近分离的心脏开始的定时停搏。递送停跳2分钟。初始剂量后停泵。确保逮捕心脏舒张和接近所需的CP温度。 注:足够的泡沫陷阱是必要的，以避免在空气中心脏栓子。可替代地用于交换通过一个活塞既储层的递送系统可以简单地构成，但是，我们发现按照荷兰国际集团的小气泡被交换储层之间插管时引入的定时的CP递送一种简单的方法给定实验之间可能不同的泵速（ 即，由于小鼠/大鼠，灌注压力，过滤器电阻等 ）。 </li><li>请停跳的心脏为2小时，在约20ºC。 CP期间每半小时转动泵回1分钟，以便另一剂量给药。 注:间歇剂量的CP，每30分钟的结果的心脏功能障碍的无组织的坏死。剂量CP（ 即 &gt; 45分钟）之间的较长可能导致坏死和缺血性挛缩，并会更适合于无保护缺血坏死的相关的损伤模型。 </li></ol> 6.再灌注<ol><li>在CP的端部，传送回储插管含氧KHB。连接的温度控制电路，以加热循环器，并转动泵以启动再灌注。此时搏冲刷，观察体温升高和心脏开始再灌注后2-4分钟再次跳动。当心脏开始心脏跳动缓慢，往往心律失常。 注:通常不是一个大的上升舒张LVP（ 即 ≤10毫米汞柱上升），这往往是在纯缺血模型的特点。心律失常可以持续顺利进入再灌注偶尔心脏将需要除颤。这可以通过使用一个刺激设置为更高的电压（〜10-50 V）和电极放置在心脏的基部和顶点来实现。 </li><li>允许心脏到reperfuse 30分钟，并且在这段时间内取冠状动脉流量测量是必要的。通过收集与量筒的流出物为30-60秒测量冠脉血流量。 </li></ol> 7.收集组织<ol><li>放气的气球，并从左心室卸下。就拿心脏关闭套管和称重。在最初的实验中，心脏的至少一个横向片应采取和TTC染色评估坏死，以确保心脏是无任何坏死伤害。 </li><li>收集心脏切片显微镜研究，湿/干重等 ，其余的或整个心脏应该放置到液氮快速冷冻。冷冻保存的心在-80ºC冰箱。 注:对于高能磷酸化合物（ 即三磷酸腺苷，磷酸肌酸）测量，心中应立即冷冻。 </li></ol>

<ol>
	<li>Kloner, R. a., Jennings, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Brief+Ischemia:+Stunning,+Preconditioning,+and+Their+Clinical+Implications:+Part+1.">Consequences of Brief Ischemia: Stunning, Preconditioning, and Their Clinical Implications: Part 1.</a> Circulation. 104 (24), 2981-2989 (2001).</li><li>Mentzer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+protection+in+heart+surgery.">Myocardial protection in heart surgery.</a> J Cardiovasc Pharmacol Ther. 16 (3-4), 290-297 (2011).</li><li>Chambers, D. J., Fallouh, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioplegia+and+cardiac+surgery:+pharmacological+arrest+and+cardioprotection+during+global+ischemia+and+reperfusion.">Cardioplegia and cardiac surgery: pharmacological arrest and cardioprotection during global ischemia and reperfusion.</a> Pharmacol Ther. 127 (1), 41-52 (2010).</li><li>Bolli, R., Marb&#225;n, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+of+myocardial+stunning.">Molecular and cellular mechanisms of myocardial stunning.</a> Physiol Rev. 79 (2), 609-634 (1999).</li><li>Kloner, R. a., Bolli, R., Marban, E., Reinlib, L., Braunwald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+and+Cellular+Implications+of+Stunning,+Hibernation,+and+Preconditioning+An+NHLBI+Workshop.">Medical and Cellular Implications of Stunning, Hibernation, and Preconditioning An NHLBI Workshop.</a> Circulation. 97 (18), 1848-1867 (1998).</li><li>Mersmann, J., Latsch, K., Habeck, K., Zacharowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measure+for+measure-determination+of+infarct+size+in+murine+models+of+myocardial+ischemia+and+reperfusion:+a+systematic+review.">Measure for measure-determination of infarct size in murine models of myocardial ischemia and reperfusion: a systematic review.</a> Shock (Augusta, Ga). 35 (5), 449-455 (2011).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+the+Langendorff+technique+of+isolated+heart+perfusion).">Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion).</a> J Mol Cell Cardiol. 50 (6), 940-950 (2011).</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Sutherland, F. J., Shattock, M. J., Baker, K. E., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+isolated+perfused+heart+characteristics+and+cautions.">Mouse isolated perfused heart characteristics and cautions.</a> Clin Exp Pharmacol Physiol. 30 (11), 867-878 (2003).</li><li>Miller, A., Wright, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Murine+Ventricular+Balloons+for+the+Langendorff+Heart+Preparation.">Fabrication of Murine Ventricular Balloons for the Langendorff Heart Preparation.</a> J Biotecnol Biomater. 1 (101), 1-4 (2011).</li><li>Curtis, 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=Characterisation,+utilisation+and+clinical+relevance+of+isolated+perfused+heart+models+of+ischaemia-induced+ventricular+fibrillation.">Characterisation, utilisation and clinical relevance of isolated perfused heart models of ischaemia-induced ventricular fibrillation.</a> Cardiovasc Res. 39 (1), 194-215 (1998).</li><li>Clements, R. T., Feng, J., Cordeiro, B., Bianchi, C., Sellke, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p38+MAPK-dependent+small+HSP27+and+&#945;B-crystallin+phosphorylation+in+regulation+of+myocardial+function+following+cardioplegic+arrest.">p38 MAPK-dependent small HSP27 and &#945;B-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest.</a> American journal of physiology. Heart and circulatory physiology. 300 (5), H1669-H1677 (2011).</li><li>Clements, R. T., Cordeiro, B., Feng, J., Bianchi, C., Sellke, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rottlerin+increases+cardiac+contractile+performance+and+coronary+perfusion+through+BKCa+++channel+activation+after+cold+cardioplegic+arrest+in+isolated+hearts.">Rottlerin increases cardiac contractile performance and coronary perfusion through BKCa++ channel activation after cold cardioplegic arrest in isolated hearts.</a> Circulation. 124 (11 Suppl), S55-S61 (2011).</li></ol>

The authors have nothing to disclose.

前款协议细节的方法来评价心肌顿抑中学与心脏停搏有关全球缺血。在我们手中这个协议产生心脏功能近似〜减少40％（LVDP +/- DP / dt）的同在30分钟再灌注后的时间点最小的变化在心脏速率。由于心脏再灌注时复温及心功能的所有参数20分钟至30稳定之前减少在一个大大缩减的心脏速率初始时间点。冠脉流量一般在很大早期再灌注因充血增加，然后下降到小于〜20-30％，比下面的30分钟再灌注控制水...

心肌顿抑定义为缺血或缺血性侮辱与心脏1,2,3,4,5的时间延长了短暂的可逆降低收缩活动，尽管恢复充足的血流量。提出的方法是专门用于模拟临床上相关的缺血性损伤可导致收缩功能可逆损伤（ 即与心脏手术利用心脏停搏，局部缺血，咽峡炎， 等等的短时间相关缺血性损伤）。在对比严重缺血的研究（心肌梗死，坏死）此协议的开发是为了评估心肌功能恢复和心肌无组织损伤，重塑，和细胞死亡。大多数纸的讨论类似，使用低温和间歇性停搏交付心脏手术元素的标准心脏停搏协议。 心肌PROT大多数心脏手术期间挠度依靠停跳和体外循环。虽然停搏（CP）解决方案和策略差异很大（血液，晶体，冷，暖等 ）的最常见的元素是:1）高钾血症和/或其他战略逮捕的心脏舒张，从而限制了能源利用率从心肌收缩产生的， 2）低温减缓新陈代谢，帮助维持ATP和其他能源储备而被逮捕。目前心麻痹液提供保护心脏缺血对侮辱，否则将被证明是致命的。然而，在手术缺血损伤的心脏保护的策略是不完美的，并且所得到的轻度缺血性损伤可导致可逆的心脏收缩功能障碍，尽管足够的血液流量（心肌顿抑），酸中毒，心肌损伤，血管作用包括减少冠状动脉灌注和血管痉挛。 此协议不同从标准的离体心脏缺血模型评估心肌梗死和，它的计算结果较温和的缺血性损伤严重缺血可导致心脏功能受损下面简要缺血或心脏停搏相关缺血性侮辱。 （审查上的Langendorff灌流技术和I / R研究看6 - 8）。对于一般准则，与小鼠离体心脏相关的实验参数进行透彻的分析看萨瑟兰（E T） 人，2003年9这里介绍的方法详细介绍了必要的设备，试剂，步骤，策略和技巧，以可靠诱发惊人的小鼠心脏。小的修改是必要的，该技术适用于老鼠。 简要地离体小鼠心脏被的Langendorff经由递送高钾hypotherm的灌注与生理的Krebs-Henseleit缓冲液（KHB）约30分钟，随后冷保护心脏骤停IC停搏液。被捕之后，心功能恢复复温，并与科华生物的心脏再灌注期间监测。变化的心脏收缩功能的恢复程度可以评价以评估心脏剂和不同的心脏保护策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Cardioplegia Solution (St Thomas II)</td>
			<td ></td>
			<td ></td>
			<td >Symbol / Concentrations (mM)</td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td ></td>
			<td ></td>
			<td>NaCl; 110</td>
		</tr>
		<tr >
			<td >Potassium chloride</td>
			<td ></td>
			<td ></td>
			<td>KCl; 16</td>
		</tr>
		<tr >
			<td >Calcium Chloride</td>
			<td ></td>
			<td ></td>
			<td>CaCL2; 1.5</td>
		</tr>
		<tr >
			<td >Magnesium Chloride</td>
			<td ></td>
			<td ></td>
			<td>MgCL2; 16</td>
		</tr>
		<tr >
			<td >Sodium Bicarbonate</td>
			<td ></td>
			<td ></td>
			<td>NaHCO3; 10</td>
		</tr>
		<tr >
			<td >Krebs-Heinslet Buffer</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td ></td>
			<td ></td>
			<td>NaCl; 118</td>
		</tr>
		<tr >
			<td >Potassium Chloride</td>
			<td ></td>
			<td ></td>
			<td>KCl; 4.8</td>
		</tr>
		<tr >
			<td >Magnesium Sulfate</td>
			<td ></td>
			<td ></td>
			<td>MgSO4; 1.7</td>
		</tr>
		<tr >
			<td >Sodium Bicarbonate</td>
			<td ></td>
			<td ></td>
			<td>NaHCO3; 24.9</td>
		</tr>
		<tr >
			<td >Potassium Phosphate (monobasic)</td>
			<td ></td>
			<td ></td>
			<td>KH2PO4; 1.2</td>
		</tr>
		<tr >
			<td >Calcium Chloride</td>
			<td ></td>
			<td ></td>
			<td>CaCL2; 1.4</td>
		</tr>
		<tr >
			<td >Sodium Pyruvate</td>
			<td ></td>
			<td ></td>
			<td>Na pyruvate; 2</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td ></td>
			<td ></td>
			<td>C6H12O6; 6</td>
		</tr>
		<tr >
			<td >Balloon reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Corn Syrup</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Spaghetti</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Silicon Dispersion Gel</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >styrofoam block</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >lab oven/incubator ( 50C)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Langendorff Perfusion equipment</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Isolated perfused heart sytem (IH-SR (Hugo-Sachs) or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Data acquisition system (DSI, ADinstruments or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Heated water circulator</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cooling water circulator</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Perfusion pump capable of 2-30 ml/min</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Inline perfusion filters - 1 um glass fiber</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Pressure sensors and amplifiers for LVP and perfusion pressure</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Small graduated cylinder (~10 mL)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Small temperature probe and thermometer (Werner or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >perfusion resevoir (1L)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >cardioplegia resevoir (~200 mL)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >gas bubbler</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >95/5 O2/CO2 mix</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical tools and reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Metzenbaum and Potz surgical scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont size 5 forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >ketamine</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >xylazine</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >heparin</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >small clamp with soft sides to hold aorta (i.e. terminal clamp with taped ends)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Silk 2-0 and 4-0 sutures</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine isolated heart model of myocardial stunning associated with cardioplegic arrest

下面的协议是用来评估心脏功能受损或心肌顿抑以下中度缺血性侮辱。该技术是模拟与众多临床相关现象，包括心脏手术心脏停搏和体外循环，非体外循环冠状动脉搭桥术，移植，心绞痛，缺血短暂， 等相关的缺血性损伤。该协议提出了模拟低温高钾心脏停搏及再灌注啮齿动物的心专注于心肌收缩功能测定的一般方法。简言之，小鼠心脏灌注在Langendorff离模式下，仪表用脑室气球，和基线心脏功能参数被记录下来。稳定之后，心脏随后经历介绍输液的心脏停跳低温解决方案，以启动舒张逮捕。停跳间歇交付了2小时。心脏是再灌注和WÄRMEd可正常体温的温度和心肌功能的恢复进行监控。使用此协议的结果可靠郁闷心脏收缩功能无心肌组织损伤总值啮齿动物。

图1显示了从一个鼠标实验的典型结果。 LVP（红线），DP / DT（绿线）和温度（紫色线）的continusouly录得超过3〜小时。字母表示 - 基线测量，B，C，D，E - 交货停搏液，女 - 再灌注，G开始，H，I，J - 再灌注测量在兔冠状动脉流量。注意，郁闷LVDP和DP / dt的再灌注后比较基准。 图1B包括从录得超过〜2秒的数据。请注意，在LVP下降和DP / DT类似，略有减少人力资源和略有增加LV...

Watch this Scientific Journal Video about Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest at JoVE.com

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

心肌顿抑相关的小鼠离体心脏模型心脏停搏

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

murine-isolated-heart-model-myocardial-stunning-associated-with

Research

JoVE Journal

Medicine

11.6K 次观看.  Brown University. The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

视频: Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

科研

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

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.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

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

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

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.

从孤立的大鼠心脏的间质渗出物收集的倒置心脏模型

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

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

建立干细胞治疗心肌梗塞心力衰竭的猪模型

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

猪模型中经皮栓塞线圈部署的心肌梗死

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...