我们感谢卡塔琳娜哈伯兰进行图像编辑。资金来源:该资深作者收到了KKF格兰特从慕尼黑工业大学成立一个新的研究实验室。

注:在实施之前所提出的模型，相应的动物保护法必须进行协商和权限必须从当地政府获得。在这项工作中，所有实验符合进行与涉及动物研究的指导原则和保护动物的德国立法。实验是经当地动物保护委员会。 1.动物准备和皮瓣手术高程<ol style="margin-left: 40px;"><li>饲养动物在单个笼中，在22-24℃的室温，并在60-65％，采用12小时日 - 和 - 夜循环的相对湿度。让小鼠自由获取饮用水和标准实验室食物。始终保持在生存手术无菌条件。 </li><li>通过腹膜内麻醉的小鼠（IP）的注射的每个含90毫克/千克体重盐酸氯胺酮和25mg /10克体重0.1毫升生理盐水溶液公斤体重dihydroxylidinothiazine盐酸盐。麻醉是必要的动物的准备，外科手术和随后的显微镜。通过检查响应疼痛刺激，确认正确的麻醉。在麻醉的时候，涂抹于眼兽医软膏，以防止干燥。 </li><li>诱导足以麻醉后，脱毛用电动剃须刀的背面。此后，应用脱毛霜剃光区域以除去剩余的毛发。 </li><li>在的〜7-10分钟，脱毛霜的停留时间，准备工具，包括皮肤镊子和微型镊子，剪刀和微型剪刀，缝合材料和记号笔。消毒并通过应用两个螺钉与螺母在所述腔室的基座，并通过固定自粘泡沫（ 图1的AC）围绕所述腔室的对应的窗口，以保证腔室系统的气密性制备的钛室。 </li><li>从后面洗涤去除所有脱毛霜它关闭用温水。然后干燥和消毒用含醇溶液中的皮肤。 </li><li>鼠标放置在俯卧位置，并定义了背部的中线。掌握肌肤集中地，抬不起来，以创建一个倍（ 图2A）。通过使用任何种类的光源的反式照明，决定在哪里provisorily放置钛帧（ 图2B）。确保胸外侧动脉（LTA）颅和旋髂深动脉（DCIA）当然尾端通过该室的窗口中心的远端分支（ 图2B-D）。一旦该腔室的定位完成时，穿孔两个层的皮肤在折叠的钛架的后固定的最底部。现在除去钛架，鼠标放置在俯卧位，并在必要时重新背部的中线。 </li><li>概述瓣垂直于脊柱处开始以15×1的宽度与长度比率的中线1mm至导致横向基于随机灌注瓣。然后概括为2毫米延伸到对侧的额外的皮肤区域（ 图2C，D）。该区域采摘的镊子和缝合后的钛架而不可见瓣区域的干扰。 </li><li>下面最后的标记，切开皮瓣。在这样做时，横切两个LTA和DCIA和提升翼片（ 图2E）。没有必要对凝血横断的血管或结扎。 </li><li>通过先前制造的皮肤穿孔（ 图2F）将腔室的螺杆和升高的挡板的使用腔室的框架和孔5-0的框架的背面部分的腔室的框架内固定的皮褶既向前和向后间断缝合（ 图2G）。 </li><li>由5-0间断缝合装置缝合皮瓣的垂直肢体回到周围的皮肤（ <strong&gt;的图。 2H，Ⅰ）。 </li><li>切除皮肤侧的半圆形区域到2毫米， 也就是小皮带，在翼片的另一侧观察到瓣的脉管系统中。这允许通过对具有90 mm ​​2的表面上的腔室的观察窗直接观察。 </li><li>除去从横纹肌采用显微仪器和光学放大镜或显微镜的明胶样疏松蜂窝组织，以提高显微镜中的图像质量。尽量不要肌肉组织层内的血管损伤。 </li><li>最后，通过安装先前已赋予了泡沫的自粘合条前方的帧的对应物，颅和向后，沾湿外用双苯甲亚胺和盐水（ 图2J），翼片密封所述腔室。此后，应用该观察窗与使用的卡环的盖玻璃。尽量不要坑害玻璃下的空气气泡（ 图2K，L）。皮肤会坚持以盖玻片通过粘附力。 </li></ol> 2.活体荧光显微镜<ol style="margin-left: 40px;"><li>等待第显微镜分析为理想光学质量外科准备后24小时。这个分析表示显微镜的基线，并且重复在第3天，5，7和10在手术后。每一次，如在步骤1.2中所述麻醉的小鼠。将动物在一个特制的有机玻璃载体上的横向褥疮位置。通过这种方式，护翼附着于玻璃盖的肌肉组织层朝上，病房。 </li><li>注0.05毫升5％的绿色荧光右旋糖酐（分子量15万）和0.05毫升1％高荧光罗丹明系列染料的尾静脉或复古球的静脉丛。绿色荧光葡聚糖弄脏血液中的非细胞成分如应用于静脉内（ⅳ）。荧光罗丹明染色白细胞和血小板，可以是DIST从其他细胞和非细胞成分inguished。最后，双苯甲亚胺渍发射更多或更少的荧光，根据缩合的状态核组分。 </li><li>接着，将鼠标在显微镜下。确保在显微镜包括一个LED系统 - 对于470＆425纳米，365及490纳米，540和580nm的LED光束的组合以及相应的过滤器组（62，HE BFP / GFP / HcRed，取值范围为1:350-390纳米激发波长，拆分395纳米/ 402-448纳米; 2范围:460-488纳米，分为495纳米/ 500-557纳米; 3范围:567-602纳米，分为610纳米/ 615纳米到20无限罗丹明，范围。 :540-552纳米，分为560纳米，发射575-640纳米）。 </li><li>记录的静止图像和利用电荷耦合器件摄像机的运动图像，并将其保存在计算机硬盘上作进一步的离线分析。 </li><li>使用不同的目标（2.5X，NA（数值孔径）= 0.06室的全景图; 5X，NA = 0.16; 10X，NA = 0.32; 20X，NA = 0.50; 50X，NA = 0.55）对感兴趣的区域的记录。 </li><li>实际上，分 ​​瓣表面是可见通过腔室的窗口分成相等的高度， 即 ，一个近端，一个中心，和一个远端面积最远离血管流入（ 图3F）的三个领域。对于图像采集，请通过以下方式在各观察时间点: <ol><li>通过扫描图像的组织形象腔的近端从窗内使用5X物镜到远端。 </li><li>在皮瓣的每个区域，选择第二或第三级小动脉及其随行静脉用易于识别的分支模式。使用5X，10X或20X物镜，使arteriolovenular束的打印输出，以便容易地在整个观察期内重新定位的捆。 </li><li>随着20X物镜和50X的目标，进一步创纪录的5-6毛细血管字段和"细胞凋亡分别每翼片的区域"字段。记录有10X和20X的目标的所有图像用的蓝色光（荧光葡聚糖）和（若丹明）滤波器，而分别绿光记录有5X和50X物镜使用蓝色光（荧光葡聚糖）和白光（双苯甲亚胺）滤波器，图像。 </li></ol></li></ol>记录的数据3.分析注:通过使用计算机辅助图像分析系统定量离线记录 ​​的所有参数如下29。 <ol style="margin-left: 40px;"><li>的面积的测定非灌注，分别坏死组织（毫米2）。 <ol><li>使用记录有5X物镜（从2.6.1）的完整扫描腔室窗口图像和荧光葡聚糖测定非灌注分别坏死组织。用"AreaBo"功能来衡量非灌注暗区:边境非灌注区和计算米的区域米2用软件的面积测量算法。 </li></ol></li><li>测量微血管直径小动脉，毛细血管和小静脉（微米）。 <ol><li>加载视频或照片从记录的AV-束或毛细管领域到软件。为获得最佳效果使用蒸馏器或视频具有最高放大率，其中所述容器的明确的边界是清晰可见的。 </li><li>使用该软件的"DiamPe"功能，以确保测量的垂直进行到容器的壁上。这样做的标记在容器的壁并与所述第三点击标记容器的垂直直径两点。该软件将在微米执行测量。 </li><li>重复在同一个容器的所有记录的测量值在同一个地方。测量多个毛细血管累积平均直径。 </li></ol></li><li>在动脉红细胞（RBC）的速度分析，毛细血管一次静脉（毫米/秒）。 <ol><li>对于动脉红细胞（RBC）的速度（毫米/秒）的测量使用该软件的"VeloLSD"功能，并选择在荧光葡聚糖窗口相同的容器的量，直径进行了测量。 </li><li>它与使用线移位方法，它是一个单独的血管内的灰度级图案的位移（毫米）的测定的基础上，在一段时间（秒）的计算机辅助图像分析系统进行分析。 </li></ol></li><li>在测量血管体积血流量（PL /秒）。 <ol><li>计算体积血流量（复/秒）在动脉，静脉和毛细血管从红细胞速度和容器的横截面面积（π* R 2）根据Gross和Aroesty，即:Q = V *π* R 2中的方程假定圆柱形容器的形状30。 </li></ol></li><li>测定红细胞灌注毛细血管的功能性毛细血管密度（营养灌注:厘米/厘米<sup&gt; 2）。 <ol><li>加载一个记录荧光毛细管场20X物镜放大倍数到软件中。使用"DensLA"功能来衡量RBC灌注微血管的功能性毛细血管密度。 </li><li>跟踪毛细血管灌流用光标和标记血管灌注。不标记非灌注毛细血管，小动脉和小静脉。该软件将测量灌注毛细血管厘米/厘米2的功能性毛细血管密度。重复上述步骤，为其他记录的毛细管领域。 </li></ol></li><li>测量微血管（微血管重塑），血管生成的早期迹象，如芽和芽，形成与新形成的微血管迂曲。 <ol><li>负载录制的视频或荧光毛细领域的帧到软件中。识别gyrose船只和使用该软件的"TorqIx"功能。跟踪的过程中，明确船舶流量和点击右键结束。该软件会画一条直线的直接方式，并设置它与跟踪路径的关系。 注:留意血管的迹象，如芽，芽和新形成的毛细血管，通常垂直发出的预先存在的毛细血管。衡量这些新形成的血管的密度在3.5步描述。 </li></ol></li><li>鉴定为凋亡性细胞死亡的特性，如核固缩，碎裂和/或边集（个/ mm 2）的。 <ol><li>加载双苯酰亚胺录制的视频（50X物镜）到软件。使用"DenseNA"功能。根据提到的特性，如核固缩，碎裂和边集用左键单击标记凋亡细胞。 </li><li>标志认证的所有特性细胞在整个视频后，点击"N / A"的软件，该软件会自动计算所有标记的​​细胞和整个区域划分它。其结果将是凋亡个/ mm 2。 </li></ol></li><li>分析白细胞粘附到血管内皮的代表炎症。白细胞的间歇加入（滚动的白细胞;粘附&lt;30秒:辊子数/ mm 2的内皮细胞表面）和白细胞的牢固附着（粘附白细胞;粘附&gt; 30秒:贴/ mm 2的内皮细胞表面的数量）。 <ol><li>通过计数罗丹明数分析的炎症反应标记的白细胞附着于毛细血管后小静脉的内皮细胞衬里为≥30秒的时间段（"贴"）和间歇地附着白细胞（&lt;30秒，"辊"） 。微静脉白细胞粘附表示为每mm 2的内皮细胞表面。 </li></ol></li><li>测量血管内和间质灰度级为微血管渗漏（大分子外渗E = E1 / E2）的参数。 <ol><li>评估大分子升eakage作为微血管通透性静脉注射荧光右旋糖酐后的参数。光密度确定的多个灰度级中的组织容器（E2）的边缘的无细胞血浆中直接相邻的毛细血管壁（E1），以及。然后计算大分子外渗（E）为E1 / E2 31的比率。 </li></ol></li></ol> 4.术后护理<ol style="margin-left: 40px;"><li>返回的动物在一个单独的笼子，以避免其他动物的公司，直到完全恢复。不要让动物无人看管，直到它已经恢复了足够的意识，保持胸骨斜卧。每天监测动物的出血，局部和全身感染的迹象和腔室的位置。 </li><li>通过观察马达的活性，体重，痛苦的迹象，耐受性敷料和自动切割评价动物的一般状态。 </li><li>请将鼠标1每笼以避免穆图人操纵室。在疼痛的迹象，适用丁丙诺啡在0.05-0.1毫克/千克体重的剂量，皮下8小时一班。 </li></ol> 5.安乐死和皮褶庭外植<ol style="margin-left: 40px;"><li>在第10天，使用麻醉药品（150毫克/千克戊巴比妥钠）的过量牺牲动物。 </li><li>取出室，品尝皮瓣组织进行免疫组化和分子蛋白分析。对于传统的组织学（福尔马林）和可量化的蛋白质分析（液氮或干冰）样品组织。 </li></ol>

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M., Intaglietta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+microhemodynamics+in+ischemic+skin+flap+tissue+by+intravital+microscopy.">Quantitative assessment of microhemodynamics in ischemic skin flap tissue by intravital microscopy.</a> Ann Plast Surg. 43, 405-414 (1999).</li><li>Roesken, F., Sch&#228;fer, T., Spitzer, W. J., Vollmar, B., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+the+microcirculation+of+osteomyocutaneous+flaps+using+fluorescence+microscopy.">In vivo analysis of the microcirculation of osteomyocutaneous flaps using fluorescence microscopy.</a> Br J Plast Surg. 52, 644-652 (1999).</li><li>Harder, Y., Amon, M., Erni, D., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+ischemic+tissue+injury+in+a+random+pattern+flap:+a+new+mouse+model+using+intravital+microscopy.">Evolution of ischemic tissue injury in a random pattern flap: a new mouse model using intravital microscopy.</a> J Surg Res. 121, 197-205 (2004).</li><li>Harder, Y., Contaldo, C., Klenk, J., Banic, A., Jakob, S. M., Erni, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+with+monophosphoryl+lipid+A+improves+survival+of+critically+ischemic+tissue.">Preconditioning with monophosphoryl lipid A improves survival of critically ischemic tissue.</a> Anesth Analg. 100, 1786-1792 (2005).</li><li>Rezaeian, F., 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=Erythropoieton+protects+critically+perfused+flap+tissue.">Erythropoieton protects critically perfused flap tissue.</a> Ann Surg. 248, 919-929 (2008).</li><li>Harder, Y., 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=Erythropoietin+reduces+necrosis+in+critically+ischemic+myocutaneous+tissue+by+protecting+nutritive+perfusion+in+a+dose-dependent+manner.">Erythropoietin reduces necrosis in critically ischemic myocutaneous tissue by protecting nutritive perfusion in a dose-dependent manner.</a> Surgery. 145, 10-1016 (2009).</li><li>Rezaeian, F., 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=Erythropoietin-induced+upregulation+of+endothelial+nitric+oxide+synthase+but+not+vascular+endothelial+growth+factor+prevents+musculocutaneous+tissue+from+ischemic+damage.">Erythropoietin-induced upregulation of endothelial nitric oxide synthase but not vascular endothelial growth factor prevents musculocutaneous tissue from ischemic damage.</a> Lab Invest. 90, 40-51 (2010).</li><li>Rezaeian, F., Ong, M. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylcysteine+attenuates+leukocytic+inflammation+and+microvascular+perfusion+failure+in+critically+ischemic+random+pattern+flaps.">N-acetylcysteine attenuates leukocytic inflammation and microvascular perfusion failure in critically ischemic random pattern flaps.</a> Microvasc Res. 82, 28-34 (2011).</li><li>Rezaeian, F., 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=Ghrelin+protects+musculocutaneous+tissue+from+ischemic+necrosis+by+improving+microvascular+perfusion.">Ghrelin protects musculocutaneous tissue from ischemic necrosis by improving microvascular perfusion.</a> Am J Physiol Heart Circ Physiol. 302, 603-610 (2012).</li><li>Rezaeian, F., 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=Long-term+preconditioning+with+Erythropoietin+reduces+ischemia-induced+skin+necrosis.">Long-term preconditioning with Erythropoietin reduces ischemia-induced skin necrosis.</a> Microcirculation. , (2013).</li><li>Harder, Y., 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=Heat+shock+preconditioning+reduces+ischemic+tissue+necrosis+by+heat+shock+protein+(HSP)-32-mediated+improvement+of+the+microcirculation+rather+than+induction+of+ischemic+tolerance.">Heat shock preconditioning reduces ischemic tissue necrosis by heat shock protein (HSP)-32-mediated improvement of the microcirculation rather than induction of ischemic tolerance.</a> Ann Surg. 242, 869-878 (2005).</li><li>Tobalem, 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=Local+shockwave-induced+capillary+recruitment+improves+survival+of+musculocutaneous+flaps.">Local shockwave-induced capillary recruitment improves survival of musculocutaneous flaps.</a> J Surg Res. 184, 1196-1204 (2013).</li><li>Lindenblatt, N., Calcagni, M., Contaldo, C., Menger, M. 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为了减少缺血并发症，从而改善临床结果，在危重灌注皮瓣组织病理生理过程的更详细的知识是必需的。的模仿急性缺血持续的新动物模型的发展，因此强制性的。因此，我们能够发展一种容易再现的和可靠的模型允许肌肉和皮肤脉管系统的各种参数，可以与所采样的皮瓣组织的免疫组织化学和分子生物学分析相关联的重复的形态，动态的和功能性的实时评价。 在外科手术?...

暴露腱，骨和植入材料在整形后的覆盖依赖于使用折翼。甲瓣是转印在其血管蒂，保证动脉流入和静脉流出的组织块。尽管有广泛的专业知识和各种皮瓣被转移的可用性，缺血引起的并发症，从伤口裂开总组织损失仍然遇到。而由二级意向保守治疗和愈合可以轻微的组织坏死后预计，显著皮瓣坏死，通常需要再次手术，包括清创，伤口空调和二次重建​​。这增加了发病率，延长住院时间，从而导致增加的医疗费用。 护翼与脉管的远端区域从动脉流入最远端未定义的图案或随机地灌注区域特别容易出现局部缺血损伤。 ACCO<html> rdingly，大量的实验和临床研究已经评估坏死的发展中都，轴型皮瓣（定义血液供应），以及随机图案的侧翼（未定义的血液供应）1-3。主要结论通常是基于坏死区域的大小的宏观评价。为了评估产生的原因和组织坏死的机制更详细地说，一些研究集中于微循环的分析。不同的技术已被用于测量组织灌流，包括用极谱电极4-5组织氧分压的分析，以及使用激光多普勒血流仪6-7，染料扩散8，和微球体9-10血流量的测量。这些技术中，然而，只允许用于测量组织灌流的间接的参数，并不的翼片的兴趣的个体区域内使microhemodynamic过程的任何形态分析。 吨"&gt;迪森已知是谁使用一个透明的腔，用于延长在体内的研究，这是他在家兔11进行第一1943 -约20年后- Algire是第一适应这样的透明腔室可以适用在小鼠中，以研究微植入肿瘤细胞12的行为。由于这样的事实，将小鼠所谓松弛皮肤的动物和在接下来的几年一些技术精炼后，莱尔和同事们能够适应这样的背皮褶腔显影更小和更轻的钛腔内，该腔室使评价用活体荧光显微镜，一种技术，它允许若干形态和微循环特性及其随时间的不同的生理和病理生理条件下的变化的直接的和重复的可视化，这样如缺血再灌注损伤13。 在PE的调查正常和病理状态下的皮肤，肌肉和骨皮瓣rfusion两个趋势出现了:首先，不使用背皮褶室，如鼠标14带蒂皮瓣耳的"急性"啪啪机型，基于横向岛状皮瓣在15仓鼠和老鼠16带蒂复合组织瓣。二，"慢性"啪啪模型，其中一个背皮褶室允许重复的微循环襟翼的组合分析了与活体荧光显微镜几天。它包括一个随机灌注肌皮瓣被集成在鼠标17的皮褶室。其宽度与长度的比值被选择了的急性缺血持续的情况下始终将导致〜50％皮瓣坏死组织瓣仰角后10至14天。组织坏死这种再现的程度使得两者，保护（ 即发展LES进一步评估s坏死）和有害的因素（ 即，在瓣病理生理发展，越来越坏死）。在过去几年里，几个实验出版物展示不同的产前，围绝经期和后期调理过程，包括组织保护性物质18-24的管理和生理应激，如热25和冲击波26的本地应用程序的效果，已经出现。 坏死，微血管形态和微循环的参数的定量分析还可以被关联到免疫组化分析和蛋白质分析。不同的蛋白质和分子，包括血管内皮生长因子（VEGF），一氧化氮合成酶（NOS），核因子κB（NF-κB）和热休克蛋白（HSP-32:血红素加氧酶-1（HO-1）和HSP- 70）已经显示出在组织保护的作用。在此基础上腔皮瓣模型，两处修改，已经开发了奥德r以在植皮愈合​​27和血管生成发展的带蒂皮瓣轴型灌注28分析新生血管和微循环。我们提出了一个可重复和可靠的模型，其中包括在老鼠皮褶室的缺血性挑战肌皮瓣。这种模式使可视化微循环及血流动力学通过活体落射荧光显微镜和定量。

<table><tbody><tr><td>C57Bl/6 mice, 6-8 w, 20-22 g</td><td>Charles River</td><td></td><td></td></tr><tr><td>Depilation cream</td><td>Balea</td><td></td><td>Any depilation cream</td></tr><tr><td>Titanium chamber</td><td>Irola</td><td>160001</td><td>Halteblech M</td></tr><tr><td>Custom-made Plexiglass mounting frame</td><td></td><td></td><td>Frame to secure chamber to avoid chamber movement due to the animal&#039;s respiration.</td></tr><tr><td>Slotted cheese head screw</td><td>Screws and More</td><td>842210</td><td>DIN84 M2x10</td></tr><tr><td>Hexagon full nut</td><td>Screws and More</td><td>93422</td><td>DIN934 M2</td></tr><tr><td>Snap ring</td><td>Schaefer-Peters</td><td>472212</td><td>DIN472 J12x1,0</td></tr><tr><td>Cover glass</td><td>Volab</td><td></td><td>Custom-made cover glass, 11.8 mm in diameter.</td></tr><tr><td>Fixing foam</td><td>tesamoll</td><td>05559-100</td><td>tesamoll Standard I-Profile</td></tr><tr><td>Ketamine hydrochloride</td><td>Parke Davis</td><td></td><td>Ketavet&amp;reg;</td></tr><tr><td>Dihydroxylidinothiazine hydrochloride</td><td>Bayer</td><td></td><td>Rompun&amp;reg;</td></tr><tr><td>Buprenorphin</td><td>Essex Pharma</td><td></td><td>Temgesic&amp;reg;</td></tr><tr><td>Saline 0.9%</td><td></td><td></td><td></td></tr><tr><td>Desinfection alcohol</td><td></td><td></td><td></td></tr><tr><td>Vicryl 5-0</td><td>Ethicon</td><td>V 490 H</td><td></td></tr><tr><td>Ethilon 5-0</td><td>Ethicon</td><td>EH 7823 H</td><td></td></tr><tr><td>1 ml syringes</td><td></td><td></td><td></td></tr><tr><td>Surgical skin marker with flexible ruler</td><td>Purple surgical</td><td>PS3151</td><td>Any surgical skin marker and flexible ruler</td></tr><tr><td>Pointed scissors</td><td></td><td></td><td></td></tr><tr><td>Micro-Scissors</td><td></td><td></td><td></td></tr><tr><td>Normal scissors</td><td></td><td></td><td></td></tr><tr><td>2 clamps</td><td></td><td></td><td></td></tr><tr><td>Fine anatomic forceps</td><td></td><td></td><td></td></tr><tr><td>Micro-forceps</td><td></td><td></td><td></td></tr><tr><td>Hex nuter driver</td><td>wiha</td><td>1018</td><td></td></tr><tr><td>Screwdriver</td><td>wiha</td><td>685</td><td></td></tr><tr><td>Snap ring plier</td><td>Knipex</td><td>4411J1</td><td>12-25 mm</td></tr><tr><td>Wire cutter</td><td>Knipex</td><td>70 02 160</td><td>Wire cutter is used to cut screws short; 160 mm</td></tr><tr><td>Trans-illumination light</td><td>IKEA</td><td>501.632.02</td><td>LED light Jansj&amp;ouml;; any light&amp;nbsp;</td></tr><tr><td>Magnification glasses</td><td></td><td></td><td></td></tr><tr><td>Intravital microscope</td><td>Zeiss</td><td>490035-0001-000</td><td>Scope.A1.Axiotech</td></tr><tr><td>LED system</td><td>Zeiss</td><td>423052-9501-000</td><td>Colibri.2</td></tr><tr><td>LED module 365nm</td><td>Zeiss</td><td>423052-9011-000</td><td></td></tr><tr><td>LED module 470nm</td><td>Zeiss</td><td>423052-9052-000</td><td></td></tr><tr><td>LED module 540-580nm</td><td>Zeiss</td><td>423052-9121-000</td><td></td></tr><tr><td>Filter set 62 62 HE BFP + GFP + HcRed</td><td>Zeiss</td><td>489062-9901-000</td><td>Range 1: 350-390 nm excitation wavelength, split 395 nm / 402-448 nm; range 2: 460-488 nm, split 495 nm / 500-557 nm; range 3: 567-602 nm, split 610 nm / 615 nm to infinite.</td></tr><tr><td>Filter set 20 Rhodamine</td><td>Zeiss</td><td>485020-0000-000</td><td>540-552 nm, split 560 nm, emission 575-640 nm</td></tr><tr><td>2.5X objective NA=0.06</td><td>Zeiss</td><td>421020-9900-000</td><td>A-Plan 2.5X/0.06</td></tr><tr><td>5X objective NA=0.16</td><td>Zeiss</td><td>420330-9901-000</td><td>EC Plan-Neofluar 5X/0.16 M27</td></tr><tr><td>10X objetive NA=0.30</td><td>Zeiss</td><td>420340-9901-000</td><td>EC Plan-Neofluar 10X/0.30 M27</td></tr><tr><td>20X objective NA=0.50</td><td>Zeiss</td><td>420350-9900-000</td><td>EC Plan-Neofluar 20X/0.50 M27</td></tr><tr><td>50X objective NA=0.55</td><td>Zeiss</td><td>422472-9960-000</td><td>LD Epiplan-Neofluar 50X/0.55 DIC 27</td></tr><tr><td>ZEN imaging software</td><td>Zeiss</td><td></td><td>ZenPro 2012</td></tr><tr><td>CapImage</td><td>Dr. Zeintl</td><td></td><td></td></tr><tr><td>Fluorescein isothiocyanate-dextran</td><td>Sigma-Aldrich</td><td>45946</td><td></td></tr><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Harmful if swallowed; causes severe skin burns and eye damage, may cause respiratory irritation.</td></tr><tr><td>Rhodamine 6G chloride</td><td>Invitrogen</td><td>R634</td><td>Harmful if swallowed; may cause genetic defects; may cause cancer; may damage fertility or the unborn child.</td></tr><tr><td>Pentobarbital</td><td>Merial</td><td></td><td>Narcoren&amp;reg;</td></tr></tbody></table>

ischemic tissue injury in the dorsal skinfold chamber of the mouse: a skin flap model to investigate acute persistent ischemia

尽管有深厚的专业知识和先进的手术技术，缺血引起的并发症，从伤口裂开了广泛的组织坏死依然存在，特别是在皮瓣重建手术。多个实验皮瓣模型已经被开发来分析潜在原因和机制，并研究治疗策略来防止缺血性并发症。大多数型号的限制因素是缺乏可能性，直接和反复可视化微血管构筑和血流动力学。该协议的目标是提出一个行之有效的小鼠模型，这些挂靠前面提到的缺少的元素。难等人已开发出一种肌皮瓣的模型与经历急性持续缺血导致〜50％坏死后10天，如果保存未处理的随机灌注模式。随着活体落射荧光显微镜的帮助下，该室模型允许重复的可视化形态及利息随时间不同区域的血流动力学。相关的过程，如细胞凋亡，炎症，微血管渗漏和新生血管能进行调查和相关的免疫组化和分子蛋白分析。迄今为止，该模型已被证明可行和可重复性在几个发表的实验研究中调查的前，围缺血性挑战的组织的作用和后处理。

坏疽 这种模式的主要终点-组织坏死以下瓣仰角（ 即，诱导急性持久性缺血） -重复地测量和图示的宏观如图3历时10天。皮瓣坏死的最终划界通常在5天和第7天之间发生手术后，其特点是一个红色条纹的血管舒张和微血管重塑的， 也就是说 ，区域，近端重要和护翼的远端坏死区（ 图3D-F之间的显影）。在灌注良好的近端，所...

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Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

鼠背皮褶室呈现的可视化窗口的肌皮瓣急性缺血持续性的区域。活落射荧光显微镜许可证微血管和血流动力学的定量直接和重复评估。形态和血流动力学的结果可以进一步与组织学和分子分析。

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

ischemic-tissue-injury-dorsal-skinfold-chamber-mouse-skin-flap-model

Research

JoVE Journal

Medicine

16.6K 次观看.  Technische Universität München. 鼠背皮褶室呈现的可视化窗口的肌皮瓣急性缺血持续性的区域。活落射荧光显微镜许可证微血管和血流动力学的定量直接和重复评估。形态和血流动力学的结果可以进一步与组织学和分子分析。 

视频: Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

科研

医学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

生物学

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 ...

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.

肠缺血再灌注损伤的小鼠模型

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

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

小鼠使用表面垫心电图心肌缺血再灌注损伤的确认

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

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 ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...