We thank Thomas Graf for providing us with the LysM-eGFP mice.

所有处理活的动物实验均按照所有相关动物的使用和保护准则和条例进行。 1.荧光记者小鼠的选择<ol><li>使用6至12周龄LysM结构-eGFP的11只小鼠（无偏爱或者男性或女性）。 注意:使用各种细胞特异性荧光报道小鼠能够在体内不同的免疫细胞的可视化。在该菌株中，循环嗜中性粒细胞（GFP 喜细胞），循环单核细胞（GFP LO细胞）和真皮的巨噬细胞（GFP LO细胞）可以被可视。与所使用的摄像参数，只能从GFP阳性嗜中性粒细胞的明亮的信号将被检测到。 注:适合这类肌肤的影像学研究的免疫细胞特异性荧光记者小鼠品系的清单可以参考8被发现。 注意:强烈建议小白鼠用于成像，猪人泰德小鼠更容易产生光损伤。这是因为着色耳朵皮肤是多少（指示组织燃烧）激光诱导斑点更敏感。其结果是，中性粒细胞募集和积累可能即使在稳态8,12被观察到。 </li><li>保持在具有12小时光照 - 黑暗周期无特定病原体（SPF）的条件下的小鼠。 </li></ol> 2.鼠标麻醉<ol><li>麻醉小鼠用腹腔注射氯胺酮-赛拉嗪（8微升克-1体重）的，溶解于无菌水中15毫克mL的混合物-1氯胺酮和1mg毫升-1甲苯噻嗪组成。 </li><li>放置在加热垫的鼠标以保持其体温在37℃下在整个制备过程。通过观察一个缺乏脚趾捏反射检查有足够的麻醉。 注意:在第一个小时后，后续季度剂量的麻醉剂将需要被皮下施用的ð将持续大约每0.5小时。晶须或尾部的抽搐还可以指示麻醉穿着关闭，并且需要补足。 </li><li>使用对眼睛的眼科润滑剂，以防止干燥时的麻醉下。 </li></ol> 3.脱毛<ol><li>仔细申请脱毛霜向背侧小鼠耳的使用棉尖涂药器的上三分之二。 </li><li>在彻底，但温和的方式使用湿棉尖涂药除去霜前3分钟 - 等待2。 注意:不要让脱毛膏停留在小鼠耳太久，因为这可能会诱发炎症13,14。 </li></ol> 4.诱导缺血再灌注损伤的<ol><li>采用镀金，N42级的钕磁铁，12毫米直径×2毫米厚，并用约3000高斯的评价诱导缺血小鼠耳部皮肤。 注意:在这种情况下，磁体的微凹面杰诺TES其北极。 </li><li>插槽磁铁成单个的塑料导轨。 注意:塑料导轨用来缓解高强度磁铁的位置和分离。由于其强大的磁力破损但抵抗力低，不要靠近各个磁体放置对方或其他金属。如果他们拉向对方可能会发生破损和分裂。 </li><li>第一（背部）磁体，使得只有边缘与所述第二（腹侧）磁体（ 图3a）接触的位置 注意:这可以防止磁铁从卡扣在一起它们已被正确地定位之前。 </li><li>定位两个磁体，使得所述腹磁铁平躺在耳朵上（ 图3a）。 注意:缺血诱导之前，确保鼠标保持在37℃和足够的麻醉维持（参见步骤2.2）。 </li><li>一旦准备就绪，精心让磁铁走到了一起（ 图3a </STRONG&gt;）。 注意:对于成像目的，夹紧只使红外和非红外区域可被观察到的耳的一半。 </li><li>缺血1.5小时后，通过加捻彼此磁体远离使用塑料导轨，从而允许再灌注发生移除磁体。 注意:必须小心，以防止耳从当磁体被置于压痕。如果肉眼可见的主要血管立即再灌流（ 即血液立即充满血管）的磁体被删除后不完全缺血是显而易见的。虽然再灌注不去除磁铁后立即发生，血管闭塞只是暂时的。因此，当务之急是要准备老鼠耳朵尽可能迅速地成像。 </li></ol> 5.注射血管贴标代理<ol><li>磁铁取出后，立即静脉给药（通过眼眶或尾静脉注射），伊文思蓝（10毫克毫升-1在PBS或盐水; 1μL摹-1体重）或选择的其他血管标记剂。 注意事项:注射前，确保有足够的麻醉仍然是通过执行温和的捏脚趾保持。 </li></ol> 6.对成像平台耳朵放置<ol><li>切2个胶带长度1.5厘米和1.8厘米宽的。 </li><li>使粘合剂双方同时留下1毫米左右的粘合剂沿其宽度粘在一起。 </li><li>切胶带两种，纵向，耳朵平台上的狭缝内适应其位置。 </li><li>插入约一半此遮蔽胶带通过狭缝，使得粘合剂面朝上。 </li><li>加热垫的鼠标的位置，这样，将被成像的耳朵旁边的遮蔽胶带条。 </li><li>通过使用两个PBS浸湿棉尖涂药，轻轻按压胶条的耳朵。 </li><li>使用带为指导，把小鼠耳通过狭缝，同时调整鼠标朝舞台更近。 </li><li>以除去胶带，第一添加PBS一滴以减少带的粘接性。 </li><li>用细的画笔轻轻地分开遮蔽胶带小鼠耳。 </li><li>通过轻轻滚动过耳湿润的棉尖涂药拼合靠近耳部平台的耳朵。 </li><li>放的PBS盖玻片下方的下降（其被保持在使用润滑脂盖玻片夹持位置; 图2），轻轻地将其放置在耳。如有必要，加满PBS多。 注意:盖玻片​​夹持成像期间增加了稳定性。 </li><li>插入直肠温度探头，并根据制造商的说明将电线连接到加热系统。 注:设置体加热垫的温度升高到37℃，并在耳阶段平台至35℃。 </li></ol> 7.多光子显微镜套装和成像参数注意:此协议使用单光束，多光子显微镜可调（680 - 1080 nm）的钛:萨激光（3.3瓦，800纳米，140飞秒脉冲长度; 80 MHz的重复率）用20X的水物镜（NA = 1.0），用于活体成像研究。 <ol><li>打开图像处理软件。 </li><li>根据制造商的说明校准激光。 </li><li>调整激发波长到950纳米。 注:GFP和伊文思蓝可在950 nm的同时兴奋。 </li><li>预览，使用以下设置:500微米2 scanfield，505点¯x505像素的分辨率，和800赫兹的在一个单一的行扫描的扫描频率。点击"预览"。 </li><li>切换衰减器（激光）电源。确保所有必需信号被拾取，而不摄像视场暴露于过量的激光的功率，这可能会引起热损伤。 注:在低功率衰减器启动，如果增加西尼亚l为暗淡。嗜中性粒细胞将不存在从在早期时间间质点 - 再灌注后，巨噬细胞可被用作一个量规来确定所需的最小的功率，因为​​前者比嗜中性粒细胞的调光器。 注意:如果该步骤是在第一时间内完成，调整在非缺血区，在这里完整血管完整性预期的设置，将促进衰减器功率的设置。在随后的实验中，这些设置不需要太多修饰，除非激光功率是不稳定的。 </li><li>调整PMT设置。 注意:检查与制造商的PMT的最大最佳增益电压。设定PMT电压超过推荐阈值将导致更高的信噪比。一般建议的PMT增益电压设置为推荐的门槛，增加了功率衰减器根据需要应信号过于模糊。 </li><li>选择是在接近成像区域缺血的边缘，其特征在于大量的伊文思蓝的泄漏。 </li><li>收集GFP和使用五十零分之五百二十五带通（BP）和655/40 BP滤波器，分别Evans蓝信号。对于二次谐波产生（SHG）信号真皮隔间内的胶原纤维，用一个四十二分之四百七十五BP滤波器。 </li><li>创建一个文件夹来保存图像的格式是可用的图像分析软件兼容。 </li><li>为了获得，使用以下设置:500微米2 scanfield，505点¯x505像素分辨率和扫描频率400赫兹在一个单一的线扫描。 </li><li>一个100微米的Z堆叠为4微米的步长可重复获得随着时间的推移，在1分钟的间隔，以监测中性粒细胞浸润。 注意:尤其对于白细胞（ 例如，中性粒细胞）具有较高的迁徙速度，即长于1分钟，可能会导致细胞跟踪分析期间困难的间隔。在这种情况下，用户既可以降低交流的厚度quisition堆叠或增大扫描频率。 注意:当在缺血区的需要采集栈可以显示为压缩（或者其特征在于较高的SHG强度）的结果小，随后再灌注会导致引起该耳朵膨胀大规模炎症。在Z方向显著漂移预期。这样，一个大的Z堆叠的采集是必要以容纳漂移。 </li><li>整个成像，补足PBS和水经常保持耳湿润和物镜浸泡在水中。 注:一个典型的实验一般持续2 - 4小时。根据不同的实验设计，并配上良好控制麻醉制度，延长成像的持续时间是可能的。 </li></ol> 8.终止实验<ol><li>根据该机构的机构动物护理和使用委员会（IACUC）通过二氧化碳窒息安乐死鼠标PRocedures。 注:由该机构的法规，规章和准则确定的批准方法总是安乐死鼠标。如果需要重复成像，按住鼠标加热垫，直到麻醉消退。一旦鼠标已经恢复足够的意识，是移动，鼠标返回到笼子里。 </li></ol> 9.图像分析注意:从成像实验产生的数据可以通过不同的软件包进行可视化。 <ol><li>打开成像分析软件。 </li><li>在超越模式，导入文件（打开→选择想要的文件夹→所有文件，点击"打开"）。 </li><li>如果默认的颜色不适用编辑伪彩色（编辑→显示调整→在通道选项卡，选择从调色板中所需的颜色）。 </li><li>调整亮度，对比度和背景（编辑→显示调整→切换最大值和最小值）。 </l我&gt; <li>确保文件的尺寸是否正确（编辑→图像属性→在几何选项卡，勾选体素的大小。在这个协议中，X = 0.99，Y = 0.99和Z = 4）。 注意:通过由像素分辨率除以scanfield尺寸派生X和Y。 Z是每个切片之间的厚度。 注意:这些数据集的输出可以表示为最大投影电影。 注意:需要跟踪分析，以了解它们的体内功能完全表征细胞活性和相互作用。使用该软件可当场功能细胞追踪详细说明可在协议参考15中找到。 注:有量化白细胞迁移的方法很多，详情可以在评论文章中引用16被发现。 </li></ol>

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The authors have nothing to disclose.

意义 IR为皮肤褥疮的主要原因之一。褥疮的早期阶段（I和II）描述的人的皮肤的状态（相对于底层皮下组织和肌肉）。然而，免疫病因的了解仍然缺乏。在这里，我们存在于小鼠耳部皮肤一个简单而强大的IR模式，以消除这一差距。我们通过夹紧两个磁铁和随后的小鼠耳研究磁铁去除（再灌注）后，下游的免疫反应模拟缺血。通过使用磁铁来产生一个固定的时间周期上每个耳朵?...

时，有一个短暂缺氧由于随后组织（再灌注）的随后重新氧合血流量（缺血）的阻塞发生缺血再灌注损伤（IRI）。在皮肤中，缺血 - 再灌注（IR）被认为是起作用的因素，压力性溃疡的病理生理学，其中长期卧床易患长期医院病人受伤的一个。在这些患者中，无论是在皮肤和下面的肌肉经常暴露于施加在骨突出的区域重压力，导致局部损伤的是，如果不进行治疗，可能会变得坏死1。 参与了IRI的损失是双重的。期间缺血，血管的闭塞导致的氧气输送的急剧下降到组织。这导致的ATP和pH值，其中失活参与细胞代谢ATP酶的减少。反过来，细胞钙含量秒杀，并强调或损坏Ç厄尔发生凋亡或坏死2。细胞内的内容，或者损害相关分子模式（湿气）的发布，像HMGB1，有助于炎症反应3。再灌注过程中出现的第二个侮辱。虽然氧和pH水平在再灌注期间恢复，这会导致产生的活性氧（ROS），从而导致细胞内脂质，DNA和蛋白质的氧化。因此，促炎介质被激活，从而衬托涉及免疫细胞向炎症部位2招募次级炎症反应。而生化事件导致对炎症反应的级联已经很好地描述，所述免疫细胞活动的空间和时间调节都不能很好地理解。 在这里，我们描述了如何使用简单的磁性定位鼠耳皮肤一个强大的IR模式。再加上多光子活体成像（MP-IVM），我们建立了一个模型来研究再灌注发生后发生的体内炎症反应。开发和利用这种技术的基本原理是试图实时了解两者间质和浸润细胞对IR的反应。 使用夹紧技术在皮肤侧面的IR的现有模型是高度侵入性的，因为它们需要在皮肤侧面钢板的手术植入，使其低于理想免疫学研究4。类似的非侵入性的夹紧技术已在小鼠皮肤侧翼5,6进行了描述。但是，由于在该方法中，活体成像成分的掺入，我们改为选择耳朵皮肤作为目标的IR部位，因为它绕过由于呼吸运动和成像7,8-期间提供稳定性。此外，跨越间质白细胞亚群是耳朵皮肤和皮肤侧面之间是相同的，虽然数字和比例可能会略有变化9。从而，耳朵皮肤表示理想成象部位。 此外，从这些IRI模型检索大多数数据是有限的，以宏观评价（溃疡分级）和端点炎症指标10微观分析。利用该模型，嗜中性粒细胞中的荧光报道小鼠的皮肤再灌注后的细胞反应的实时可视化被启用。以前发表的活耳成像模型被利用8与另外的修饰（ 图1，图2）。

<table border="0" cellpadding="0" cellspacing="0" width="1506">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Mice strains</td>
			<td style="width:333px;"></td>
			<td style="width:125px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Lysozyme-GFP C57BL/6</td>
			<td style="width:333px;">Thomas Graf, Center for Genomic Regulation</td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">C57BL/6-C2J</td>
			<td style="width:333px;">Jackson Laboratories</td>
			<td style="width:125px;">000058</td>
			<td>To be crossed with Lysozyme-GFP to generate albino Lysozyme-GFP for skin imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viaflex 0.9% (wt/vol) saline</td>
			<td>Baxter Healthcare</td>
			<td>F8B1323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine (100 mg ml&#8722;1 ketamine hydrochloride</td>
			<td>Parnell</td>
			<td></td>
			<td>Ketamine is a controlled drug and all relevant local regulations should be followed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ilium Xylazil-20 (20 mg ml&#8722;1 xylazine hydrochloride)</td>
			<td>Troy Laboratories</td>
			<td></td>
			<td>Xylazil-20 is a controlled drug and all relevant local regulations should be followed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans blue (10 mg ml&#8722;1 in PBS or saline)</td>
			<td>Sigma-Aldrich</td>
			<td>46160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapurified water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin syringe with needle</td>
			<td>BD</td>
			<td>328838</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Biologix Research Company</td>
			<td>30-0135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M paper masking tape</td>
			<td>3M</td>
			<td>2214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deckglaser microscope cover glass (22 mm &#215; 32 mm)</td>
			<td>Paul Marienfeld</td>
			<td>101112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved splinter forceps</td>
			<td>Aesculap, B. Braun Melsungen</td>
			<td>BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veet hair removal cream</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medical cotton-tipped applicators</td>
			<td>Puritan Medical Products Company</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-fold towels</td>
			<td>Kimberly-Clark</td>
			<td>20311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes delicate task wipes</td>
			<td>Kimtech Science</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold-plated, N42-grade neodymium magnets, 12mm in diameter and 2mm thick&#160;</td>
			<td>first4magnets</td>
			<td>F656S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic guide, 10cm by 1.5cm (polyvinyl chloride material)</td>
			<td></td>
			<td></td>
			<td>fold in half lengthwise, bind with masking tape and slot magnet in</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TriM Scope II single-beam two-photon microscope</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tunable (680&#8211;1,080 nm) Coherent Chameleon Ultra II One Box Ti:sapphire laser (&#8805;3.3 W at 800 nm; pulse length of 140 fs, 80 MHz repetition rate)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water-dipping objectives (20&#215;, NA = 1.0)</td>
			<td>Olympus</td>
			<td>XLUMPLFLN20xW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miscroscope filter and mirror sets (for imaging GFP, SHG, Evans Blue)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">495 long-pass</td>
			<td>Chroma</td>
			<td>T495LPXR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">560 lomg-pass</td>
			<td>Chroma</td>
			<td>T560LPXR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">475/42 band-pass</td>
			<td>Semrock</td>
			<td>FF01-475/42-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">525/50 band-pass</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">655/40 band-pass</td>
			<td>Chroma</td>
			<td>NC028647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skin-imaging stage platform (refer to diagram for assembly)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A metal base plate (126 mm &#215; 126 mm &#215; 1 mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">A brass platform for the ear (79 mm &#215; 19 mm; 1 mm thickness at side, 0.5 mm thickness in the middle; Fig. 1) with slit (1.7 mm &#215; 1 mm; 1.5 mm away from long edge)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two plastic blocks (10 mm in height)&#8212;for heat insulation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved holder, for positioning the control thermistor on the ear platform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interface cable CC-28 with DIN connector and thermistors, one for the temperature control and the other for the temperature monitor</td>
			<td>(Warner Instruments (Harvard Apparatus)</td>
			<td>640106</td>
			<td>connect the interface cable to both resistive heater blocks set at 35&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resistive heater blocks RH-2</td>
			<td>(Warner Instruments (Harvard Apparatus)</td>
			<td>640274</td>
			<td>Resistive heater blocks can heat the brass ear platform up to over 100 &#176;C within minutes. Ensure that the control thermistor has been properly secured in the holder in order to avoid overheating.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller TC-344B for the ear platform</td>
			<td>(Warner Instruments (Harvard Apparatus)</td>
			<td>640101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller TR-200 for mouse heating pad</td>
			<td>Fine Science Tools</td>
			<td>21052-00</td>
			<td>Unit is no longer for sale. Ask manufacturer for alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply for TR-200</td>
			<td>Fine Science Tools</td>
			<td>21051-00</td>
			<td>Unit is no longer for sale. Ask manufacturer for alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tools</td>
			<td>21060-00</td>
			<td>Unit is no longer for sale. Ask manufacturer for alternatives.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animal rectal probe&#160;</td>
			<td>Fine Science Tools</td>
			<td>21060-01</td>
			<td>Unit is no longer for sale. Ask manufacturer for alternatives. After connecting the rectal probe and heating pad to the temperature controller TR-200, set the temperature to 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslip holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 plastic rods, 1 cm in diameter, 10 cm in length</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 plastic adaptor with holes drilled to accommodate rods (refer to diagram)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 plastic tightening screws for keeping plastic rods in place</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">1 metal plate, 6 cm x 2.5 cm, with a 2 cm square cut at 1 end, 2 mm edge away from short edge</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 pair of nut and bolt for attaching metal plate to plastic rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">1 acrylic base (4 cm x 5 cm x 1.5 cm) with magnet to hold coverslip holder on skin-imaging stage platform. 1 rod is permanently fixed onto base.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:565px;">Name</td>
			<td style="width:333px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging analysis software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris v8.1.2</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

该协议使用一个定制的耳部皮肤成像平台， 如图1。这个平台的几个特点专门设计，以方便成像，同时保持生理的设置。放置耳加热黄铜平台上不仅保持在35℃的生理温度耳，但它也隔离由于呼吸不可避免动作耳。在加入黄铜平台上的金属夹的创建一个间隙，以防止在盖玻片固定器从施加在耳朵上的重量，从而保持不间断的血流量。阶段平台也被设计成容纳一?...

Watch this Scientific Journal Video about Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses at JoVE.com

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

这个协议描述了在使用磁铁的夹紧小鼠耳部皮肤缺血再灌注（IR）模型的诱导。使用定制的活体成像模型，我们研究的体内炎症反应再灌注后。这项技术的发展背后的基本原理是延长白细胞如何皮肤缺血再灌注损伤反应的认识。

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 ...

inducing-ischemia-reperfusion-injury-mouse-ear-skin-for-intravital

Research

JoVE Journal

Medicine

11.2K 次观看.  Agency for Science, Technology and Research (A*STAR), Biopolis. 这个协议描述了在使用磁铁的夹紧小鼠耳部皮肤缺血再灌注（IR）模型的诱导。使用定制的活体成像模型，我们研究的体内炎症反应再灌注后。这项技术的发展背后的基本原理是延长白细胞如何皮肤缺血再灌注损伤反应的认识。 

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术:缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

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, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

脑缺血再灌注的双血管闭塞小鼠模型

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

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

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

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

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

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

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...