Name: inducing ischemia-reperfusion injury in the mouse ear skin for intravital multiphoton imaging of immune responses
Uploaded: 2016-12-22T15:00:00
Description: 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

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...

<ol>
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G., 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=Visualizing+the+neutrophil+response+to+sterile+tissue+injury+in+mouse+dermis+reveals+a+three-phase+cascade+of+events.">Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events.</a> J Invest Dermatol. 131, 2058-2068 (2011).</li><li>Li, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+multiphoton+imaging+of+immune+responses+in+the+mouse+ear+skin.">Intravital multiphoton imaging of immune responses in the mouse ear skin.</a> Nat Protoc. 7, 221-234 (2012).</li><li>Tong, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin+immune+atlas:+three-dimensional+analysis+of+cutaneous+leukocyte+subsets+by+multiphoton+microscopy.">The skin immune atlas: three-dimensional analysis of cutaneous leukocyte subsets by multiphoton microscopy.</a> J Invest Dermatol. 135, 84-93 (2015).</li><li>Saito, 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=The+loss+of+MCP-1+attenuates+cutaneous+ischemia-reperfusion+injury+in+a+mouse+model+of+pressure+ulcer.">The loss of MCP-1 attenuates cutaneous ischemia-reperfusion injury in a mouse model of pressure ulcer.</a> J Invest Dermatol. 128, 1838-1851 (2008).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Roediger, B., Ng, L. G., Smith, A. L., Fazekasde de St Groth, B., Weninger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dendritic+cell+migration+within+the+skin.">Visualizing dendritic cell migration within the skin.</a> Histochem Cell Biol. 130, 1131-1146 (2008).</li><li>Kikushima, K., Kita, S., Higuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+imaging+for+the+in+vivo+tracking+of+high-speed+vesicle+transport+in+mouse+neutrophils.">A non-invasive imaging for the in vivo tracking of high-speed vesicle transport in mouse neutrophils.</a> Sci Rep. 3, 1913 (2013).</li><li>Ng, L. G., 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=Migratory+dermal+dendritic+cells+act+as+rapid+sensors+of+protozoan+parasites.">Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.</a> PLoS Pathog. 4, e1000222 (2008).</li><li>Soohoo, A. L., Bowersox, S. L., Puthenveedu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+clathrin-mediated+endocytosis+of+G+protein-coupled+receptors+at+single-event+resolution+via+TIRF+microscopy.">Visualizing clathrin-mediated endocytosis of G protein-coupled receptors at single-event resolution via TIRF microscopy.</a> J Vis Exp. , e51805 (2014).</li><li>Beltman, J. B., Maree, A. F., de Boer, R. 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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 ...

The overall goal of this experiment is to understand how immune cells respond to ischemia reperfusion through intravital multiphoton imaging of the mouse ear skin. This method can help to address some of the very important questions in the field of Immunology and Dermatology to understand the dynamic behavior of immune cell. The main advantage of this technique is that it provide a robust imaging model for studying cellular activities in the skin under inflamed or a resting state.Though this method may provide insight into ischemic reperfusion injuries, this can also be applied to other systems such as hypersensitivity reactions as well as infections. To begin this procedure, place the mouse on a heating pad to maintain its body temperature at 37 degrees Celsius throughout the preparation procedure. Confirm the depth of anesthesia by the absence of toe pinch reflex.Next, apply ophthalmic lubricant on the animal's eyes to prevent dryness while under anesthesia. Afterward, carefully apply depilatory cream to the upper two thirds of the dorsal ear and wait for two to three minutes. Then, remove the cream using wet cotton tip applicators in a thorough but gentle manner.Now, induce ischemia in the mouse ear skin using the neodymium magnets. To do so, first slot the magnets into their individual plastic guides. Position one magnet such that only the edge will contact with a second ventral magnet, which should be placed below the ventral side of the ear with the ear laying flat on the magnet.Once ready, carefully let the magnets come together. After 1.5 hours of ischemia, remove the magnets by twisting them away from each other using the plastic guides, allowing reperfusion to take place. Immediately after magnet removal, administer Evans Blue or another blood vessel labeling agent of choice intravenously.In this step, cut two pieces of masking tape. Allow the adhesive sides to stick together whilst leaving about one millimeter of adhesive along its width. Then, cut the masking tape in two to accommodate its placement within the slit on the ear platform.Insert this masking tape about halfway through the slit such that the adhesive side faces up. After that, position the mouse on the heating pad such that the ear to be imaged is next to the masking tape strip. By using two PBS moistened cotton tip applicators, gently press the ear against the adhesive strip.Using the strip as a guide, bring the mouse ear through the slit while simultaneously adjusting the mouse closer towards the stage. As the ear skin is very fragile, this step must be done very carefully to avoid any preparation-induced injuries. To remove the sticky tape, first add a drop of PBS to reduce the adhesiveness of the tape.Then, separate the mouse ear from the masking tape as gently as possible using a fine paint brush. Flatten the ear against the ear platform by gently rolling a moist cotton tip applicator over the ear. Subsequently, put a drop of PBS underneath the coverslip and gently place it over the ear.Afterward, insert the rectal temperature probe and connect the wires to the heating system according to manufacturer's instructions. To acquire images, place the animal under the multiphoton microscope. Start with an imaging region that is in close proximity to the edge of the ischemia characterized by massive Evans Blue leakage.The schematic diagram here shows the positioning of the magnets. These two photos show a pre-ischemic and a post-ischemic ear after magnet clamping. In this schematic of the ear indicates the region imaged with respect to where the magnet had been placed.This time lapse sequence of maximum projection, shows the neutrophil infiltration in response to reperfusion in LysM-eGFPM albino mouse ear skin after 1.5 hours of ischemia. At earlier time points, the lack of Evans Blue signal indicates temporary vessel occlusion after ischemia. Once mastered, preparations for the ischemic reperfused ear for imaging can be done in less than 30 minutes if it is performed properly.While performing this procedure, it is important to constantly check the depth of anesthesia and the temperature of the mouse. Following this procedure, other method like whole mouse staining of the mouse ear skin can be performed to have a global view of the cellular localizations. After the developments of this protocol, it paved the way for researcher in the field of Immunology and Dermatology to explore innate immunity in the mouse ear skin.After watching these videos, you should have a good understanding of how to establish a mouse ear skin imaging model to study ischemic reperfusion injury. Don't forget that working with multiphoto microscope can be hazardous. And precautions such as laser safety training should be undertaken.

Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

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

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

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

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, ...