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.

살아있는 동물을 다루는 모든 실험은 모든 관련 동물 사용 및 관리 지침 및 규정에 따라 실시 하였다. 형광 리포터 생쥐 1. 선택 <ol><li> 6 ~ 12 주 된 LysM-eGFP는 11 마우스 (남성 또는 여성 중 하나에 대한 더 선호)를 사용합니다. 주 : 다양한 세포 관련 형광 리포터 생쥐의 사용은 생체 내에서 서로 다른 면역 세포의 가시화를 가능하게한다. 이 균주에 가시화 ?...

<ol>
	<li>Black, J., 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=National+Pressure+Ulcer+Advisory+Panel's+updated+pressure+ulcer+staging+system.">National Pressure Ulcer Advisory Panel's updated pressure ulcer staging system.</a> Adv Skin Wound Care. 20, 269-274 (2007).</li><li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, R. 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Y., Oskoui, P., Whittaker, M. S., Lanzafame, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple,+noninvasive,+clinically+relevant+model+of+pressure+ulcers+in+the+mouse.">Development of a simple, noninvasive, clinically relevant model of pressure ulcers in the mouse.</a> J Invest Surg. 17, 221-227 (2004).</li><li>Tsuji, S., Ichioka, S., Sekiya, N., Nakatsuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+ischemia-reperfusion+injury+in+a+microcirculatory+model+of+pressure+ulcers.">Analysis of ischemia-reperfusion injury in a microcirculatory model of pressure ulcers.</a> Wound Repair Regen. 13, 209-215 (2005).</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=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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+immune+cell+migration.">Analysing immune cell migration.</a> Nat Rev Immunol. 9, 789-798 (2009).</li></ol>

의미 IR 피부 욕창의 주요 원인 중 하나이다. (배후 피하 조직 및 근육에 비해) 욕창의 초기 단계 (I 및 II)은 인간의 피부의 상태를 설명한다. 그러나, 면역 학적 원인에 대한 이해는 여전히 부족하다. 여기서,이 간극을 해결하기 위하여 마우스 귀 피부에 간단하고 강력한 IR 모델을 제시한다. 우리는 자석 제거 (재관류) 후 하류 면역 반응을 연구 두 개의 자석과 이후의 마우스 귀?...

과도 저산소증 인해 조직 (재관류)의 후속 재 - 산소화이어서 혈류 (허혈)의 폐색에있을 때의 허혈 - 재관류 손상 (IRI)가 발생한다. 피부, 허혈 재관류 (IR)의 연장 상 나머지 부상 장기 입원 환자 걸리기 욕창의 병태 생리에 기여 요인 중 하나라고 생각된다. 이러한 환자에서 피부와 기본 근육 모두 지속적으로 치료 왼쪽 경우, 괴사 하나가 될 수 지역화 부상의 결과로, 뼈 굴지의 영역에 걸쳐 작용 무게 압력에 노출되어있다. IRI에 관련된 손해 배상은 두 가지입니다. 허혈, 혈관 폐색 조직 산소 전달의 급격한 저하로 연결. 이것은 세포 대사에 관여 -ATPase를 불 활성화 ATP 및 산도의 감소를 초래한다. 차례로, 세포의 칼슘 농도는 스파이크, 그리고 스트레스 또는 c를 손상ELL 학생은 세포 사멸 또는 괴사 (2)를받을. 세포 내 내용이나 손상 관련 분자 패턴 (DAMP)의 출시, HMGB1 같은 염증 반응 3에 기여한다. 두 번째 모욕은 재관류시에 발생합니다. 산소와의 pH 수치가 재관류 기간 동안 복원되지만,이 세포 내 지질, DNA 및 단백질의 산화를 유도 활성 산소 종의 발생 (ROS)을 초래한다. 따라서, 염증성 매개 물질은 염증 사이트 2 면역 세포의 채용을 포함하는 이차 염증 반응을 설정하는 활성화됩니다. 염증 반응에 이르기까지의 생화학 적 사건의 케스케이드 잘 설명 하였지만, 면역 세포 활동의 시공간적 조절은 잘 이해되지 않는다. 여기서 우리는 간단한 자석 클램프를 사용하여 마우스 귀 피부에 강력한 IR 모델에 대해 설명합니다. 광자 생체 내에 영상 (MP-IVM)와 결합하여, 우리재관류가 발생 후 발생하는 생체 내 염증 반응을 연구하는 모델을 설립했다. 이 기술의 개발과 사용의 뒤에 이론적 근거는 간질 및 침투 세포가 모두 실시간으로 IR에 어떻게 반응하는지 이해하려고 노력하는 것입니다. 그들은 면역 연구 4 이하보다 유용하게 활용할 피부 측면에서 강판의 외과 적 이식을 필요로 피부 측면에 클램핑 기술을 사용하여 IR 기존의 모델은 매우 침습적이다. 유사한 비 침습적 클램핑 기술은 마우스 피부 플랭크 5,6- 설명되었다. 이 호흡에 의한 움직임을 회피하고 -7,8- 촬상시 안정성을 제공하지만,이 때문에 방법에서 생체 내에 이미징 성분의 혼입, 우리는 대신, 표적 IR 사이트와 귀의 피부를 선택 하였다. 또한, 간질 걸쳐 백혈구의 서브 세트는 있지만, 귀 피부와 피부 사이의 동일 측면숫자와 비율은 약간 9 다를 수 있습니다. 따라서, 귀 피부 이상적인 촬상 부위를 나타낸다. 또한, 이러한 IRI 모델에서 검색 대부분의 데이터는 거시적 평가 (궤양의 등급) 및 엔드 포인트 염증 지표 (10)의 현미경 분석에 제한됩니다. 이 모델을 사용하여, 형광 리포터 마우스 피부 재관류 후 호중구의 세포 반응의 실시간 시각화를 사용할 수있다. 이전에 출판 생체 내에 귀 이미징 모델은 추가 수정 (도 1, 2)와 (8) 사용된다.

<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).

그림 1과 같이이 프로토콜은 맞춤형 귀 피부 이미징 플랫폼을 사용합니다. 이 플랫폼의 몇 가지 기능은 특히 생리 설정을 유지하면서 영상을 용이하게하기 위해 설계되었습니다. 가열 황동 플랫폼에 귀를 배치하는 것은 35 ° C의 온도에서 생리 귀에 유지뿐만 아니라 호흡으로 인해 불가피 움직임로부터 귀를 분리. 황동 플랫폼에 금속 클립을 첨가하여 중단 혈...

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

이 프로토콜은 자석 클램프를 사용하여 마우스 귀 피부에 허혈 - 재관류 (IR) 모델의 유도를 설명합니다. 맞춤 제작 된 생체 내에 이미징 모델을 사용하여, 우리는 생체 내 염증 반응 후 재관류에서 공부합니다. 이러한 기술의 발달 배후의 이론적 근거는 백혈구 IR 피부 손상에 반응하는 방법에 대한 이해를 확장하는 것이다.

면역 반응의 생체 내에 다중 광자 이미징을위한 마우스 귀 피부에 유도 허혈 - 재관류 손상

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

Normothermic 심장 마비와 심폐 소생술의 : 국소 빈혈 - Reperfusion 상해의 마우스 모델

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 ...

연구

의학

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

쥐 Cremaster 근육의 국소 빈혈 reperfusion의 - 상해에서 백혈구 - 내피 상호 작용의 실시간 디지털 이미징 (IRI)

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

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

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

창자 허혈 - 재관류 손상의 쥐 모델

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

안압 상승을 통해 망막 허혈 - 재관류 손상의 마우스 모델

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

대뇌 국 소 빈 혈 reperfusion의 2 배 폐색 마우스 모델

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

쥐의 심근 허혈 재관류 후 관상 동맥 흐름 예비의 평가

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

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

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

뮤린 모델에서 허혈혈 재관류 부상 후 줄기 세포의 지연 된 자궁 내 납품

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

쥐 간 이식 모형에 있는 혈역학적인 변경을 시뮬레이션하여 기관 허혈-재관류 상해

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