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.

Todos os experimentos que lidam com animais vivos foram conduzidos de acordo com todo uso de animais relevantes e diretrizes e regulamentos de cuidados. 1. Escolha do Mice repórter fluorescentes <ol><li> Use de 6 a 12 semanas de idade LysM-eGFP 11 ratos (sem preferências para machos ou fêmeas). Nota: O uso de vários ratinhos repórter fluorescente específico de células permite a visualização de diferentes células imunitárias in vivo. Nesta estirpe, os n...

<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+ischemia/reperfusion+injury.">Cell biology of ischemia/reperfusion injury.</a> Int Rev Cell Mol Biol. 298, 229-317 (2012).</li><li>Huebener, P., 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+HMGB1/RAGE+axis+triggers+neutrophil-mediated+injury+amplification+following+necrosis.">The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.</a> J Clin Invest. 125, 539-550 (2015).</li><li>Wassermann, E., 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=A+chronic+pressure+ulcer+model+in+the+nude+mouse.">A chronic pressure ulcer model in the nude mouse.</a> Wound Repair Regen. 17, 480-484 (2009).</li><li>Stadler, I., Zhang, R. 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>

Significado IR é uma das principais causas de úlceras de pressão pele. Os estágios iniciais (I e II) de úlceras de pressão descrevem a condição da pele humana (em comparação com os tecidos subcutâneos subjacentes e músculos). No entanto, uma compreensão da etiologia imunológica ainda está em falta. Aqui, apresentamos um modelo de IR simples e robusto na pele de orelha de rato, a fim de resolver esta lacuna. Nós simular a isquemia por clampeamento da orelha de rato entre dois ím...

lesão de isquemia-reperfusão (IRI) ocorre quando há uma hipoxia transitória devido à obstrução do fluxo sanguíneo (isquemia), seguido por uma subsequente re-oxigenação dos tecidos (reperfusão). Na pele, isquemia-reperfusão (IR) é pensado para ser um dos fatores que contribuem para a fisiopatologia das úlceras de pressão, onde repouso prolongado predispõe os pacientes do hospital de longo prazo para a lesão. Nesses pacientes, a pele e os músculos subjacentes são constantemente expostos a pressão do peso exercido sobre áreas de proeminência óssea, resultando em lesões localizadas que, se não tratada, pode tornar-se necrótica 1. Os danos envolvidos em um IRI são duas. Durante a isquemia, a oclusão de vasos sanguíneos conduz a uma redução drástica da entrega de oxigénio aos tecidos. Isto resulta numa diminuição de ATP e pH, que inactiva ATPases envolvidas no metabolismo celular. Por sua vez, os níveis de cálcio celular pico, e salientou ou danificado cells sofrem apoptose ou necrose 2. A liberação de conteúdo intracelular ou padrões moleculares danos associados (DAMP), como HMGB1, contribui para a resposta inflamatória 3. O segundo insulto ocorre durante a reperfusão. Embora os níveis de oxigénio e de pH são restaurados durante a reperfusão, isto resulta na geração de espécies reactivas de oxigénio (ROS), o que leva à oxidação de lípidos intracelulares, DNA e proteínas. Por conseguinte, os mediadores pró-inflamatórios são activados, o que desencadeia uma resposta inflamatória secundária que envolve o recrutamento das células imunes para o local inflamatório 2. Enquanto a cascata de acontecimentos bioquímicos que conduzem à resposta inflamatória tem sido bem descrito, a regulação espacial e temporal das actividades celulares imunitárias não são bem compreendidos. Aqui, descrevemos um modelo IR robusta na pele de orelha de rato usando simples aperto ímã. Juntamente com a imagem multiphoton intravital (MP-IVM), nósestabeleceu um modelo para estudar as respostas inflamatórias in vivo que ocorrem após a reperfusão ocorre. A lógica por trás do desenvolvimento e uso desta técnica é tentar entender como ambas as células intersticiais e infiltrando responder a IR em tempo real. Modelos existentes de IR utilizando a técnica de fixação na lateral da pele são altamente invasivo, uma vez que exigem a implantação cirúrgica de placas de aço no flanco da pele, tornando-os menos do que o ideal para estudos imunológicos 4. Uma técnica de fixação não-invasiva semelhante foi descrito na pele do rato 5,6 flanco. No entanto, devido à incorporação da componente de imagem intravital neste método, que em vez escolheu a pele da orelha como o local de IR alvo, uma vez que contorna os movimentos devidos à respiração e oferece estabilidade durante a imagiologia de 7,8. Além disso, subconjuntos de leucócitos que se estendem por o interstício são idênticos entre a pele da orelha e o flanco da pele, embora onúmeros e proporções podem variar ligeiramente 9. Assim, a pele da orelha representa um local de imagem ideal. Além disso, a maioria dos dados obtidos a partir destes modelos IRI são limitadas a avaliações macroscópicas (classificação de úlceras) e análises microscópicas de endpoint indicadores inflamatórios 10. Utilizando este modelo, a visualização em tempo real, da resposta celular de neutrófilos após a reperfusão na pele de um rato repórter fluorescente é activado. Um modelo de imagem da orelha intravital publicado anteriormente é utilizado 8 com modificações adicionais (Figuras 1, 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).

Esse protocolo utiliza uma pele de orelha de plataforma de imagem-construído sob encomenda, como se mostra na Figura 1. Várias características desta plataforma são projetados especificamente para facilitar a imagem, mantendo as configurações fisiológicas. Colocar a orelha na plataforma de bronze aquecida não só mantém a orelha a uma temperatura fisiológica de 35 ° C, mas também a partir de isolados do ouvido movimentos inevitáveis ​​devidas à respiraç...

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

Este protocolo descreve a indução de um modelo de isquemia-reperfusão (IR) em pele de orelha de rato usando íman de fixação. Usando um modelo de imagens intravital custom-built, estudamos nas respostas inflamatórias in vivo pós-reperfusão. A lógica por trás do desenvolvimento desta técnica é ampliar a compreensão de como leucócitos responder a uma lesão IR pele.

Induzindo a lesão de isquemia-reperfusão na pele da orelha de rato por Intravital Multiphoton imagem de respostas imunes

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

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