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.

Toutes les expériences portant sur des animaux vivants ont été menées conformément à toute utilisation des animaux et directives pertinentes et règlements soins. 1. Choix de la souris de rapporteur fluorescent <ol><li> Utilisez 6 à 12 semaines d&#39;âge LysM-eGFP 11 souris (pas de préférence pour les mâles ou les femelles). Remarque: L&#39;utilisation de diverses souris rapporteurs fluorescents spécifiques de cellules permet la visualisation des différentes ...

<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>

Importance IR est l&#39;une des principales causes des ulcères de pression de la peau. Les premiers stades (I et II) des escarres de décubitus décrivent l&#39;état de la peau humaine (par rapport aux tissus sous-cutanés sous-jacents et les muscles). Cependant, la compréhension de l&#39;étiologie immunologique fait encore défaut. Ici, nous présentons un modèle simple et robuste IR sur la peau de l&#39;oreille de la souris afin de combler cette lacune. Nous simulons l&#39;ischémie par ...

Ischémie- reperfusion (IRI) se produit lorsqu&#39;il y a une hypoxie transitoire due à l&#39;obstruction de la circulation sanguine (ischémie), suivie d&#39;une ré-oxygénation subséquente des tissus (reperfusion). Dans la peau, l&#39;ischémie-reperfusion (IR) est considéré comme l&#39;un des facteurs qui contribuent à la physiopathologie des ulcères de pression, où alitement prolongé prédispose les patients hospitalisés à long terme à des blessures. Chez ces patients, la peau et les muscles sous - jacents sont constamment exposés à la pression du poids exercé sur les zones de proéminence osseuse, entraînant des blessures localisées qui, l&#39; absence de traitement, peuvent devenir nécrotiques 1. Les dommages-intérêts impliqués dans un IRI sont doubles. Au cours de l&#39;ischémie, l&#39;occlusion des vaisseaux sanguins conduit à une baisse drastique de l&#39;apport d&#39;oxygène aux tissus. Cela se traduit par une diminution de l&#39;ATP et du pH, qui inactive les ATPases impliqués dans le métabolisme cellulaire. À son tour, le taux de calcium cellulaires pic et stressés ou c endommagésaunes subissent une apoptose ou une nécrose 2. La libération des contenus intracellulaires ou des motifs moléculaires de dommages associés (DAMP), comme la HMGB1, contribue à la réponse inflammatoire 3. La seconde insulte se produit lors de la reperfusion. Bien que les niveaux d&#39;oxygène et de pH sont restaurées au cours de la reperfusion, cela se traduit par la génération d&#39;espèces réactives de l&#39;oxygène (ROS), ce qui conduit à l&#39;oxydation des lipides intracellulaires, l&#39;ADN et les protéines. Par conséquent, les médiateurs pro-inflammatoires sont activés, ce qui déclenche une réponse inflammatoire secondaire qui comprend le recrutement de cellules immunitaires vers le site inflammatoire 2. Alors que la cascade d&#39;événements biochimiques conduisant à la réponse inflammatoire a été bien décrite, la régulation spatiale et temporelle des activités de cellules immunitaires ne sont pas bien compris. Ici, nous décrivons un modèle IR robuste sur la peau de l&#39;oreille de souris à l&#39;aide de serrage simple aimant. Couplé avec l&#39;imagerie multiphotonique intravitale (MP-IVM), nousmis en place un modèle pour étudier les réponses inflammatoires in vivo qui se produisent après la reperfusion a lieu. La raison derrière le développement et l&#39;utilisation de cette technique est d&#39;essayer de comprendre comment les cellules interstitielles et infiltrant répondent à IR en temps réel. Les modèles existants de IR en utilisant la technique de serrage sur le flanc de la peau sont très envahissantes, car elles nécessitent l&#39;implantation chirurgicale de plaques d&#39;acier dans le flanc de la peau, ce qui les rend moins-que-idéal pour les études immunologiques 4. Une technique de serrage non invasive similaire a été décrite dans le flanc de souris 5,6 de la peau. Toutefois, en raison de l&#39;incorporation de la composante d&#39;imagerie intravitale dans cette méthode, nous avons choisi à la place de la peau de l&#39; oreille que le site IR ciblé, car il évite les mouvements dus à la respiration et offre la stabilité lors de l&#39; imagerie 7,8. Par ailleurs, les sous-ensembles de leucocytes qui enjambent interstitiel sont identiques entre la peau de l&#39;oreille et le flanc de la peau, même si lales nombres et les proportions peuvent varier légèrement 9. Ainsi, la peau de l&#39;oreille représente un site d&#39;imagerie idéal. En outre, la plupart des données récupérées à partir de ces modèles IRI sont limités à des évaluations macroscopiques (de classement des ulcères) et les analyses microscopiques des indicateurs inflammatoires d&#39;extrémité 10. En utilisant ce modèle, la visualisation en temps réel de la réponse cellulaire des neutrophiles après la reperfusion dans la peau d&#39;une souris rapporteur fluorescent est activé. Un modèle d&#39;imagerie de l&#39; oreille intravitale précédemment publié est utilisé 8 avec des modifications supplémentaires (figures 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).

Ce protocole utilise une peau de l&#39; oreille plate - forme d&#39;imagerie intégré personnalisé, comme représenté sur la figure 1. Plusieurs caractéristiques de cette plate-forme sont spécifiquement conçus pour faciliter l&#39;imagerie tout en conservant les paramètres physiologiques. Placer l&#39;oreille sur la plate-forme en laiton chauffé maintient non seulement l&#39;oreille à une température physiologique de 35 ° C, mais il isole aussi l&#39;oreille d...

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

Ce protocole décrit l&#39;induction d&#39;un modèle d&#39;ischémie-reperfusion (IR) sur la peau de l&#39;oreille de la souris utilisant un aimant de serrage. En utilisant un modèle d&#39;imagerie intravitale sur mesure, nous étudions dans les réponses inflammatoires in vivo post-reperfusion. La raison derrière le développement de cette technique est d&#39;étendre la compréhension de la façon dont les leucocytes réagissent aux blessures IR de la peau.

Induire ischémie-reperfusion chez la souris Ear Skin pour Intravitale multiphotonique Imaging des réponses immunitaires

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

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Medicine

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