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.

जीवित पशुओं के साथ काम कर सभी प्रयोगों सभी प्रासंगिक पशु का उपयोग और देखभाल के दिशा निर्देशों और नियमों के अनुसार आयोजित की गई। 1. फ्लोरोसेंट संवाददाता चूहों की पसंद <ol><li> 6 से 12 सप्ताह पुराने Lys...

<ol>
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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. 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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. 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महत्व आईआर त्वचा दबाव अल्सर के प्रमुख कारणों में से एक है। दबाव अल्सर के प्रारंभिक दौर (मैं और द्वितीय) मानव त्वचा की हालत का वर्णन (अंतर्निहित चमड़े के नीचे के ऊतकों और मांसपेशियों की तुलना म?...

Ischemia-reperfusion चोट (आईआरआई) तब होता है जब वहाँ रक्त प्रवाह (ischemia) ऊतकों (reperfusion) के एक बाद फिर से ऑक्सीजन के द्वारा पीछा की बाधा के कारण एक क्षणिक हाइपोक्सिया है। त्वचा में, ischemia reperfusion-(आईआर) दबाव अल्सर के pathophysiology, जहां लंबे समय तक बिस्तर पर आराम की चोट के लिए लंबी अवधि के अस्पताल में मरीजों predisposes के लिए योगदान कारकों में से एक माना जाता है। इन रोगियों में, दोनों त्वचा और अंतर्निहित मांसपेशियों लगातार वजन बोनी प्रमुखता के क्षेत्रों के ऊपर लगाए गए दबाव को उजागर कर रहे हैं, स्थानीय चोटों कि, अगर अनुपचारित छोड़ दिया, परिगलित 1 हो सकता है, जिसके परिणामस्वरूप। हर्जाना एक आईआरआई में शामिल दुगना कर रहे हैं। ischemia के दौरान, रक्त वाहिकाओं का रोड़ा ऊतकों को ऑक्सीजन वितरण की भारी गिरावट की ओर जाता है। इस एटीपी और पीएच, जो सेलुलर चयापचय में शामिल ATPases निष्क्रिय की कमी का परिणाम है। बदले में, सेलुलर कैल्शियम का स्तर स्पाइक, और जोर दिया है या क्षतिग्रस्त गएल apoptosis या परिगलन 2 से गुजरना। Intracellular सामग्री या क्षति जुड़े आणविक पैटर्न (नम) की रिहाई, HMGB1 की तरह, भड़काऊ प्रतिक्रिया से 3 योगदान देता है। दूसरी अपमान reperfusion के दौरान होता है। ऑक्सीजन और पीएच स्तर reperfusion के दौरान बहाल कर रहे हैं हालांकि, इस प्रतिक्रियाशील ऑक्सीजन प्रजातियों की पीढ़ी (आरओएस), जो intracellular लिपिड, डीएनए और प्रोटीन के ऑक्सीकरण की ओर जाता है में परिणाम है। नतीजतन, समर्थक भड़काऊ मध्यस्थों सक्रिय कर रहे हैं, जो एक माध्यमिक भड़काऊ प्रतिक्रिया है कि भड़काऊ साइट 2 के लिए प्रतिरक्षा कोशिकाओं की भर्ती शामिल है बंद सेट। जबकि भड़काऊ प्रतिक्रिया करने के लिए अग्रणी जैव रासायनिक घटनाओं का झरना अच्छी तरह से वर्णित किया गया है, प्रतिरक्षा सेल गतिविधियों के स्थानिक और लौकिक विनियमन अच्छी तरह से समझ नहीं रहे हैं। यहाँ, हम साधारण चुंबक का उपयोग कर clamping माउस कान त्वचा पर एक मजबूत आईआर मॉडल का वर्णन है। multiphoton intravital इमेजिंग (MP-IVM) के साथ मिलकर, हमइन विवो भड़काऊ प्रतिक्रियाओं है कि हो reperfusion की जगह लेता है के बाद अध्ययन करने के लिए एक मॉडल की स्थापना की। विकास और इस तकनीक के उपयोग के पीछे तर्क यह समझने के लिए कि कैसे दोनों मध्य और घुसपैठ की कोशिकाओं को वास्तविक समय में आईआर का जवाब कोशिश है। त्वचा पार्श्व पर clamping तकनीक का उपयोग आईआर के मौजूदा मॉडलों, अत्यधिक आक्रामक हैं, क्योंकि वे त्वचा दिशा में स्टील प्लेट की शल्य आरोपण की आवश्यकता होती है, उन्हें प्रतिरक्षा अध्ययन के 4 के लिए कम-से-आदर्श बना रही है। इसी तरह की एक गैर-आक्रामक clamping तकनीक माउस त्वचा पार्श्व 5,6 में वर्णित किया गया है। हालांकि, इस पद्धति में intravital इमेजिंग घटक के समावेश के कारण, हम बजाय लक्षित आईआर साइट के रूप में कान त्वचा, चुना क्योंकि यह साँस लेने के कारण आंदोलनों में गतिरोध उत्पन्न और इमेजिंग 7.8 के दौरान स्थिरता प्रदान करता है। इसके अलावा, ल्युकोसैट सबसेट कि interstitium अवधि, कान त्वचा और त्वचा पार्श्व के बीच समान हैं, हालांकिसंख्या और अनुपात थोड़ा 9 भिन्न हो सकते हैं। इस प्रकार, कान त्वचा एक आदर्श इमेजिंग साइट का प्रतिनिधित्व करता है। इसके अलावा, सबसे डेटा इन आईआरआई मॉडल से लिया गया स्थूल मूल्यांकन (अल्सर की ग्रेडिंग) और समापन बिंदु भड़काऊ संकेतक 10 का सूक्ष्म विश्लेषण करने के लिए सीमित कर रहे हैं। इस मॉडल का उपयोग करना, एक फ्लोरोसेंट रिपोर्टर माउस की त्वचा में reperfusion के बाद neutrophils के सेलुलर प्रतिक्रिया के वास्तविक समय दृश्य सक्षम है। एक पहले प्रकाशित intravital इमेजिंग कान मॉडल अतिरिक्त संशोधनों (आंकड़े 1, 2) के साथ 8 उपयोग किया जाता है।

<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).

के रूप में चित्र 1 में दिखाया इस प्रोटोकॉल, एक कस्टम निर्मित कान त्वचा इमेजिंग मंच का उपयोग करता है। इस मंच के कई सुविधाओं विशेष इमेजिंग की सुविधा के लिए शारीरिक सेटिंग्स को बनाए रखते...

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

इस प्रोटोकॉल चुंबक का उपयोग कर clamping माउस कान त्वचा पर एक ischemia reperfusion-(आईआर) मॉडल की प्रेरण का वर्णन है। एक कस्टम निर्मित intravital इमेजिंग मॉडल का उपयोग करना, हम विवो भड़काऊ प्रतिक्रियाओं के बाद reperfusion में अध्ययन करते हैं। इस तकनीक के विकास के पीछे तर्क कैसे ल्यूकोसाइट्स त्वचा आईआर चोट का जवाब की समझ का विस्तार करने के लिए है।

प्रतिरक्षा प्रतिक्रिया के intravital multiphoton इमेजिंग के लिए माउस कान त्वचा में उत्प्रेरण ischemia-reperfusion चोट

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

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

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Murine Model of Intestinal Ischemia-reperfusion Injury

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Intraocular दबाव की ऊंचाई के माध्यम से रेटिना ischemia-reperfusion चोट का एक माउस मॉडल

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Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

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

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