This work was supported by a grant of the &#x201C;Deutsche Forschungsgemeinschaft&#x201D; to S.U. Eisenhardt (EI 866/1-1).

1. Anesthesia and Monitoring
<ol>
 <li>Appropriate national and institutional ethics should be in place before performing animal experiments. Following approval from the ethics committee anesthetize male Sprague Dawley rats with a body weight from 120 - 180 g. Deliver 2 - 3 vol% isoflurane to a Plexiglas box via isoflurane vaporizer and place the rat inside. </li>
 <li>As soon as the appropriate level of anesthesia is achieved (lack of reaction to toe or tail pinch) the rat is weighted and shaved on the ventral ce...

<ol>
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Physiol. 255, H1269-H1275 (1988).</li><li>Lazarus, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+ischaemia-reperfusion+injury+in+murine+skeletal+muscle.">The role of mast cells in ischaemia-reperfusion injury in murine skeletal muscle.</a> J Pathol. 191, 443-448 (2000).</li><li>van den Heuvel, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Ischaemia-reperfusion+injury+in+flap+surgery.">Review: Ischaemia-reperfusion injury in flap surgery.</a> J. Plast. Reconstr. Aesthet. Surg. 62, 721-726 (2009).</li><li>Rosen, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+lectins+in+the+immune+system.">Cell surface lectins in the immune system.</a> Semin. 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Res. 78, 1100-1104 (1996).</li><li>Dobschuetz, E. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+intravital+fluorescence+microscopy--a+novel+method+for+the+assessment+of+microvascular+permeability+in+acute+pancreatitis.">Dynamic intravital fluorescence microscopy--a novel method for the assessment of microvascular permeability in acute pancreatitis.</a> Microvasc Res. 67, 55-63 (2004).</li><li>Vutskits, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+methylene+blue+on+the+central+nervous+system.">Adverse effects of methylene blue on the central nervous system.</a> Anesthesiology. 108, 684-692 (2008).</li><li>Takasu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+survival+time+with+combined+early+blood+transfusion+and+fluid+administration+in+uncontrolled+hemorrhagic+shock+in+rats.">Improved survival time with combined early blood transfusion and fluid administration in uncontrolled hemorrhagic shock in rats.</a> J. Trauma. 8, 312-316 (2010).</li><li>Proctor, K. G., Busija, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+arteriolar,+regional,+and+whole+organ+blood+flow+in+cremaster+muscle.">Relationships among arteriolar, regional, and whole organ blood flow in cremaster muscle.</a> Am. J. Physiol. 249, 34-41 (1985).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mouse+Cremaster+Muscle+Preparation+for+Intravital+Imaging+of+the+Microcirculation.">The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation.</a> J. Vis. Exp. (52), e2874 (2011).</li><li>Kanwar, S., Hickey, M. J., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postischemic+inflammation:+a+role+for+mast+cells+in+intestine+but+not+in+skeletal+muscle.">Postischemic inflammation: a role for mast cells in intestine but not in skeletal muscle.</a> Am. J. Physiol. 275, 212-218 (1998).</li><li>Leoni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflamed+phenotype+of+the+mesenteric+microcirculation+of+melanocortin+type+3+receptor-null+mice+after+ischemia-reperfusion.">Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion.</a> FASEB J. 22, 4228-4238 (2008).</li><li>Simoncini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+oestrogen+receptor+with+the+regulatory+subunit+of+phosphatidylinositol-3-OH+kinase.">Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase.</a> Nature. 407, 538-541 (2000).</li><li>Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+levels+of+soluble+P-selectin+mediate+adhesion+of+leukocytes+to+the+endothelium+through+Mac-1+activation.">Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.</a> Circ. Res. 103, 1128-1138 (2008).</li><li>Mori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+induced+microvascular+responses+in+LDL-receptor+-/-+mice.">Ischemia-reperfusion induced microvascular responses in LDL-receptor -/- mice.</a> Am. J. Physiol. 276, H1647-H1654 (1999).</li><li>Eisenhardt, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Molecular+Changes+Induced+by+Ischemia/Reperfusion+in+Human+Free+Muscle+Flap+Tissue+Samples.">Monitoring Molecular Changes Induced by Ischemia/Reperfusion in Human Free Muscle Flap Tissue Samples.</a> Ann. Plast. Surg. , (2011).</li><li>Eisenhardt, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+activation-specific+human+anti-{alpha}M{beta}2+single-chain+antibodies+as+potential+diagnostic+tools+and+therapeutic+agents.">Generation of activation-specific human anti-{alpha}M{beta}2 single-chain antibodies as potential diagnostic tools and therapeutic agents.</a> Blood. 109, 3521-3528 (2007).</li><li>Eisenhardt, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+pentameric+to+monomeric+C-reactive+protein+on+activated+platelets+localizes+inflammation+to+atherosclerotic+plaques.">Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques.</a> Circ Res. 105, 128-137 (2009).</li><li>Eisenhardt, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-reactive+protein:+how+conformational+changes+influence+inflammatory+properties.">C-reactive protein: how conformational changes influence inflammatory properties.</a> Cell Cycle. 8, 3885-3892 (2009).</li><li>Granger, D. 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No conflicts of interest declared.

Leukocyte-endothelial interaction, production of reactive oxygen species and activation of the complement system are the key features of IRI-induced tissue dysfunction.26 The microcirculation of the affected tissue is considered as the integral site for the inflammatory onset. Apart from ex vivo experiments such as flow chamber assays27, 28 it is mandatory to provide well-established models of intravital imaging to further evaluate the in vivo relevance. Though IRI has been implica...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the equipment:</td>
 <td>Company:</td>
 <td>Catalogue No.:</td>
 <td>Comments:</td>
 </tr>
 <tr>
 <td>Forene 100% (V/V)</td>
 <td>Abbot</td>
 <td>B506</td>
 <td>API isoflurane</td>
 </tr>
 <tr>
 <td>Terylene Suture</td>
 <td>Serag Weissner</td>
 <td>OC108000</td>
 <td></td>
 </tr>
 <tr>
 <td>Portex Fine Bore Polythene Tubing</td>
 <td>Smiths Medical</td>
 <td>800/100/100</td>
 <td>0.28 mm inner Diameter</td>
 </tr>
 <tr>
 <td>0,9% saline solution</td>
 <td>Fresinus Kabi</td>
 <td>808771</td>
 <td></td>
 </tr>
 <tr>
 <td>Change-A-tip deluxe cautery kit</td>
 <td>Bovie Medical</td>
 <td>DEL1</td>
 <td></td>
 </tr>
 <tr>
 <td>Abbocath -T 14G</td>
 <td>Venisystems</td>
 <td>G713 - A01</td>
 <td>used as lens tube</td>
 </tr>
 <tr>
 <td>Servo Ventilator 900C</td>
 <td>Maquet</td>
 <td></td>
 <td>used as animal ventialtor</td>
 </tr>
 <tr>
 <td>Logical pressure transducer</td>
 <td>Smiths Medical</td>
 <td>MX1960</td>
 <td></td>
 </tr>
 <tr>
 <td>Sirecust 404 Monitor</td>
 <td>Siemens</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ABL 700 Benchtop Analyzer</td>
 <td>Radiometer</td>
 <td></td>
 <td>for blood gas measurement</td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>Effenberger</td>
 <td>8319</td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum stage</td>
 <td>Alfun</td>
 <td>AW7022</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical microscope OPMI 6-SDFC</td>
 <td>Carl Zeiss</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Microsurgical instruments lab set</td>
 <td>S&amp;T</td>
 <td>767</td>
 <td></td>
 </tr>
 <tr>
 <td>Biemer vessel clip</td>
 <td>Diener</td>
 <td>64.562</td>
 <td></td>
 </tr>
 <tr>
 <td>Applying forceps</td>
 <td>Diener</td>
 <td>64.568</td>
 <td>for Biemer vessel clip</td>
 </tr>
 <tr>
 <td>Rhodamine 6G</td>
 <td>Sigma-Aldrich</td>
 <td>R4127</td>
 <td></td>
 </tr>
 <tr>
 <td>Vaseline white DAB</td>
 <td>Winthrop</td>
 <td>2726853</td>
 <td></td>
 </tr>
 <tr>
 <td>Cover glasses</td>
 <td></td>
 <td></td>
 <td>32x32 mm</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Intravital setup</td>
 </tr>
 <tr>
 <td>Zeis Axio Scope A-1 MAT</td>
 <td>Carl Zeis</td>
 <td>490036</td>
 <td>epifluorescence microscope</td>
 </tr>
 <tr>
 <td>470 nm LED</td>
 <td>Carl Zeis</td>
 <td>423052</td>
 <td>fluorescence light source</td>
 </tr>
 <tr>
 <td>Colibri 2 System</td>
 <td>Carl Zeis</td>
 <td>423052</td>
 <td></td>
 </tr>
 <tr>
 <td>W Plan-Apochromat 20x/1,0 DIC</td>
 <td>Carl Zeis</td>
 <td>421452</td>
 <td>water immersion objective</td>
 </tr>
 <tr>
 <td>AxioCam MRm Rev. 3 FireWire</td>
 <td>Carl Zeis</td>
 <td>426509</td>
 <td>high resolution digital camera</td>
 </tr>
 <tr>
 <td>Axio vision LE software</td>
 <td>Carl Zeis</td>
 <td>410130</td>
 <td>use for offline analysis</td>
 </tr>
 </tbody>
</table>

real-time digital imaging of leukocyte-endothelial interaction in ischemia-reperfusion injury (iri) of the rat cremaster muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Watch this Scientific Journal Video about Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle at JoVE.com

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

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

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

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

real-time-digital-imaging-leukocyte-endothelial-interaction-ischemia

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15.0K vues.  University of Freiburg Medical Centre. Numérique intravitale microscopie à épifluorescence des veinules post-capillaires de la microcirculation crémastérien est une méthode pratique pour obtenir un aperçu de leucocytes-endothélial interaction In vivo En cas d'ischémie-reperfusion (IRI) du tissu musculaire strié. Nous avons ici de fournir un protocole détaillé pour effectuer en toute sécurité de la technique et de discuter de ses applications et ses limites.