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 cervical area. </li>
 <li>Place the rat in dorsal recumbency on a heating pad to maintain body temperature at 37 &deg;C and apply isoflurane at 2 vol% using a silicone mask. </li>
 </ol>
 The following preparation steps are best accomplished using a surgical microscope.
 <ol start="4">
 <li>For the preparation of the trachea, the carotid artery and the jugular vein, perform a 2 cm horizontal skin incision in the area of the suprasternal notch and mobilize the salivary glands laterally. </li>
 <li>You now face the ventral neck muscles. Carefully separate them in the midline and find the trachea. Expose 1 - 2 cm of the trachea and place a micro forceps under it so as to raise it up. </li>
 <li>Now slit about halfway through the ventral side of the trachea. Be careful not to cut all the way through, if you do, the cut end of the trachea will slip back into the chest and be very difficult to work with. </li>
 <li>Insert an Abbocath tube (14G) used as tracheal tube into the lower part of the trachea, which has previously been connected to an animal ventilator. Tip: Firm fixation on magnet clamps as well as suturing of the trachea around the tube is essential to keep the tracheal tube in place. Generally we use Terylene 5/0 suture, however similar suture material can be used. </li>
 <li>Respiration can then be volume controlled (frequency, 35 - 45 breaths/minute; tidal volume, 4.5 - 5 ml; FiO2, 0.35 - 0.50; Isoflurane 1.5 - 2 vol%).14 Atelectasis can be prevented by maintaining a positive end-expiratory pressure of 5 to 10 mm of H2O.15 </li>
 <li>For the carotid canulation, the right sternohyoid muscle is separated by blunt dissection to locate the carotid artery.</li>
 <li>Separate the vagus nerve carefully from the carotid artery and mount the artery on an angled micro forceps to stop blood flowing from the heart. </li>
 <li>Pass two pieces of equal length of suture beneath the carotid artery. In reference to the heart, the more distal suture is tied tightly to occlude blood flowing from the head region whereas the proximal suture is tied loosely around the carotid artery. </li>
 <li>Using micro scissors a small cut is made into the carotid artery between the two ligatures.</li>
 <li>Insert a polyethylene catheter (0.28 mm inner diameter) filled with normal saline solution that is connected to a pressure transducer. </li>
 <li>Remove the micro forceps and thread the catheter further into the artery. Then tighten the proximal ligature around the artery and catheter. Tip: Application of a second proximal suture prevents bleeding after removing the micro forceps. </li>
 <li>Tie the distal ligature around the carotid and the catheter for additional anchorage. </li>
 <li>Monitor anesthesia continuously by monitoring heart rate and arterial pressure. Perform intermittent arterial blood gas analyses using a blood gas analyzer.16 A heart rate lower than 300 bpm or exceeding 360 bpm as well as a mean arterial pressure dropping below 80 mmHg for longer than 5 minutes are criteria for exclusion.17, 18 Maintain blood pH within physiological limits (7.35 - 7.45). In case the experiment has to be terminated due to abnormal monitoring rates, scarify the animal by neck dislocation or exanguation. </li>
</ol>
For the intravenous application of fluorescent dyes or other drugs of interest, perform the following:
<ol start="17">
 <li>Focus the area of the left jugular vein with the surgical microscope. </li>
 <li>With forceps in each hand, tear the thin fascia to reveal the jugular vein. Mount the vein on an angled micro forceps. Blood flow will then stop. </li>
 <li>Using forceps, two pieces of equal length of suture are passed under the jugular vein concordant to the carotid canulation. Tie the distal suture tightly and the proximal suture loosely around the jugular vein. </li>
 <li>Make a small cut into the jugular vein and insert a polyethylene catheter (0.28 mm inner diameter) that has been flushed with saline. Mark that incision and canulation can be more demanding and time consuming than that of the carotid. </li>
 <li>Thread the catheter toward the heart and tighten the ligature closest to the heart around the vein and the catheter. </li>
 <li>After conforming patency, tie distal ligature around the catheter. Tip: Additional fixation of both catheters with tape may prevent dislodgment. </li>
 <li>Rinse catheters frequently to prevent intraluminal clotting. </li>
</ol>
2. Preparation of the Cremaster Muscle
<ol>
 <li>We use an aluminium stage of 1.5 - 2 cm thickness for cremasteric imaging. The stage rapidly adopts the desired temperature of the heating pad thus alleviating thermal control of the cremasteric tissue. This is crucial for inflammation studies. Alternatively a Plexiglas platform may be used though regulation of cremasteric temperature is more difficult. Place the scrotum in the centre of the stage. </li>
 <li>The initial incision is made in the skin and external spermatic fascia above the scrotum in the very distal end followed by cautious dilatation of the subdermal space using fine scissors. Avoid touching the underlying tissue with the instruments. </li>
 <li>As soon as the tissue is exposed it is moistened with pre-heated (37 &deg;C) phosphate buffered saline solution with calcium and magnesium. Apply the solution regularly to any exposed tissue.</li>
 <li>Connective tissue between the external spermatic fascia and the cremaster muscle is carefully removed to free the cremaster muscle from surrounding tissue. </li>
 <li>The exterior surface of the cremaster muscle is then cautiously cleared of connective tissue. Continuous superfusion during dissection hydrates the connective tissue, facilitating visibility and removal. </li>
 <li>Suture the distal end of the cremaster sack to hold down and slightly extend the end of the sack. </li>
 <li>Incise the distal end of the sack on the ventral aspect and elongate the incision proximally using micro scissors. Carefully cauterize bleeding vessels along the incision lines utilizing a thermal cautery. Avoid unnecessary cauterization to limit additional inflammatory stimuli. Tip: Blood flow dynamics near the periphery of the preparation are altered by this tissue damage.19 Therefore, to minimize these affects on data collection, blood vessels near the center of the preparation should be used for imaging. </li>
 <li>The open cremaster lies flat on the aluminum pedestal though still connected by a thin ligament to the epididymis underneath the testicle. Reflecting the testicle to one side exposes this ligament including a small artery and vein which connect to the epididymis. Use the cautery to seal the vessels and employ the micro scissors to cut the connective ligament between testicle and cremasteric tissue.</li>
 <li>Gently push back the isolated testicle into the inguinal canal. Other authors resect the testicle after proximal ligature (orchiectomy) along with the associated inguinal fad pad.20 In a sensitive model as IRI induced leukocyte activation we try to avoid all additional surgical stimuli, therefore we refrain from resecting the testis, which is usually not necessary to get sufficient exposure of the cremaster muscle. </li>
 <li>In addition to the first fixation suture four edges of tissue (two on each side) and attach the threads to tapes to cautiously spread the cremasteric tissue radially on the aluminum stage. Leaving a gap between the aluminum stage and the external entrance of the inguinal canal simplifies subsequent cremasteric clipping for ischemia. </li>
 <li>Place two ends of polyethylene catheter (0.28 mm inner diameter) close to the cremasteric tissue for the superfusion setup. Make sure that the lumen is free of air to avoid air bubbles in the eventual fluid chamber. </li>
 <li>Draw a line of petroleum jelly (Vaseline) around the cremaster muscle using a syringe. The line must extend the size of the cover slip that is used for coverage. </li>
 <li>To create a fluid chamber, place a square cover slip (32&times;32 mm) over the cremasteric tissue and firmly attach the edges to the Vaseline line. </li>
 <li>In case the cremasteric tissue is not superfused with a specific drug, continuous replacement of the surrounding fluid is nonessential. A single application of phosphate buffered saline solution with calcium and magnesium into the created chamber may then be sufficient. Local stimulation with drugs however should be performed continuously via micro-perfusion pump at a rate of 3 ml per hour. </li>
 <li>The cremasteric tissue is now ready for microscopic imaging. </li>
</ol>
3. Intravital Setup
The basic intravital setup may vary. For epifluorescence imaging the experiments should be executed in a dark room.
<ol>
 <li>The animal is transferred to the stage of an intravital epifluorescence microscope equipped with a 470 nm LED luminous source for epi-illumination. Use a water immersion objective (20&times;/1.0) to achieve magnification of approximately 800&times;. Record observations by means of a high resolution digital camera and store records on a personal computer for offline evaluation. </li>
 <li>For leukocyte labeling, inject rhodamine 6G intravenously via the jugular catheter at concentrations of 0.4 mg/kg body weight.</li>
 <li>Choose a postcapillary venule for observation. Vessel size should range between 20-60 &mu;m and blood flow should be sufficient. To minimize the influence of pre-activation of the tissue, only vessels in which leukocyte rolling is &lt;20 cells/30 seconds and the number of adherent cells &lt;10 cells/200&mu;m of venular endothelium may be utilized for further analysis. </li>
 <li>If possible, up to three postcapillary venules can be used for observation; however they should be located in a corresponding section of the cremasteric tissue to avoid confusing the vessels at different time points. </li>
</ol>
4. Ischemia-reperfusion Injury (IIR)
<ol>
 <li>Let tissue stabilize for 30 minutes. </li>
 <li>Perform a recording of 30 seconds to establish basal values for leukocyte rolling and adherence. Ideally, generate in total three basal recordings to verify cell numbers and to obtain standard deviations. </li>
 <li>Gently place a Biemer vessel clip around the very proximal end of the exposed cremasteric tissue by using the application forceps. Stasis should occur immediately and can be visualized by epifluorescence microscopy in the observed vessel section. </li>
 <li>A variety of time courses can be utilized depending on the level of damage required. We apply 30 minutes of ischemia time as described by other authors.21-23 Remove the vessel clamp afterwards. Allow blood stream to stabilize for another 15 minutes. </li>
 <li>Subsequent recordings of 30 seconds duration can be made throughout the reperfusion period (e.g. 30 seconds every 15 minutes). Save recordings digitally for offline analysis. </li>
 <li>For the termination of the experiment, the rat is euthanized by cervical dislocation under sufficient anesthesia by pressing a blunt instrument such as the dull edge of a scissor blade at the base of the skull. With the other hand, the base of the tail is quickly pulled, causing separation of the cervical vertebrae from the skull. </li>
</ol>
5. Offline Video Playback Analysis
<ol>
 <li>For offline video playback analysis it is helpful to use software that allows selection of distinct pictures and observation of the video sequence in slow motion. Adjust contrast and brightness appropriately. We use software provided by the manufacturer of the microscope that allows length measurements and digital magnification. </li>
 <li>For the quantification of leukocyte rolling, define a virtual line that intersects the vessel vertically that is consistent in all records. Count the number of rolling leukocytes that pass the line within 30 seconds manually. </li>
 <li>For the quantification of adherent leukocytes, define a 200 &mu;m vessel section that is consistent in all records.23 Count the number of clearly visible leukocytes that remain static during 30 seconds - thus defined as adherent.24 </li>
</ol>
6. Representative Results
IRI of the cremaster muscle has no effect on mean arterial pressure (MAP), heart rate and blood pH
Using the aforementioned setup (Figure 1), we investigated the microcirculation in IRI over a two hours protocol; however a much longer observation time up to 6 hours is possible. As shown in Figure 2, IRI of the cremaster muscle has no significant macrohemodynamic effects on the rat circulation as mean arterial pressure and heart rate remain stable throughout the investigation period. Furthermore we monitored homeostasis by frequent measurements of the arterial blood-pH that ranged within physiological limits and showed no significant inter-group differences. 
IRI induces leukocyte rolling in the cremasteric circulation
Leukocyte endothelial interaction is a key event in the acute inflammation. By means of intravital microscopy we found a time-dependent increase in the number of rolling leukocytes in IRI of the cremaster muscle (Figure 3A) concordant to previous data 21, 25. Rolling mounted over the two hours observation time to a maximum of 137.63 &plusmn; 22.55 % of the baseline value after 120 minutes of reperfusion time and then reached statistical significance compared to sham operated animals (137.63 &plusmn; 22.55 vs 99.43 &plusmn; 14.04 % of baseline value).
IRI induces leukocyte adhesion in the cremasteric circulation
For further evaluation of leukocyte-endothelial interaction we analyzed leukocyte adhesion in a 200 &mu;m vessel section. IRI induced an increase in the number of adherent leukocytes that significantly exceeded values of sham operated animals after 60 minutes (118.33 &plusmn; 6.83 vs. 96.27 &plusmn; 5.78 % of baseline value) and further ascended by 120 minutes of reperfusion (Figure 3B).
In summary the described in vivo model supplies consistent data of acute leukocyte activation in IRI whilst animal survival and circulation stability is warranted.
<img src="/files/ftp_upload/3973/3973fig1.jpg" alt="Figure 1" /> 
Figure 1. A. Flow chart for intravital epifluorescence microscopy performance in ischemia-reperfusion injury of the rat cremaster muscle. Following required preparations for anesthesia and monitoring, the rat cremaster muscle is exposed for imaging. Records of leukocyte activation are taken before and after tissue ischemia. Subsequent video analysis is best performed offline. B. Schematic figure on the proposed intravital setup. <a href="https://www.jove.com/files/ftp_upload/3973/3973fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3973/3973fig2.jpg" alt="Figure 2" /> 
Figure 2. IRI of the cremaster muscle has no effect on mean arterial pressure (MAP), heart rate and blood pH. Mean arterial pressure (A) and heart rate (B) were monitored every 30 minutes throughout the experiment via pressure transducer after canulation of the right carotid artery. Measurement of the arterial blood-pH (C) was performed after 0, 60, and 120 minutes. Values are mean &plusmn;SEM of 6 different rats and ranged on physiological levels without significant inter-group differences. <a href="https://www.jove.com/files/ftp_upload/3973/3973fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3973/3973fig3.jpg" alt="Figure 3" /> 
Figure 3. IRI increases leukocyte-endothelial interaction in the cremasteric circulation. After labeling of leukocytes with rhodamine 6G (0.4 mg/kg body weight) intravital epifluorescence microscopy was used to determine leukocyte-endothelial interaction in reperfusion injury over a 120 minutes protocol after 30 minutes of tissue ischemia. Records were taken at magnification of approximately &times;800. A. IRI significantly increases the number of rolling leukocytes in postcapillary venules of the cremaster muscle by 120 minutes of reperfusion whereas leukocyte rolling remains stable throughout the experiment in cremasteric tissue that did not undergo ischemia. Values are mean &plusmn; SEM of 6 observed rats. # p&lt;0.05 using the unpaired t test. B. The number of adherent leukocytes is significantly increased in a randomly chosen 200&mu;m postcapillary vessel section after 30 minutes of cremasteric ischemia and subsequent 60 minutes of tissue reperfusion. Results get even more pronounced after a two hours reperfusion period. Values are mean &plusmn; SEM of 6 observed rats. # p&lt;0.05 using the unpaired t test. C. Representative pictures of a postcapillary venule prior to ischemia (left) and 120 minutes after IRI (right). <a href="https://www.jove.com/files/ftp_upload/3973/3973fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiology and pathophysiology of leukocyte adhesion. , 520 (1995).</li><li>Baatz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+white+blood+cell+staining+by+intravascular+administration+of+rhodamine+6G.">Kinetics of white blood cell staining by intravascular administration of rhodamine 6G.</a> Int. J. Microcirc. Clin. Exp. 15, 85-91 (1995).</li><li>Mempel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr. Opin. Immunol. 16, 406-417 (2004).</li><li>Abbitt, K. B., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fluorescent+dyes+on+selectin+and+integrin-mediated+stages+of+adhesion+and+migration+of+flowing+leukocytes.">Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes.</a> J. Immunol. Methods. 239, 109-119 (2000).</li></ol>

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

Research

JoVE Journal

Medicine

15.1K Views.  University of Freiburg Medical Centre. الرقمية intravital المجهري epifluorescence من الوريدات شعيرة وريدية في دوران الأوعية الدقيقة مشمري هو وسيلة مريحة للحصول على نظرة ثاقبة الكريات البيض البطاني تفاعل في الجسم الحي في الإسكيمية-ضخه الاصابة (IRI) من الأنسجة العضلية المخططة. ونحن نقدم هنا بروتوكولا مفصلة...

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

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

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

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

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

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

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

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...