The research project was performed at the Jes&#250;s Us&#243;n Minimally Invasive Surgery Center (CCMIJU), part of the ICTS Nanbiosis. The study was performed with assistance from the following Nanbiosis units: U21, experimental operating room; U22, animal housing; and U14, cell therapy. This work was supported by ISCIII project PI16/02164 and by European Regional Development Fund (FEDER), a way to make Europe. The funder had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. Special thanks are extended to Mar&#237;a P&#233;rez for preparing the figures and to Fernanda Carrizosa for providing constant encouragement and supporting the scientific bibliography.

All procedures were conducted in accordance with the ethical committee of the Jes&#250;s Us&#243;n Minimally Invasive Surgery Centre and the welfare guidelines of the regional government that are based on European legislation.
1. Presurgical and Surgical Preparation
<ol>
	<li>House Wistar rats weighing 290&#8211;350 g in cages at 22&#8211;25 &#176;C with free access to food and water. Acclimate for 1 week prior to the surgery to prevent stress-induced problems.</li>
	<li>Place a rat in an anesthetic induction chamber, deliver 5 min of oxygen (0.5-1 L/min), and use a vaporizer to deliver 5% sevoflurane to induce anesthesia.</li>
	<li>Take the rat out of the chamber once anesthesia is induced. Place the inhalation facemask on the rat and provide a flow rate of 2% sevoflurane to maintain anesthesia. Check for the lack of response to a toe pinch.</li>
	<li>Use an eye protection ointment to prevent corneal drying and damage.</li>
	<li>Monitor the animal under general anesthesia as follows: Place a rectal thermometer (35.9&#8211;37.5 &#176;C), check the mucous membrane color, and position a rodent pulse oximeter to check for the O2 saturation (&#62;95%) and heart rate (250&#8211;450 bpm).</li>
	<li>Use a heat support (electric heating pads or circulating water blankets) to avoid hypothermia and improve post-procedural anesthesia recovery.</li>
	<li>Inject 5 mL of warm subcutaneous physiological saline solution to maintain proper hydration.</li>
	<li>Provide analgesic and anti-inflammatory drugs (meloxicam 1 mg/kg/day) and prophylactic antibiotics (enrofloxacin 7.5 mg/kg/day) subcutaneously before the procedure and for 5 days postoperatively.</li>
	<li>Shave the animal&#8217;s abdominal and inguinal areas.</li>
	<li>Apply topical povidone-iodine, followed by 70% ethanol. Cover the animal with a sterile drape.</li>
</ol>
2. Free Skin Flap Model Surgery
<ol>
	<li>Using a surgical marker, draw a 3 cm x 6 cm flap matching one of the 6 cm sides with the abdomen midline. Next, make a 6 cm skin incision at the midline of the abdomen and two perpendicular 3 cm incisions at the upper and lower part of the 6 cm midline incision.</li>
	<li>To start dissecting the designed 3 cm x 6 cm skin flap, use scissors and Adson forceps to raise the flap (rather than a scalpel) due to the skin&#8217;s mobility.</li>
	<li>Gently pull the flap from the cranial area towards the caudal area to help with the dissection and identify the epigastric pedicle surrounded by the abundant loose connective tissue.</li>
	<li>Dissect the flap pedicle without touching it or by grasping the adventitia as little as possible to avoid damaging the vessel wall.</li>
	<li>Use 8/0 nylon sutures to occlude by ligatures the proximal caudal femoral vessels, lateral circumflex femoral vessels, and saphenous vessels. Thereby, the perfusion of the flap is provided by the femoral artery and continues directly through the superficial caudal epigastric artery, while the venous drainage is performed by the superficial caudal epigastric vein toward the femoral vein.</li>
	<li>Clamp the vascular pedicle and then cut it to start the 8 h ischemia period.&#160;During the procedure use electric blankets to maintain temperature.&#160;Two 5 ml injections of warm (25&#176;C) 0.9% saline solution are administered subcutaneously. The first administration is performed 2 h after the beginning of the procedure; and the second at the end of the procedure to obtain a proper recovery of the animal.</li>
	<li>Use heparinized saline solution (100 U/mL) to perfuse the flap and remove the stagnant blood from the microcirculation.</li>
	<li>Use 10/0 nylon sutures to perform the microsurgical anastomoses.</li>
	<li>After 8 h of ischemia, reperfuse the flap by removing the microvascular clamps and check the vascular patency as described below.</li>
</ol>
3. Intraoperative Assessment
<ol>
	<li>Perform a manual patency test (empty and refill test) for the vein and artery. To do this, use two microsurgical forceps, position them distal to the anastomosis and perform the milking. Release the closest forceps to the anastomosis site first. If the blood flow continues after a vascular section is emptied, then the anastomosis is patent.</li>
	<li>Assess the blood flow using a transit-time ultrasound flowmeter and microsurgical probes.
	<ol>
		<li>Measure the diameter of the pedicle vessels in order to choose the proper size for the flow probes. 
		NOTE: A 0.7 mm flow probe can measure vessels ranging from 0.4 mm to 0.7 mm; a 1.0 mm flow probe can measure vessels ranging from 0.7 mm to 1.0 mm; a 1.5 mm flow probe can measure vessels ranging from 1.0 mm to 1.5 mm.</li>
		<li>Place the target vessel in the ultrasonic sensing window (between the reflector and the transducers) of the flow probe to quantify flow volume. 
		NOTE: Hold the probe neutral to the plane of the vessel, to avoid any tension or pulling.</li>
		<li>Check the quality of the acoustic coupling by observing that all the bars are green on the display. 
		NOTE: If difficult to get good acoustic coupling, use ultrasonic gel or physiological saline solution topically.</li>
		<li>When good coupling is achieved and the vessel is placed in the acoustic window without any tension, click on the Record button on the display to store the data. 
		NOTE: To obtain a reliable and correct measurement, make sure that the waveform pattern is constantly repeatable.</li>
	</ol>
	</li>
	<li>Once done, use Polyglycolic Acid (PGA) 4-0 absorbable braided sutures (16mm 3/8 triangular needle)&#160;&#160;to close the skin. Use a simple interrupted pattern to maintain strength and tissue position if part of the suture is bitten off by the rat postoperatively.</li>
	<li>Assess the flap&#8217;s microcirculation using laser speckle contrast analysis (LASCA).
	<ol>
		<li>Make a new recording for each animal and for each follow-up of the study. For this, click File/New Recording. A new window opens, and the Setup Panel is displayed. Then edit the information of the project name, subject, operator and recording name.</li>
		<li>For maximum reproducibility, standardize the following parameters: working distance, measurement area, point density, frame rate, and ambient light conditions.
		<ol>
			<li>Adjust the Working Distance by moving the laser in relation to the tissue. Zoom in or out the laser head towards the tissue of interest. To check for the measured value, click Image Setup. Here, set at 12.0 cm.</li>
			<li>Standardize the Measurement Area by entering the desired Width and Height at the Image Setup. The designed flap measures 3 cm x 6 cm. For this measurement, select a width of 4.0 cm and a height of 7.0 cm to have some extra space.</li>
			<li>Set the Point Density as high in the Image Setup. High, medium, and low are the three options.</li>
		</ol>
		</li>
		<li>At the Image Capture Setup, select the Frame Rate (10 images/s) for the recording and the Duration (1 min) of the recording. 
		NOTE: Have the same ambient light condition in the surgery room while operating or performing the assessments.</li>
		<li>Click the Record button to start recording. The Setup Panel is replaced by the Recording Panel. Data is saved automatically. Take snapshots during the procedure to enable further comparison. 
		NOTE: The perfusion scale can be changed to improve visualization (Click Tools | Filters and Color Scales | Perfusion Scale | Manual 0 - 150), but the measured perfusion values will be unaffected. Before and after the recording, different regions of interest (ROIs) can be created to measure the perfusion within them. Here, we evaluated just the area of the practiced flap (3 cm x 6 cm).</li>
	</ol>
	</li>
	<li>Use ImageJ software to measure the survival and necrosis areas.
	<ol>
		<li>Locate a ruler at the side of the flap and then take control pictures for macroscopic measurements of the flap survival area.</li>
		<li>To evaluate pictures, open the ImageJ user interface. Click File and Open the image to be measured.</li>
		<li>Select Straight Line at the toolbox and draw a straight line over 1 cm of the ruler. Click on Analyze | Set Scale and introduce in the text box for known distance the value of 1 cm.</li>
		<li>Click on the Polygon Selection Tool and draw the polygon lines over the flap to calculate the viable area. Ultimately, click on Analyze | Measure to obtain the area value.</li>
	</ol>
	</li>
	<li>Place a postoperative dressing on the animal before housing to prevent self-mutilation of the surgical area.&#160;After the procedures, animals are&#160;housed in cages individually, in a room with temperature control (22&#176;C to 25&#176;C).</li>
</ol>
4. Postoperative Assessment and Tissue Sampling
<ol>
	<li>Anesthetize the rat at 7 postoperative days for the flap assessment and tissue sampling by following the same steps previously described in this protocol (steps 1.2 and 1.3). Check for the depth of anesthesia by the lack of response to the toe pinch.</li>
	<li>Photograph the surgical area to enable macroscopic measurements of the flap survival and necrosis areas. Make the postoperative macroscopic measurements following the same steps of the intraoperative assessment that have been explained before in the protocol (step 3.5). 
	NOTE: Pay attention while using the Polygon Selection Tool by drawing the lines on the flap delimiting the viable area (measured in cm2). The percentage of the viable area can be calculated as (cm2 of viable area/cm2 of total flap area) &#215; 100.</li>
	<li>Assess the flap&#8217;s microcirculation using the LASCA technique (step 3.4) to visualize and quantify perfusion differences</li>
	<li>After the macroscopic analysis, remove the 4/0 sutures and rise the flap to reassess the vascular pedicle blood flow by using the transit-time ultrasound.</li>
	<li>Perform tissue sampling by longitudinally dividing the flap into two parts of 1.5 cm x 6 cm.
	<ol>
		<li>Immerse one part in a biopsy container with 4% paraformaldehyde at room temperature for further histological analyses.</li>
		<li>Introduce the other part of the tissue in a cryopreservation tube, immerse it in liquid nitrogen and then, cryopreserve the tube by storing it at -80 &#176;C for future molecular analyses.</li>
	</ol>
	</li>
	<li>Euthanize the rat under general inhalation anesthesia using a rapid intracardiac injection of 2 M KCl/kg according to the ethical committee recommendations.</li>
</ol>

<ol>
	<li>Siemionow, M., Arslan, E. <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+injury:+a+review+in+relation+to+free+tissue+transfers.">Ischemia/reperfusion injury: a review in relation to free tissue transfers.</a> Microsurgery. 24 (6), 468-475 (2004).</li><li>Wang, W. Z., Baynosa, R. C., Zamboni, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+ischemia-reperfusion+injury+for+the+plastic+surgeon:+2011.">Update on ischemia-reperfusion injury for the plastic surgeon: 2011.</a> Plastic and Reconstructive Surgery. 128 (6), 685-692 (2011).</li><li>Chen, K. T., 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=Timing+of+presentation+of+the+first+signs+of+vascular+compromise+dictates+the+salvage+outcome+of+free+flap+transfers.">Timing of presentation of the first signs of vascular compromise dictates the salvage outcome of free flap transfers.</a> Plastic and Reconstructive Surgery. 120 (1), 187-195 (2007).</li><li>Sears, E. D., Chung, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replantation+of+finger+avulsion+injuries:+a+systematic+review+of+survival+and+functional+outcomes.">Replantation of finger avulsion injuries: a systematic review of survival and functional outcomes.</a> The Journal of Hand Surgery. 36 (4), 686-694 (2011).</li><li>Caterson, E. J., Lopez, J., Medina, M., Pomahac, B., Tullius, S. G. <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+injury+in+vascularized+composite+allotransplantation.">Ischemia-reperfusion injury in vascularized composite allotransplantation.</a> The Journal of Craniofacial Surgery. 24 (1), 51-56 (2013).</li><li>Amin, K. R., Wong, J. K. F., Fildes, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+Reduce+Ischemia+Reperfusion+Injury+in+Vascularized+Composite+Allotransplantation+of+the+Limb.">Strategies to Reduce Ischemia Reperfusion Injury in Vascularized Composite Allotransplantation of the Limb.</a> The Journal of Hand Surgery. 42 (12), 1019-1024 (2017).</li><li>Barre-Sinoussi, F., Montagutelli, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+are+essential+to+biological+research:+issues+and+perspectives.">Animal models are essential to biological research: issues and perspectives.</a> Future Science OA. 1 (4), (2015).</li><li>Shaughness, G., Blackburn, C., Ballestin, A., Akelina, Y., Ascherman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Thrombosis+Formation+in+1-mm-Diameter+Arterial+Anastomoses+with+Transit-Time+Ultrasound+Technology.">Predicting Thrombosis Formation in 1-mm-Diameter Arterial Anastomoses with Transit-Time Ultrasound Technology.</a> Plastic and Reconstructive Surgery. 139 (6), 1400-1405 (2017).</li><li>Ballestin, A., 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=Ischemia-reperfusion+injury+in+a+rat+microvascular+skin+free+flap+model:+A+histological,+genetic,+and+blood+flow+study.">Ischemia-reperfusion injury in a rat microvascular skin free flap model: A histological, genetic, and blood flow study.</a> PloS One. 13 (12), 0209624 (2018).</li><li>Kochi, T., 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=Characterization+of+the+Arterial+Anatomy+of+the+Murine+Hindlimb:+Functional+Role+in+the+Design+and+Understanding+of+Ischemia+Models.">Characterization of the Arterial Anatomy of the Murine Hindlimb: Functional Role in the Design and Understanding of Ischemia Models.</a> PloS One. 8 (12), (2013).</li><li>Smit, J. M., Zeebregts, C. J., Acosta, R., Werker, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+in+free+flap+monitoring+in+the+last+decade:+a+critical+review.">Advancements in free flap monitoring in the last decade: a critical review.</a> Plastic and Reconstructive Surgery. 125 (1), 177-185 (2010).</li><li>Krag, C., Holck, 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+value+of+the+patency+test+in+microvascular+anastomosis:+Correlation+between+observed+patency+and+size+of+intraluminal+thrombus:+An+experimental+study+in+rats.">The value of the patency test in microvascular anastomosis: Correlation between observed patency and size of intraluminal thrombus: An experimental study in rats.</a> British Journal of Plastic Surgery. 34, 64-66 (1981).</li><li>Selber, J. C., 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+prospective+study+of+transit-time+flow+volume+measurement+for+intraoperative+evaluation+and+optimization+of+free+flaps.">A prospective study of transit-time flow volume measurement for intraoperative evaluation and optimization of free flaps.</a> Plastic and Reconstructive Surgery. 131 (2), 270-281 (2013).</li><li>Briers, D., 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=Laser+speckle+contrast+imaging:+theoretical+and+practical+limitations.">Laser speckle contrast imaging: theoretical and practical limitations.</a> Journal of Biomedical Optics. 18 (6), 066018 (2013).</li><li>Zotterman, J., Bergkvist, M., Iredahl, F., Tesselaar, E., Farnebo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+partial+and+full+venous+outflow+obstruction+in+a+porcine+flap+model+using+laser+speckle+contrast+imaging.">Monitoring of partial and full venous outflow obstruction in a porcine flap model using laser speckle contrast imaging.</a> Journal of Plastic, Recontructive and Aesthetic Surgery. 69 (7), 936-943 (2016).</li><li>Zotterman, J., Tesselaar, E., Farnebo, 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+use+of+laser+speckle+contrast+imaging+to+predict+flap+necrosis:+An+experimental+study+in+a+porcine+flap+model.">The use of laser speckle contrast imaging to predict flap necrosis: An experimental study in a porcine flap model.</a> Journal of Plastic, Recontructive and Aesthetic Surgery. 72 (5), 771-777 (2019).</li></ol>

The authors have nothing to disclose.

Microsurgical free tissue transfers have become the method of choice for reconstructing large defects. A period of ischemia occurs during such free tissue transfers. When this period exceeds the tissue&#8217;s tolerance, I/R injury can cause failure of the practiced free flap9. The description of the methodology to develop a cost-effective and translational preclinical model to study I/R injury in reconstructive microsurgery may help lead the study of different compounds to counteract this pathophysiological process.
In the described animal model, after the vascular ligatures were placed and the free flap was raised, no hindlimb blood flow compromises were noted, nor pain or limp. As Kochi et al.10 described, our model also left three collateral routes through intramuscular networks.
Monitoring of free flaps is of major importance11, as salvage is inversely related to the duration between ischemia onset and its clinical recognition. For this purpose, free flaps should be studied intra- and postoperatively.
Intraoperatively, the widely used empty and refill test or the acoustic Doppler enable identification but not quantification of flow presence or absence through an anastomosis12. For this reason, we used transit-time ultrasound technology, a novel method that allows surgeons to quantify the blood flow of microsurgical anastomoses13. In our study, all microsurgical anastomoses were patent after 8 h of ischemic insult as well as at the end of the study. Immediately after the creation of the microsurgical anastomoses, we noted higher blood flow volumes than the minimums recommended in literature8. This predicted good pedicle perfusion at the end of the study, demonstrating that the results were not influenced by the microsurgical technique but rather by the I/R injury cascade of events. However, this technique is not free of limitations. To obtain reliable results, the microsurgical probes must be held neutral to the plane of the vessel, not pulling it or creating any tension. A good acoustic coupling is needed to obtain a proper signal, which can be achieved using ultrasonic gel or saline. A high-quality coupling signal, provided by the equipment, is an important parameter to consider during the measurements.
We have used LASCA, also known as laser speckle contrast imaging or laser speckle imaging, postoperatively14. This technology represents a valuable technique for semi-quantitative real-time mapping of flow within free flaps as verified here. One of the limitations is that the results are provided in arbitrary units and not directly related to actual flow values. In this sense, further research is needed to validate this correlation. Laser Doppler flowmetry is more commonly used but limited by the fact that it only measures perfusion in a single point in the flap, whereas LASCA allows the detection of regional changes in skin perfusion within the flap15. Furthermore, a recent study16 indicated that LASCA may perioperatively predict the regions at high risk of postoperative flap necrosis. Our results suggest that LASCA is a promising technique for the peri- and postoperative monitoring of free flaps.

Microsurgery has become a common surgical technique for reconstruction that allows for interventions (e.g., free tissue transfers) to restore complex tissue defects, replantation of amputated limbs, and even composite tissue allotransplantations.
Microsurgical reconstructions are ideal for a wide variety of defects caused by traumatic injuries, burns, or oncological resections. However, there is a low percentage of free flap failure, among which ischemia-reperfusion (I/R) injury is one of the main responsible factors. All microsurgically transferred tissues endure a mandatory period of ischemia followed by the reperfusion. This period of primary ischemia is usually well tolerated; thus, the success rate of microsurgical procedures exceeds 90%1,2. However, only 63.7% of flaps requiring surgical revision can be completely saved3. In addition, in cases of replantation of finger avulsion injuries, the success rate is 66%4; and in cases of composite tissue allotransplantation suffering I/R injury, rejection percentages are increased since the I/R injury activates innate immunity5,6.
Therefore, the study of this pathophysiological phenomenon is of interest. Animal models are essential for investigating physiological mechanisms and assessing novel therapies before it can be applied to humans7. Vessel anatomy and the physiological similarities between rats and human beings make rats an ideal model for the investigation of biological processes such as I/R injury.
Here, we present a detailed protocol for the creation of a rat free skin flap model with I/R injury, as well as different possibilities for intra- and postoperative assessments. The overall goal of this method is to describe a useful preclinical model to study I/R injury and possible treatments to reduce its related damages.

<table border="0" cellpadding="0" cellspacing="0" style="width:681px;" width="681">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">AureFlo Unit</td>
			<td style="width:121px;">Transonic (Ithaca, USA)</td>
			<td style="width:161px;">N/A</td>
			<td style="width:167px;">Transit-time ultrasound flowmeter equipment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Commbined Basic Hand- and Reconstructive Surgery Set (round handle)</td>
			<td style="width:121px;">S&#38;T AG (Neuhausen, Switzerland)</td>
			<td style="width:161px;">RHR-SET. Art.No.00795</td>
			<td>Set of microsurgical instruments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">FLOW-i</td>
			<td style="width:121px;">Maquet Critical Care AB (Solna, Sweeden)</td>
			<td style="width:161px;">N/A</td>
			<td>Anesthesia Delivery System</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Micro clamps ABB-1</td>
			<td style="width:121px;">S&#38;T AG (Neuhausen, Switzerland)</td>
			<td style="width:161px;">00408V</td>
			<td>Double microvascular clamp with frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Micro clamps ABB-11</td>
			<td style="width:121px;">S&#38;T AG (Neuhausen, Switzerland)</td>
			<td style="width:161px;">00414V</td>
			<td>Double microvascular clamp without frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Micro clamps B-1</td>
			<td style="width:121px;">S&#38;T AG (Neuhausen, Switzerland)</td>
			<td style="width:161px;">00396V</td>
			<td>Sigle microvascular clamp</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:232px;">Nylon suture 10/0</td>
			<td style="width:121px;">Laboratorio Arag&#243; (Barcelona, Spain)</td>
			<td style="width:161px;">19921</td>
			<td>Microsurgical suture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">OPMI Pentero 800</td>
			<td style="width:121px;">Carl Zeiss AG (Oberkochen, Germany)</td>
			<td style="width:161px;">N/A</td>
			<td>Surgical microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">PeriCam PSI System</td>
			<td style="width:121px;">Perimed AB (J&#228;rf&#228;lla, Sweden)</td>
			<td style="width:161px;">N/A</td>
			<td>Laser speckle contrast analysis equipment</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:232px;">Philips Intellivue MX450</td>
			<td style="width:121px;">Philips Medizin Systeme (B&#246;blingen, Germany)</td>
			<td style="width:161px;">N/A</td>
			<td>Monitoring system</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:232px;">Protector posoperatorio para roedores</td>
			<td style="width:121px;">Fundaci&#243;n Centro de Cirug&#237;a de M&#237;nima Invasi&#243;n Jes&#250;s Us&#243;n (C&#225;ceres, Spain)</td>
			<td style="width:161px;">P201400272</td>
			<td>Postoperative protector for rodents</td>
		</tr>
	</tbody>
</table>

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.

Immediately after the creation of the microsurgical anastomoses, we obtained higher blood flow volumes than the minimum flows recommended in the literature8; thus, all microsurgical anastomoses were patent 1 week after the surgery (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60292/60292fig1.jpg" /> 
Figure 1: Transit-time ultrasound blood flow assessment. (A) Position of the microsurgical flow probe to assess blood flow. (B) Blood flow pattern and quantification obtained of the anastomosed vessels of the flap pedicle. <a href="https://www.jove.com/files/ftp_upload/60292/60292fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Observation of the microcirculatory deprivation of blood flow during the ischemic insult was possible with the LASCA technique, including the immediate hyper perfusion during the flap reperfusion, and, perioperatively, the different areas with less perfusion and a higher risk of postoperative flap necrosis that were indeed necrotized 7 days after the end of the study (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60292/60292fig2.jpg" /> 
Figure 2: Laser speckle contrast analysis technology. (A) Visualization of microcirculatory tissue blood perfusion in the physiological condition. (B) Visualization of microcirculatory tissue blood perfusion during ischemia. (C) Visualization of microcirculatory tissue blood perfusion immediately after reperfusion. <a href="https://www.jove.com/files/ftp_upload/60292/60292fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The flap survival area after 8 h of ischemia and its subsequent reperfusion was around 40%. Previously published results9 showed statistically significant differences when this model was compared with flaps where no ischemic insult was inflicted.

Watch this Scientific Journal Video about A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery at JoVE.com

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

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 ...

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7.2K Views.  Jesús Usón Minimally Invasive Surgery Centre. 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.

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

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 ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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 ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

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, ...