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><tbody><tr><td>AureFlo Unit</td><td>Transonic (Ithaca, USA)</td><td>N/A</td><td>Transit-time ultrasound flowmeter equipment</td></tr><tr><td>Commbined Basic Hand- and Reconstructive Surgery Set (round handle)</td><td>S&amp;amp;T AG (Neuhausen, Switzerland)</td><td>RHR-SET. Art.No.00795</td><td>Set of microsurgical instruments</td></tr><tr><td>FLOW-i</td><td>Maquet Critical Care AB (Solna, Sweeden)</td><td>N/A</td><td>Anesthesia Delivery System</td></tr><tr><td>Micro clamps ABB-1</td><td>S&amp;amp;T AG (Neuhausen, Switzerland)</td><td>00408V</td><td>Double microvascular clamp with frame</td></tr><tr><td>Micro clamps ABB-11</td><td>S&amp;amp;T AG (Neuhausen, Switzerland)</td><td>00414V</td><td>Double microvascular clamp without frame</td></tr><tr><td>Micro clamps B-1</td><td>S&amp;amp;T AG (Neuhausen, Switzerland)</td><td>00396V</td><td>Sigle microvascular clamp</td></tr><tr><td>Nylon suture 10/0</td><td>Laboratorio Arag&amp;oacute; (Barcelona, Spain)</td><td>19921</td><td>Microsurgical suture</td></tr><tr><td>OPMI Pentero 800</td><td>Carl Zeiss AG (Oberkochen, Germany)</td><td>N/A</td><td>Surgical microscope</td></tr><tr><td>PeriCam PSI System</td><td>Perimed AB (J&amp;auml;rf&amp;auml;lla, Sweden)</td><td>N/A</td><td>Laser speckle contrast analysis equipment</td></tr><tr><td>Philips Intellivue MX450</td><td>Philips Medizin Systeme (B&amp;ouml;blingen, Germany)</td><td>N/A</td><td>Monitoring system</td></tr><tr><td>Protector posoperatorio para roedores</td><td>Fundaci&amp;oacute;n Centro de Cirug&amp;iacute;a de M&amp;iacute;nima Invasi&amp;oacute;n Jes&amp;uacute;s Us&amp;oacute;n (C&amp;aacute;ceres, Spain)</td><td>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. Ischemie-reperfusie letsel is de belangrijkste oorzaak van flap falen in reconstructieve microchirurgie. Deze video beschrijft een preklinisch diermodel voor de studie van deze pathofysiologie. Dit gratis huidflapmodel op basis van de oppervlakkige caudale epigastrische vaten in de rat kan de evaluatie van verschillende therapieën en verbindingen mogelijk maken om ischemie-reperfusieletselgerelateerde schade tegen te gaan.Microchirurgische reconstructies zijn ideaal voor een breed sca...

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Neuroscience

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 ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

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

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