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. This video describes a pre-clinical animal model for the study of this pathophysiology. This free skin flap model based on the superficial caudal epigastric vessels in the rat may allow the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury related damages.

Microsurgical reconstructions are ideal for a wide variety of defects. However, these procedures ensure a mandatory period of ischemia followed by the reperfusion. This period is usually well-tolerated, and the success rate of microsurgical procedures exceeds 90%However, only 73.7%of flaps requiring surgical revision can be completely safe.

In addition, in cases of replantation of finger avulsion injuries, the success rate is about 60%and in cases of composite tissue re-transplantation, suffering ischemia-reperfusion injury projection percentages are increased since injury activates innate immunity. Therefore, the study of this pathophysiological phenomenon is of interest. To begin this procedure, use a surgical marker to draw a three centimeter by six centimeter flap, making sure that one of the six centimeter sides matches the abdomen midline.

Next, make a six centimeter skin incision at the midline of the abdomen, two perpendicular three centimeter incisions, as well as one of six centimeters parallel to the first incision. To start dissecting the designated skin flap, use scissors and Adson forceps to raise the flap. Gently pull the flap from the cranial area towards the caudal area.

Dissect the flap pedicle without touching it or by grasping the adventitia as little as possible to avoid damaging the vessel wall. Use 8-0 nylon sutures to occlude by ligatures the saphenous vessels, lateral circumflex femoral vessels, and the proximal caudal femoral vessels. Clamp the vascular pedicle and then cut it to start the eight hour ischemia period.

Use heparinized saline solution to perfuse the flap, then remove the stagnant blood from the microcirculation. Use 10-0 nylon sutures to perform the microsurgical anastomoses. After eight hours of ischemia, reperfuse the flap by removing the microvascular clamps, and check the vascular patency.

First, assess the blood flow using a transit-time ultrasound flowmeter and microsurgical probes. Place the target vessel in the ultrasonic sensing window of the flow probe to quantify flow volume. 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.

After this, use 4-0 absorbable sutures to close the skin. To assess the flap's microcirculation, use laser speckle contrast analysis. Make a new recording for each sample and for each followup study by clicking on file, new recording.

A new window will open which displays the setup panel. Here, edit the information for the project name, subject, operator, and recording name. Adjust the working distance by moving the laser in relation to the tissue.

Zoom the laser head in or out towards the tissue of interest. In the image setup, standardize the measurement area by entering the desired width and height. Set the point density to high.

At the image capture setup, select the frame rate and duration for the recording. Then, click the record button to start recording. The setup panel is replaced by the recording panel and data is saved automatically.

Take snapshots during the procedure to enable further comparison. After this, use ImageJ software to measure the survival and necrosis areas by first placing a ruler at the site of the flap and taking control pictures for macroscopic measurement. In the ImageJ toolbox, select straight line and draw a line over one centimeter of the ruler.

Click on analyze, set scale, and in the textbox for known distance, enter the value of one centimeter. Click on the polygon selection tool and draw the polygon lines over the flap to calculate the viable area. Click on analyze, measure to obtain the area value.

Then, place a postoperative dressing on the animal before housing to prevent self-mutilation. Seven days after the surgery, photograph the surgical area to enable macroscopic measurements of the flap survival and necrosis areas. Using the previously described laser speckle contrast analysis technique, visualize and quantify perfusion differences to assess the flap's microcirculation.

After the macroscopic analysis, remove the 4-0 sutures, raise the flap, and use transit-time ultrasound to reassess the vascular pedicle blood flow. Perform tissue sampling by longitudinally dividing the flap into two parts that measure 1.5 centimeters by six centimeters. Immediately after the creation of the microsurgical anastomoses, we obtained higher blood flow values than the minimum flows recommended in the literature.

Thus, all microsurgical anastomoses were patent one week after the surgery. Observation of the microcirculatory depravation of blood flow during the ischemic insult was possible with the laser speckle contrast analysis technique. This includes the immediate hyperperfusion during the flap reperfusion and perioperatively, the different areas with less perfusion had a higher risk of postoperative flap necrosis that were indeed necrotized seven days after the end of the study.

The flap survival area after eight hours of ischemia and its subsequent reperfusion was around 40%Previously published results show statistically significant differences when this model is compared with flaps where no ischemic insult is inflicted. Remember to dissect the flap pedicle without touching it or by grasping the adventitia as little as possible to avoid damaging the vessel wall, as well as perform proper microsurgical anastomoses in order to avoid complications due to the surgical technique. Intraoperatively, transit-time ultrasound technology allows us to quantify the blood flow of microsurgical anastomoses and thus predict flap perfusion.

Postoperatively, laser speckle contrast analysis allows a semi-continuity of real-time mapping of flow within free flaps. It is a promising technique, however further research is needed. This video has described the protocol of a suitable model for evaluating therapeutic agents to counteract ischemia-reperfusion injury in the field of reconstructive microsurgery.