The most important aspect to obtain consistent flap survival is paying attention to detail in various steps of the microsurgical technique. For example, to obtain good visualization of the vessels, of the surgical instruments and of the fine suture lines, it is very helpful to place underneath the vessels, a sterilized colored plastic background. As many researchers, we prefer to use sterilized fragments of yellow or green balloons (Figures 7 and 11). This background provides the additional advantage of minimizing adherence of suture lines to the adjacent structures, which sometimes leads to the need of pulling the suture line with too much tension, which may in turn lead to vascular tearing. Finally, the use of a background has the additional advantage of decreasing the probability of inadvertently dragging potential thrombogenic tissue debris to the anastomosis site.

Considering that the flap’s vessels are very fine and fragile, it is important not to pinch the entire width of the vessels, in order to avoid intimal lesion that, in turn, will lead to intravascular thrombosis and flap failure. To prevent inadvertent injury to both the flap’s vessels and to the recipient site’s vessels, it is safer to liberally ligate and divide neighboring tributaries, which will allow an easier manipulation of these vessels.

Before starting the anastomoses, it is vital to place the vessels in their definitive position, striving to prevent vascular kinking or torsion of the flap’s pedicle. Given the small caliber and delicate consistency of the vessels, these are often difficult to exclude unequivocally. One helpful trick is to secure the flap in its final position with 3 stitches placed away from the site of the anastomoses. Next, if in doubt, temporarily open the vascular clamps placed at the flap’s pedicle, and fill the vessels’ lumen with heparinized normal saline in a concentration of 10 IU/mL until they become engorged. This leads vessels to assume the configuration they will present after being perfused by blood, as when the clamps are removed after anastomoses completion.

Moreover, it is of paramount importance to detect any air bubbles, even if small, inside the vessels during the entire procedure and particularly before tying the final stitches. If these bubbles are distant from the vascular section, the vessels can be milked gently with microsurgical forceps. If they are located close to the anastomotic sites, simple irrigation leads the less dense bubbles to be easily expelled from the vascular lumen. Failure to acknowledge the presence of air bubbles can cause irreversible flap ischemia and necrosis, no doubt due to the fine caliber of the flap vessels.

Additionally, it cannot be overemphasized the need for meticulous care while passing and tying the stitches, in order to: include the three layers of the vessels (intima, media and adventitia); obtain good vessel eversion to ensure adequate intimal contact, which is vital to anastomosis sealing and endothelial regrowth; avoid loose vascular contact, which will result in anastomotic incompetence, i.e., bleeding; and avoid grabbing too much vascular tissue, which will lead to anastomosis stenosis and proclivity to thrombosis, which in turn will result in venous congestion or poor flap perfusion, if the vein or artery are involved, respectively.

Finally, it is essential to ensure perfect hemostasis, during the entire procedure, especially when raising the flap in its deep surface. Otherwise hematoma formation and rat death are likely to ensue.

Modifications and troubleshooting of the technique

The authors observed that making a transverse incision in the middle portion of the SCM using an electric cautery, not only allows a better exposure of the carotid artery, but also minimizes the risk of undue tension over the future arterial anastomosis.

Another important technical tip is to start the anastomosis from the vessels’ back wall, in order to minimize the risk of unwillingly catching this wall when placing the stitches in the more easily exposed front wall. If the back wall is sutured to the anterior aspect of the anastomosis, lack of vascular patency will almost invariably result either immediately due to mechanical reasons or after only a few hours as a result of thrombosis⁸.

If the anastomoses of the epigastric vessels of the rat are considered too technically challenging due to the small caliber of these vessels, the femoral vessels can be ligated distal to the origin of the epigastric vessels and used as the vascular pedicle of the epigastric flap. In this way, larger vessels will be used (the femoral artery has a caliber of 1.0 to 1.2 mm; and the femoral vein has a caliber of 1.2 to 1.5 mm). Moreover, by dissecting and ligating the other tributaries of the femoral vessels, a vascular pedicle length of over 2 cm can be obtained, which will facilitate flap insetting^18,34,35.

Reproducibility

Our experience of more than ten years of using this flap for teaching and research purposes strongly suggests that the rat epigastric flap is a reproducible model of free tissue transfer^{11,13,17,18,26}. It can be easily incorporated in microsurgical courses, as it is a good teaching and training model for microsurgery trainees^{11,13,17,18,26}. In our experience, although technically challenging in the beginning for the novice in microsurgery, after some training, the free epigastric flap can be successfully transferred to the neck of the rat with minimal to no subsequent necrosis in 70 to 80% of cases. These results concur with those generally reported in the literature^13,18,36.

Significance with respect to existing methods

Numerous free flaps have been described in the rat^{10,16,18,37-39}. The most commonly used for teaching and research purposes have been the transverse rectus abdominis myocutaneous flap, the latissimus dorsi and serratus anterior muscle flaps, the hind limb replantation model, and the epigastric (groin) flap^18,35. These flaps have been favored, due to their consistent anatomy and sizeable vascular pedicle. The epigastric flap is arguably the one associated with lesser donor site morbidity, as it dissected above the muscle fascia¹⁸. Moreover, the epigastric flap, described in 1967, was the first flap to be described in rats^34,35. This occurred only 4 years after the first description of an experimental flap in an animal by Goldwyn. Interestingly, this flap was a groin flap in the dog³⁴.

Limitations of the technique

The two main limitations of this model are the need for microsurgical skills in order to carry out the surgery, and the presence of significant necrosis in 20 to 25% of cases, according to different authors^13,18,36. Another potential limitation of the model herein presented is the auto cannibalism of the flap. However, as the authors above, this is an infrequent finding that almost only occurs in cases of total flap necrosis.

Future applications of the technique

The rat epigastric free flap can be used in experimental studies of tissue perfusion, tissue repair and surgical wound infection^40,41. Its nutrient vessels are particularly suitable for intravascular injection of solutions containing substances of interest, namely drugs, viral vectors or liposomes, that will mostly produce a local or regional effect^30,31. In addition, beneath the flap, pathogens, foreign bodies, radioactive seeds or chemicals can also be placed, mimicking several disease processes and potential treatments^30,31.