This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.
Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat’s neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.
Transfert de tissu libre a été de plus en plus utilisé dans la pratique clinique pour la reconstruction de tissus manquants depuis les années 1970 1-5. Cela a permis la reconstruction de défauts complexes et autrement incurables résultant de extirpation de la tumeur, les traumatismes, les infections, malformations ou des brûlures 1-7. Lambeaux libres de ce type sont particulièrement utiles pour la reconstruction des régions anatomiques très complexes, comme ceux de la tête et du cou, la main, le pied, et le périnée 1,4.
Cependant, aujourd'hui encore les stagiaires chirurgicales sont souvent découragés par la complexité de plusieurs étapes de soulever, transférer et encartage un lambeau libre avec l'utilisation de techniques et d' instruments de microchirurgie 8,9. En outre, il est largement admis que , pour devenir un microsurgeon compétent, une vaste pratique expérimentale dans un modèle animal est obligatoire 4,8-13.
la recherche ailleurs, fondamentale et translationnelledans le domaine du transfert de tissu libre est d' une grande 8,14-16 potentiel clinique. Nonobstant, les chercheurs sont souvent dissuadés d'utiliser des modèles de microchirurgie de transfert de tissu en raison du manque d'informations concernant les aspects techniques impliqués dans les procédures opératoires 4,8-14. Le rat est un bon modèle animal pour la recherche et la formation de microchirurgie, car il est relativement peu coûteux, facile à maintenir, et se prêtent à la manipulation fréquente 8,11,13,14,17,18.
Bien que plusieurs os libre, les muscles et la peau volets ont été décrits chez le rat 18-24, le lambeau fascio épigastrique libre est le plus largement utilisé à des fins d'enseignement 9,12,13,18,25. Ce lambeau libre a été décrite en 1967 par Strauch et Murray et a acquis une popularité croissante depuis, en raison de plusieurs facteurs, l'anatomie à savoir constante vasculaire, la facilité relative de dissection, vaisseaux nourriciers importants, et la redondance de la peau dans la zone donneuse, which permet la fermeture primaire du défaut résultant de l'élévation de lambeau 4,9-11,13,17,18,25-28.
Flap anatomie et histologie
Le volet épigastrique est fourni par l'artère épigastrique superficielle et la veine (Figure 1). Ces navires proviennent et se déversent dans l'artère et la veine fémorales, respectivement. En moyenne , le calibre de la veine épigastrique superficielle est de 0,6 à 0,8 mm, ce qui contraste avec les 0,3 à 0,5 mm de l'artère épigastrique superficielle (figure 2) 17,18. L'artère épigastrique superficielle dégage deux branches principales: une latérale et une branche médiane qui, à son tour diviser plusieurs fois, en provenance des réseaux capillaires qui alimentent la plupart des téguments de la région épigastrique. Ces capillaires se déversent dans les affluents des veines épigastriques superficielles qui ont un cours parallèle à l'arbre artériel (Figure 2) 13,17,18. Le diagramme de la figure 3 représente la région de la paroi abdominale ventrolatérale fournie par les vaisseaux épigastriques superficiels qui peuvent être mobilisées dans le rabat épigastrique. Ce volet peut être jusqu'à 5 cm de long et 3 cm de largeur 13,17,18.
Sur le plan histologique, le clapet se compose de tégument qui recouvre les muscles abdominaux ventrolatérales de paroi (figure 4) 13,17,18. Il contient une couche superficielle de la peau, formée par le derme et l'épiderme. Sous la peau il y a une couche de tissu adipeux nommé pannicule adiposus. En dessous de cette couche il y a une autre couche de muscle strié connu sous le nom pannicule carnosus 18,28,29. Ci – dessous l'carnosus pannicule il est tissu cellulaire lâche qui est superficielle au fascia profond qui couvre les muscles abdominaux plus importants. Par conséquent, le volet est un bloc composite de tissus, contenant toutes les couches, à l' exception de l'aponévrose du muscle profond (figure 5) 13,17,18,27-31.
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 insetting18,34,35.
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 transfer11,13,17,18,26. It can be easily incorporated in microsurgical courses, as it is a good teaching and training model for microsurgery trainees11,13,17,18,26. 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 literature13,18,36.
Significance with respect to existing methods
Numerous free flaps have been described in the rat10,16,18,37-39. 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) flap18,35. 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 rats34,35. 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 authors13,18,36. 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 infection40,41. 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 effect30,31. In addition, beneath the flap, pathogens, foreign bodies, radioactive seeds or chemicals can also be placed, mimicking several disease processes and potential treatments30,31.
The authors have nothing to disclose.
L'un des auteurs (Diogo Casal) a reçu une subvention du programme pour l'éducation médicale avancée, qui est parrainé par Fundação Calouste Gulbenkian, Fundação Champalimaud, Ministério da Saúde e Fundação para a Ciência e Tecnologia, Portugal.
Les auteurs tiennent à remercier l'aide technique de M. Alberto Severino dans le tournage et montage de la vidéo. Les auteurs sont également reconnaissants à M. Octávio Chaveiro, M. Marco Costa et M. Carlos Lopes pour leur aide dans la préparation des spécimens d'animaux présentés dans ce document.
Enfin, les auteurs tiennent à remercier Mme Gracinda Menezes pour son aide dans tous les aspects logistiques relatifs à l'acquisition et l'entretien des animaux.
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