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.
Gratis væv overdragelsen er i stigende grad anvendt i klinisk praksis til rekonstruktion forsvundne væv siden 1970'erne 1-5. Det har gjort rekonstruktion af komplekse og ellers uhelbredelige fejl som følge af tumor udryddelse, traumer, infektioner, misdannelser eller forbrændinger 1-7. Frie flige af denne art er særligt anvendelige til rekonstruktion meget komplekse anatomiske områder, som dem i hoved og hals, hånden, foden, og perineum 1,4.
Men selv i dag kirurgiske praktikanter ofte skræmt af kompleksiteten i flere trin, der er involveret i at hæve, overførsel og insetting en fri klap med brug af mikrokirurgiske teknikker og instrumenter 8,9. Desuden er det almindeligt accepteret, at for at blive en dygtig microsurgeon, omfattende eksperimentel praksis i en dyremodel er obligatorisk 4,8-13.
Desuden grundlæggende og translationel forskningpå området for fri væv overførsel er af stor klinisk potentiale 8,14-16. Uanset, er forskerne ofte afskrækkes fra at bruge mikrokirurgiske modeller af væv overførsel på grund af manglende oplysninger om de tekniske aspekter, der er involveret i de operative procedurer 4,8-14. Rotten er en god dyremodel for mikrokirurgisk forskning og uddannelse, da det er relativt billigt, let at holde, og medgørlige til hyppige manipulation 8,11,13,14,17,18.
Selv om flere gratis knogler, muskler og hud flaps er blevet beskrevet i rotten 18-24, den frie epigastriske fasciocutaneous flap er den mest udbredte til undervisningsformål 9,12,13,18,25. Denne gratis klap blev første gang beskrevet i 1967 af Strauch og Murray og har fået stigende popularitet lige siden, skyldes flere faktorer, nemlig konstant vaskulær anatomi, relativt lette dissektion, betydelige fartøjer næringsstoffer, og redundans i huden i donor zone, which tillader primær lukning af defekten følge af klap elevation 4,9-11,13,17,18,25-28.
Flap Anatomi og Histologi
Epigastriske flap leveres af den overfladiske epigastriske arterie og vene (figur 1). Disse skibe stammer fra og løbe ned i femorale arterie og vene, hhv. I gennemsnit kaliber af den overfladiske epigastriske vene er 0,6 til 0,8 mm, i modsætning til de 0,3 til 0,5 mm fra den overfladiske epigastriske arterie (figur 2) 17,18. Den overfladiske epigastriske arterie afgiver to hovedgrene: en lateral og en medial gren, som igen deler flere gange, oprindelse kapillære netværk, der forsyner det meste af Integument epigastriske region. Disse kapillærer dræne ind i bifloder de overfladiske epigastriske vener, der har en parallel kurs til arteriel træet (figur 2) 13,17,18. Diagrammet i figur 3 reviser området af den ventrolaterale bugvæggen leveret af de overfladiske epigastriske fartøjer, der kan mobiliseres i epigastriske flap. Denne klap kan være op til 5 cm i længden og 3 cm i bredden 13,17,18.
Histologisk er klappen består af Integument der dækker ventrolaterale bugvægsmuskulatur (figur 4) 13,17,18. Den indeholder en overfladisk lag af huden, dannet af dermis og epidermis. Under huden er der et lag af fedtvæv navngivet panniculus adiposus. Under dette lag er der endnu et lag af tværstribede muskler kaldet panniculus carnosus 18,28,29. Nedenfor panniculus carnosus der er løs areolar væv, der er overfladisk til den dybe fascia, der dækker de større abdominale muskler. Derfor klappen er en sammensat blok af væv, som indeholder alle disse lag, bortset fra den dybe muskelfascie (figur 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.
En af forfatterne (Diogo Casal) modtaget et tilskud fra programmet for Advanced Medical Education, som er sponsoreret af Fundação Calouste Gulbenkian, Fundação Champalimaud, Ministério da Saúde e Fundação para a Ciência e Tecnologia, Portugal.
Forfatterne vil gerne takke teknisk hjælp af Mr. Alberto Severino i filme og redigere videoen. Forfatterne er også taknemmelige til Mr. Octávio Chaveiro, Mr. Marco Costa og Mr. Carlos Lopes for deres hjælp med at forberede dyret prøver præsenteret i dette papir.
Endelig vil forfatterne gerne takke Ms. Gracinda Menezes for hendes hjælp i alle de logistiske aspekter vedrørende anskaffelse og vedligeholdelse dyr.
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