Un modello di trasferimento del tessuto libero: The Rat Epigastrica libero Flap
Summary
This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.
Abstract
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.
Introduction
Trasferimento tessuto libero è stato utilizzato sempre più nella pratica clinica per ricostruire i tessuti mancanti dal 1970 1-5. Questo ha permesso la ricostruzione di difetti complessi e altrimenti incurabili derivanti da tumore estirpazione, traumi, infezioni, malformazioni o ustioni 1-7. Lembi liberi di questo tipo sono particolarmente utili per ricostruire regioni anatomiche altamente complessi, come quelli della testa e del collo, la mano, il piede, e il perineo 1,4.
Tuttavia, ancora oggi gli allievi chirurgici sono spesso scoraggiati dalla complessità delle diverse fasi del processo di raccolta, trasferimento e insetting un lembo libero con l'uso di tecniche e strumenti 8,9 microchirurgia. Inoltre, è ampiamente accettato che per diventare un abile microsurgeon, vasta pratica sperimentale in un modello animale è obbligatoria 4,8-13.
la ricerca, inoltre, di base e traslazionalenella zona di trasferimento panno che è di grande potenzialità 8,14-16 clinica. Nonostante ciò, i ricercatori sono spesso dissuasi dal utilizzando modelli di microchirurgia di trasferimento dei tessuti a causa della mancanza di informazioni riguardanti gli aspetti tecnici coinvolti nelle procedure operative 4,8-14. Il ratto è un buon modello animale per la ricerca e la formazione microchirurgico, in quanto è relativamente poco costoso, facile da mantenere, e suscettibili di frequenti manipolazioni 8,11,13,14,17,18.
Anche se diversi lembi ossei liberi, muscoli e la pelle sono stati descritti nel ratto 18-24, il lembo fasciocutaneo epigastrica libero è il più utilizzato a scopo didattico 9,12,13,18,25. Questo lembo libero è stato descritto nel 1967 da Strauch e Murray e ha guadagnato crescente popolarità da quando, a causa di diversi fattori, vale a dire l'anatomia costante vascolare, relativa facilità di dissezione, vasi di nutrienti consistenti, e la ridondanza della pelle nella zona donatrice, which permette la chiusura primaria del difetto derivante dalla elevazione del lembo 4,9-11,13,17,18,25-28.
Flap Anatomia e Istologia
Il lembo epigastrica è fornita dal arteria epigastrica superficiale e vena (Figura 1). Questi vasi provengono da e scarico nell'arteria femorale e la vena rispettivamente. Mediamente il calibro della vena epigastrica superficiale è di 0,6 a 0,8 mm, in contrasto con i 0,3 a 0,5 mm di arteria epigastrica superficiale (Figura 2) 17,18. L'arteria epigastrica superficiale emette due rami principali: una laterale e un ramo mediale che a sua volta dividere più volte, originari reti capillari che forniscono la maggior parte del tegumento della regione epigastrica. Questi capillari scaricano nelle affluenti delle vene epigastrici superficiali che hanno un percorso parallelo all'albero arteriosa (Figura 2) 13,17,18. Il diagramma della figura 3 representa la regione della parete addominale ventrolaterale fornite dai vasi epigastrici superficiali che possono essere mobilizzati nel lembo epigastrico. Questo lembo può essere fino a 5 cm di lunghezza e 3 cm di larghezza 13,17,18.
Istologicamente, il lembo è composto dal tegumento che copre le ventrolateral muscoli della parete addominale (Figura 4) 13,17,18. Esso contiene uno strato superficiale della pelle, formata dal derma e dell'epidermide. Sotto la pelle vi è uno strato di tessuto grasso chiamato pannicoli adiposi. Sotto questo strato c'è un altro strato di muscolo striato noto come pannicolo carnosus 18,28,29. Sotto la carnosus pannicolo c'è tessuto areolare sciolto, che è superficiale alla fascia profonda che copre i muscoli addominali più grandi. Quindi, il lembo è un blocco composto di tessuti, contenente tutti questi strati, tranne per la fascia muscolare profondo (Figura 5) 13,17,18,27-31.
Protocol
Representative Results
Discussion
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.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Uno degli autori (Diogo Casal) ha ricevuto una sovvenzione dal programma di Advanced Medical Education, che è sponsorizzato da Fundação Calouste Gulbenkian, Fundação Champalimaud, Ministério da Saúde e Fundação para a Ciência e Tecnologia, Portogallo.
Gli autori desiderano ringraziare l'aiuto tecnico di Alberto Severino nella ripresa e di montaggio del video. Gli autori sono anche grato a Mr. Octávio Chaveiro, Marco Costa e il signor Carlos Lopes per il loro aiuto nella preparazione dei provini, presentato in questo documento.
Infine, gli autori desiderano ringraziare la signora Gracinda Menezes per il suo aiuto in tutti gli aspetti logistici relativi alla acquisizione e il mantenimento degli animali.
Materials
| Skin Skribe Surgical Skin Marker | Moore Medical | 31456 | https://www.mooremedical.com/index.cfm?/Skin-Skribe-Surgical-Skin-Marker/ &PG=CTL&CS= HOM&FN=ProductDetail& PID=1740&spx=1 |
| Micro retractor | Fine Science Tools | RS-6540 | http://www.finescience.de |
| Graeffe forceps 0.8 mm tips curved | Fine Science Tools | 11052-10 | http://www.finescience.de |
| Acland clamps | Fine Science Tools | 00398 V | http://www.merciansurgical.com/aclandclamps.pdf |
| Clamp applicator | Fine Science Tools | CAF-4 | http://www.merciansurgical.com/acland-clamps.pdf |
| High-Temperature Cautery | Fine Science Tools | AA03 | http://www.boviemedical.com/products_aaroncauteries_high.asp |
| Micro-vessel dilators 11 cm 0.3 mm tips 00124 | Fine Science Tools | D-5a.2 | http://www.merciansurgical.com |
| Micro Jewellers Forceps 11cm angulated 00109 | Fine Science Tools | JFA-5b | http://www.merciansurgical.com |
| Micro Jewellers Forceps 11 cm straight 00108 | Fine Science Tools | JF-5 | http://www.merciansurgical.com |
| Acland Single Clamps B-1V (Pair) | Fine Science Tools | 396 | http://www.merciansurgical.com |
| Micro Scissors Round Handles 15 cm Straight | Fine Science Tools | 67 | http://www.merciansurgical.com |
| Iris Scissors 11.5 cm Curves EASY-CUT | Fine Science Tools | EA7613-11 | http://www.merciansurgical.com |
| Mayo Scissors 14 cm Straight Chamfered Blades EASY-CUT | Fine Science Tools | EA7652-14 | http://www.merciansurgical.com |
| Derf Needle Holders 12 cm TC | Fine Science Tools | 703DE12 | http://www.merciansurgical.com |
| Monosyn 5-0 | B.Braun | 15423BR | http://www.mcfarlanemedical.com.au/ 15423BR/ SUTURE-MONOSYN-5_or_0-16MM-70CM-(C0023423)-BOX_or_36/pd.php |
| Ethilon 5-0 | Ethicon | W1618 | http://www.farlamedical.co.uk/category_Ethilon-Suture-1917/Ethilon-Sutures/ |
| Dafilon 10-0 | B.Braun | G1118099 | http://www.bbraun.com/cps/rde/xchg/bbraun-com/hs.xsl/products.html?prid=PRID00000816 |
| Veet Sensitive Skin Hair Removal Cream Aloe Vera and Vitamin E 100 ml | Veet | http://www.veet.co.uk/products/creams/creams/veet-hair-removal-cream-sensitive-skin/ | |
| Instrapac – Adson Toothed Forceps (Extra Fine) | Fine Science Tools | 7973 | http://www.millermedicalsupplies.com |
| Castroviejo needle holders | Fine Science Tools | 12565-14 | http://s-and-t.ne |
| Straight mosquito forcep | Fine Science Tools | 91308-12 | http://www.finescience.de |
| Cutasept F skin disinfectant | Bode Chemie | http://www.productcatalogue.bode-chemie.com/products/skin/cutasept_f.php | |
| Lacri-lube Eye Ointment 5g | Express Chemist | LAC101F | http://www.expresschemist.co.uk/lacri-lube-eye-ointment-5g.html |
| Normal saline for irrigation | Hospira, Inc. | 0409-6138-22 | http://www.hospira.com/en/search?q=sodium+chloride+irrigation%2C+usp&fq=contentType%3AProducts |
| Heparin Sodium Solution (5000IU/ml) | B.Braun | http://www.bbraunusa.com/products.html?prid=PRID00006982 | |
| Meloxicam Metacam | Boehringer Ingelheim | http://www.bi-vetmedica.com/species/pet/products.html | |
| Heat Lamp HL-1 | Harvard Apparatus | 727562 | https://www.harvardapparatus.com/webapp/wcs/stores/servlet/ haisku3_10001_11051_39108_-1_ HAI_ProductDetail_N_ 37610_37611_37613 |
| Homeothermic Blanket System with Flexible Probe | Harvard Apparatus | 507220F | https://www.harvardapparatus.com/webapp/wcs/stores/servlet/ haisku3_10001_11051_39108_-1_ HAI_ProductDetail_N_ 37610_37611_37613 |
| Dry heat sterilizer | Quirumed | 2432 | http://www.quirumed.com/pt/material-de-esterilizac-o/esterilizadores |
| Surgical drapes | Barrier | 800430 | http://www.molnlycke.com/surgical-drapes/ |
| Biogel Surgical Gloves | Medex Supply | 30465 | https://www.medexsupply.com |
| Operating microscope | Leica Surgical Microsystems | 10445319 | http://www.leica-microsystems.com/products/surgical-microscopes/ |
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