Chapter 5. Orthotopic vascularized groin flap transplantation model in rats

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1 Chapter 5. Orthotopic vascularized groin flap transplantation model in rats 1. Rationale of model development Nicolae Ghetu Codrin-Nicolae Dobreanu Victor George Ilie Vlad Ionut Ilie Alain-Ali Mojallal Dragos Pieptu Fuelled by the advent of the research in allotransplantation, the inguinal flap emerged as an alternative to more complicated hindlimb flap. When simple mechanistic and general pathways are investigated, the inguinal flap reveals its noteworthy advantages: easier harvesting technique, straightforward and expedite inset and anastomosis; variation from the standard flap - adipofascial flap without skin paddle or the addition of other types of tissues such as muscle and bone. The extreme versatility of the flap allows for a wide array of research directions from microsurgical training and assessing skills, and techniques in microsurgery (i.e. the perforator flaps), to ischemia reperfusion, microcirculatory hemodynamics, pharmacological studies, pulse magnetic field effects, tissue engineering and the extensive studies in allotransplantation. When compared to the heterotopic hindlimb vascularized flap, the herein model is simpler in composition, as important tissues are missing: vascularized bone marrow, joints, muscles, tendons and nerves. However, the vascularized groin flap 79

2 retains entirely the highly immunogenic skin and the fat tissue (in the extended groin flap version even more skin and fat than the hindlimb flap), thus it is a valid vascularized composite flap fully eligible for research in vascular composite allotransplantation (VCA). The orthotopic transplantation of the groin flap into similar size recipient defect demands microsurgical technique, only slightly more difficult than the classic femoral artery and vein microsurgical repair. Therefore it is a nice passage from basic microsurgical techniques to the more advanced level of tissue transplantation. Orthotopic transplants needs special handling for follow-up due to inconspicuous position and sometimes requires shortterm anesthesia, for clinical evaluation or histological sampling. Obviously, in its heterotopic version, the follow-up (by direct vision) is easier with no need for rat handling outside the cage, unless biopsy is needed. The experimental orthotopic groin flap in rats is our preferred model of adipofascial-cutaneous vascularized composite transplantation and represents our personal experience in the Center for Simulation and Training in Surgery, University of Medicine and Pharmacy "Grigore T. Popa" Iasi, Romania. We use this model in agreement with national and international accepted ethical regulations, principles and guidelines for animal research. 80

3 2. Steps in model development/implementation The preparatory steps regarding the operating team and rat anesthesia, prepping and draping, as well as rat positioning are the same as in the previous chapter "Heterotopic vascularized hindlimb composite flap transplantation model in rats". We strongly recommend getting through the previous chapter before starting this model, as most of the tips and trick apply to the groin flap. Groin flap harvesting in donor rat Preoperatory drawing: With the rat supine and with the hindlimbs unrestrained, draw the operative plan as in Figures 1 and 2. Mark the inguinal ligament and the femoral vessels on the inner side of the thigh. Slightly proximal to the knee there is the branching point of the superficial epigastric vessels that course cranial, to supply the overlying skin and the fat pad of the groin area. More distally the epigastric vessels divide into a medial and a lateral branch, respectively, to supply different areas of the fat pad. The design of the skin paddle can be centered on the femoral and epigastric vessels, or it can be extended more cranially to include the skin or only the adipofascial tissue overlying the abdominal wall (Figure 2). 81

4 Fig. 1. Standard groin flap design, centered on the inguinal ligament, femoral and epigastric pedicles Fig. 2. On the right side of the rat is depicted the standard groin flap. On the left side is depicted the extended groin flap with skin paddle and adipofascial tissue harvested more cranially, up to the rib cage 82

5 For the common adipofascio-cutaneous version, draw the skin island round shaped, about 4 cm diameter (Figure 3). Fig. 3. The skin island of the standard groin flap, 4 cm long (left) and 4 cm wide (right) Skin incision: With sharp scissors or surgical blade incise the skin paddle until you reach the tissue situated immediately underneath the skin. After skin retraction, one can notice the flimsy appearance of the thigh fascia in the distal half of the incision, with minimal contribution from the fat tissue. On the cranial half of the incision, the fat pad is slightly thicker and the underlying abdominal wall muscles are less visible, when compared to the thigh counterpart (Figure 4). 83

6 Fig. 4. Skin retraction, after incision, delineating the skin paddle: the fascial plane and the fat pad are visible Subcutaneous dissection: Using dissection scissors, start harvesting from the caudal part, over the muscle fascia, and course medially and laterally to circumvallate the skin paddle (Figure 5). Fig. 5. Adipofascial-cutaneous groin flap rose superficial to the muscle fascia, with the adipofascial component harvested cranially over the skin paddle, higher on the abdominal wall 84

7 On the lateral abdominal wall the fat pad can be harvested more cranially. Use the cautery to severe the perforators from the rectus abdominis muscles, as well as the communication branches to internal mammary and lateral thoracic pedicles, cranially. The plan of dissection must always remain on top of the muscle fascia over all undersurface of the flap (Figure 6). Fig. 6. The undersurface of the groin flap, pedicled on the superficial epigastric vessels. Dissecting all the fibrous attachments of the flap from the underlying muscle fascia, in a centrifugal manner, one will soon notice the vertically oriented epigastric vessels branching out from the main pedicle of the hindlimb and coursing into the flap. At this point it is wise not to cut all attachments of the flap as, during the following dissection, the flap can be tensioned and the superficial epigastric vessels elongated and injured. 85

8 NOTA BENE: as the groin flap is round shaped, with same thickness and no other obvious anatomical landmarks (such as the femoral bone or muscle mound for the hindlimb flap), during the inset the flap position can be easily mistaken. To prevent this, place a mark on the flap (in our case the drawing of the pedicle is the mark to prevent rotational displacement). Vessels dissection: With the most caudal part of the flap reflected, one can see the main pedicle of the thigh, the femoral vessels and nerve and the branching point of the epigastric pedicle proximal to the knee joint. Dissect individually and skeletonize the femoral vessels from proximal to distal until the branching point of the epigastric vessels, ligating the sizable branches with 9/0 suture, or cauterizing the small muscle branches with the bipolar. Around and distal to the branching point the dissection is tedious due to the abundance of vessels branching. The terminal branches of the femoral artery (descending genicular, saphenous, and popliteal arteries) must be ligated and divided, and leave only the epigastric artery in continuity with the femoral artery. Do the same for the femoral vein and the corresponding venous branches. Cut the sensory nerve at this point, too. Once the pedicle skeletonized, divide the remaining attachments of the flap, thus the flap becomes pedicled on the femoral vessels (Figures 7 and 8). 86

9 Fig. 7. The groin flap pedicled on the femoral vessels, after distal branches ligation and skeletonization. Superficial view of the skin paddle Fig. 8. The groin flap pedicled on the femoral vessels. View of the undersurface of the flap If heterotopic transplant is intended, ligate the vessels individually, as proximal as possible, past the divided inguinal ligament; vessels extra-length might be needed in order to reach for the recipient vessels and to place the anastomosis site in a more favorable position. Cut vessels immediately distal from ligature site and record the ischemia starting time. Once both femoral vessels are ligated and divided, the flap is 87

10 completely separated from the donor rat. If orthotopic transplantation is the aim of the operation, vessels length is not critical, they should be cut long enough to reproduce the original femoral pedicle after the anastomosis (or only slightly longer); if too long, complications due to vessels length redundancy such as kinking or torsion will occur. Under microscope magnification, irrigate the femoral artery with heparin saline solution (10units/mL), using a 24G angiocatheter sheath; if the catheter is too big, dilate progressively the artery with the micro forceps or specially designed dilator. Handle the artery only by the adventitia and insert partially the catheter in the lumen (around 1 cm), advancing parallel to the vessel walls, preventing thus intima injuries. Inject slowly, with constant pressure, 10mL of heparinized saline. Initially blood will flow out through the femoral vein, and slowly there will be clear saline coming out. This step concludes the groin flap harvesting (Figures 9 and 10). Fig. 9. The groin flap back-table after flushing with heparin saline. On the background lies the femoral pedicle, artery and vein, individually skeletonized 88

11 Fig. 10. The groin flap back-table, aspect of the undersurface of the flap after flushing with heparin saline If one proceeds to transplantation directly, transfer the groin flap as it is, to the operator. If transplantation is delayed, wrap the flap in wet saline gauze and place it on ice, avoiding direct contact of wet gauze and ice by interposition of dry gauze on top of the ice. After the groin flap harvesting, the donor rats will be euthanized, using recommended protocols. Orthotopic groin flap transplantation Preparation of the recipient site: In the right groin area of the recipient rat create the same size defect as the groin flap harvested from the donor rat (roughly 4 cm diameter). Skeletonize the femoral pedicle, individually the artery and the vein, up to the inguinal ligament, ligating all the side branches. Place the approximator on the artery stump and a single clamp on the femoral vein. Keep in mind to create the 89

12 pedicle length, avoiding redundancy that can lead to complications. Inset of the groin flap: Bring the flap in the previously created orthotopic defect on top of the groin area; place the flap with the skin paddle outside. Check for the positional mark on the skin paddle and place the flap in the correct position; make sure the pedicle is not twisted. Fix the flap to the defect with 3-4 absorbable 3/0 or 4/0 skin stitches on the most remote part of the flap, opposite to the operator. Take care not to injure the pedicle during this step. Bring the donor and recipient pedicles together and check the alignment. Slight vessels redundancy is helpful as it makes the pedicle anastomosis easier. Microvascular anastomosis: For the orthotopic transplantation, the advantage is the same parallel disposition of both sets of vessels (with the artery situated more cranially), that allows for unrestricted handling of the vessels, with no torsion or twisting of the vessels around each other. The pedicles are coursing from top right to bottom left, which makes the ideal setting for the microvascular anastomosis. The anastomosis technique is described elsewhere and it is not our purpose here to get through the detailed technique. Start with the artery, place the approximator clamps uneven, with longer end for the recipient artery. At the end of the anastomosis place a single clamp immediately proximal to the anastomosis site, remove the approximator and place in on the 90

13 vein, avoiding the crowding of the clamps. Use the green or blue color background for this step, especially for the vein, which is more demanding technically; place the background on top of the artery, thus it can separate working space for vein anastomosis and it maintains the water media in which the vein is more visible and easy to work on. Irrigate the vessels stumps (with heparin saline solution), remove adventitia and dilate before starting the suturing. Separate 6 to 9 stitches of 10/0 micro suture are placed to complete the anastomosis. Remove background, release approximator from the vein and single clamp from the artery. Observe for the reflow, artery pulsation, and vein fullness and perform Acland's anastomosis patency test. Check the position of the vessels at ease and during hindlimb movements and before closing make sure there will be no kinking, torsion, elongation and tension in the anastomosis site. Observe flap for signs of revascularization like fullness, capillary refill and fresh bleeding from wound edges (Figure 11). Fig. 11. After the microvascular anastomosis of the femoral artery and vein, respectively, the revascularized flap shows full epigastric pedicle with fresh bleeding spots from the fat pad edges. 91

14 When the transplanted flap size is comparable to the orthotopic defect, the skin stitches properly fix the flap paddle; therefore the flap itself acts like an external splinter, holding the underlying pedicle in place pretty well. Therefore, even in the absence of adipo-fascial component or the undersurface of the flap fixation, we did not encounter any vascular problems. However, for the heterotopic transplantation, the local situation may ask for some anchoring, to prevent pedicle misplacement that would put the flap at vascular risk. Closure: Complete the flap inset using the same absorbable material. Clean rat skin from all traces of blood that can trigger autophagia. Place the rats individually in cages, with analgesics according to guidelines (Figure 12). Fig. 12. Groin flap inset in orthotopic position, fixed to the defect with absorbable stitches; notice the skin mark (the draw of the pedicle) positioned correctly. 92

15 Keep detailed evidence of operating times, accidents, incidents, operation abandon or failure, as well as the causes. Keep also record of all important facts throughout the follow-up period. Follow-up: Immediate postoperatively follow the waking up process, resuming of the ambulation and feeding habits. Daily clinical evaluation is recommended to assess the evolution of the transplant. Different from the heterotopic hindlimb vascularized flap, the orthotopic groin flap daily inspection is slightly more difficult, due to rat ambulating pattern and position of the flap on the inner aspect of the thigh, hidden from direct view. The research assistants must have proper skills for correct handling and safe restraining of the rats, in order not to damage the flap during inspection. If no such skills, use special gloves or specially designed restraining devices for rats. Also, short-term inhalation anesthesia is suitable, for agitated rats, to protect them from self-injury and flap damage. In the early postoperative period clinical signs express anastomosis-related complications. When the appearance of the flap is doubtful, perform pin-prick test or scoring of the flap s skin. The most important physical signs are: color, temperature, quality of capillary filling, bleeding from a cut edge of the flap and tissue turgor. A pink color with 1-to-2- second refill is consistent with good perfusion. When the flap exhibits arterial insufficiency, it looks pale, flaccid, without capillary refill or bleeding and is cooler. When vein occlusion with venous insufficiency occurs, the flap looks congested, 93

16 blue, with brisk capillary refill and dark bleeding at pin-prick. Compressive hematoma may induce same clinical signs. Later on, check for signs of infection or wound healing complications like dehiscence. When the groin flap is allotransplanted, it should be inspected daily for clinical signs of acute or chronic rejection, mentioned in the previous chapter. Benefits and limitations Technically, the groin flap vascularized composite transplantation is a more straightforward model as compared with the previous one, and it is a faster procedure. In terms of difficulty, we recommend this model to the trainees who want to commence the advance level training in microsurgery, i.e. the tissue transfer, after mastering the basics. With the technical demands of flap harvesting and transfer met, researcher can shift focus and explore the multiple advantages of the flap mentioned in the introductory chapter and throw this flap to work in the wide stream of research opportunities. Regarding the outcome, the references prove that the orthotopic vascularized groin flap transplantation model is a very good example how a technically relatively simple tool, in experienced hands, can afford and expand sound meaningful research and allow breakthrough results. References: 1. Acland R.D. Practice Manual for Microvascular Surgery, Mosby-Year Book; 2 Sub edition (January 1989) 94

17 2. Bartelmann U, Wolf N, Engelhardt W et al. The vascularized isolated groin flap in rats: a suitable tool for the study of burns. Arch Dermatol Res 1981; 270: Cheng HY, Ghetu N, Huang WC, et al. Syngeneic adiposederived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats. Cytotherapy Oct 9. pii: S (13) doi: /j.jcyt Chang KP, Huang SH, Lin CL et al. An alternative model of composite tissue allotransplantation: groin-thigh flap. Transpl Int 2008; 21: Demirseren ME, Yenidunya MO, Yenidunya S. Island rat groin flaps with twisted pedicles. Plast Reconstr Surg 2004; 114: Herold C, Reimers K, Allmeling C et al. A normothermic perfusion bioreactor to preserve viability of rat groin flaps extracorporally. Transplant Proc 2009; 41: Ilie VG, Ilie VI, Dobreanu C et al. Training of microsurgical skills on nonliving models. Microsurgery. 2008;28(7): ISSN , PMID: , DOI: /micr Ilie V, Ilie V, Ghetu N et al. Assessment of the microsurgical skills: 30 minutes versus two weeks patency. Microsurgery. 2007;27(5): ISSN PMID: , DOI: /micr Ionac M. Arterial end-to-end anastomosis. In Ionac M, Lineaweaver W, Zhang F (eds) "Practical Manual of Experimental Microsurgery" Publisher Idea Design & Print, Timisoara, 2002, ISBN Ionac M. Venous end-to-end anastomosis. In Ionac M, Lineaweaver W, Zhang F (eds) "Practical Manual of 95

18 Experimental Microsurgery" Publisher Idea Design & Print, Timisoara, 2002, ISBN Liu F, Luo X, Lan S et al. Immunosuppression with a combination of triptolide and cyclosporin A in rat vascularized groin flap allotransplantation. Plast Reconstr Surg 2013; 131: 343e-350e 11. Nazzal JA, Johnson TS, Gordon CR, et al. Heterotopic limb allotransplantation model to study skin rejection in the rat. Microsurgery. 2004;24(6): Ozkan O, Koshima I, Gonda K. A supermicrosurgical flap model in the rat: a free true abdominal perforator flap with a short pedicle. Plast Reconstr Surg 2006; 117: Shimizu F, Okamoto O, Katagiri K et al. Prolonged ischemia increases severity of rejection in skin flap allotransplantation in rats. Microsurgery 2010; 30: Strauch B, Murray DE. Transfer of composite graft with immediate suture anastomosis of its vascular pedicle measuring less than 1 mm in external diameter using microsurgical techniques. Plast Reconstr Surg 1967, oct, vol. 40, no 4: Uysal CA, Ogawa R, Lu F et al. Effect of mesenchymal stem cells on skin graft to flap prefabrication: an experimental study. Ann Plast Surg 2010; 65: Xiao B, Xia W, Zhang J et al. Prolonged cold ischemic time results in increased acute rejection in a rat allotransplantation model. J Surg Res 2010; 164: e299-e Xiao B, Xia W, Zhao K et al. Ex vivo transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of rapamycin therapy prolongs free flap allograft survival. Plast Reconstr Surg 2011; 127:

19 18. Yazici I, Siemionow M. The abdominal adipofascial flap: a new model for direct in vivo observation of microcirculatory hemodynamics of the abdominal fat tissue in rat. Ann Plast Surg 2008; 60: Weber RV, Navarro A, Wu JK et al. Pulsed magnetic fields applied to a transferred arterial loop support the rat groin composite flap. Plast Reconstr Surg 2004; 114: accessed on Nov. 13 th, Yuichi H. Skin and muscle flaps in the rat, page , chapter 6. In Tamai S, Usui M, Yoshizu T (eds) Experimental and Clinical Reconstructive Microsurgery, Springer Zdichavsky M, Jones JW, Ustuner ET et al. Scoring of skin rejection in a swine composite tissue allograft model. J Surg Res 1999;85:

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