نموذج من حر نقل الأنسجة: الجرذ شرسوفي الحرة رفرف
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
وقد استخدمت نقل الأنسجة الحرة على نحو متزايد في الممارسة السريرية لإعادة إعمار الأنسجة المفقودة منذ 1970s 1-5. وقد سمح ذلك إعادة بناء العيوب المعقدة وغير قابل للعلاج إلا الناجمة عن الورم استئصال، والصدمات النفسية، والالتهابات، والتشوهات أو حروق 1-7. اللوحات خالية من هذا النوع هي مفيدة بشكل خاص لإعادة إعمار المناطق التشريحية معقدة للغاية، كتلك الموجودة في الرأس والرقبة، واليد، والقدم، والعجان 1،4.
ومع ذلك، وحتى اليوم هي في كثير من الأحيان نغرق المتدربين الجراحية التي تعقد عدة خطوات تشارك في رفع ونقل وinsetting رفرف الحرة مع استخدام تقنيات وأدوات 8،9 المجهرية. وبالإضافة إلى ذلك، فمن المقبول على نطاق واسع أن لتصبح microsurgeon يتقن، والممارسة التجريبية واسعة في نموذج حيواني إلزامي 4،8-13.
وعلاوة على ذلك، الأساسي ومتعدية البحوثفي مجال نقل الأنسجة الحرة على جانب كبير من 8،14-16 إمكانية السريرية. ورغم ذلك، كثيرا ما يردع الباحثين من استخدام نماذج المجهرية نقل الأنسجة بسبب نقص المعلومات فيما يتعلق بالجوانب الفنية المشاركة في الإجراءات 4،8-14 المنطوق. الفئران هو نموذج حيواني جيد للبحث والتدريب المجهرية، كما أنها غير مكلفة نسبيا، وسهلة للحفاظ على، وقابلة للتلاعب المتكرر 8،11،13،14،17،18.
وعلى الرغم من وصفت عدة العظام الحرة والعضلات والجلد اللوحات في الفئران 18-24، وشرسوفي رفرف fasciocutaneous الحر هو الأكثر استخداما على نطاق واسع لأغراض التدريس 9،12،13،18،25. وقد وصفت هذه رفرف مجانية لأول مرة في عام 1967 من قبل Strauch وموراي واكتسب شعبية متزايدة منذ ذلك الحين، وذلك بسبب عدة عوامل، وعلم التشريح وهي المستمر الأوعية الدموية، والسهولة النسبية لتشريح والأوعية المغذية كبيرة، والتكرار من الجلد في منطقة المانحة، ذوي الخوذات البيضاءالتراث الثقافي غير المادي يسمح إغلاق الرئيسي للخلل الناتج عن ارتفاع رفرف في 4،9-11،13،17،18،25-28.
رفرف علم التشريح وعلم الأنسجة
ويتم تزويد رفرف شرسوفي من الشريان الشرسوفي السطحي والوريد (الشكل 1). هذه السفن تأتي من وتصب في شريان الفخذ والوريد، على التوالي. في المتوسط عيار الوريد الشرسوفي السطحي هو 0،6-0،8 مم، المتناقضة مع 0،3-0،5 ملم من الشريان الشرسوفي السطحي (الشكل 2) 17،18. الشريان الشرسوفي السطحي يعطي قبالة فرعين رئيسيين: الوحشي وفرع وسطي وهذا بدوره تقسيم عدة مرات، تنشأ شبكات الشعرية التي تغذي معظم إهاب المنطقة شرسوفي. استنزاف هذه الشعيرات الدموية في روافد الأوردة شرسوفية السطحية التي لديها مسار مواز للشجرة الشرايين (الشكل 2) 13،17،18. الرسم البياني في الشكل (3) إعادةيعرض منطقة جدار البطن بطناني توفيره من قبل سفن شرسوفي السطحية التي يمكن تعبئتها في رفرف شرسوفي. هذا رفرف يمكن أن تصل إلى 5 سم في الطول و 3 سم في 13،17،18 العرض.
تشريحيا، ويتكون رفرف من إهاب التي تغطي البطنية الوحشية عضلات جدار البطن (الشكل 4) 13،17،18. وهو يحتوي على الطبقة السطحية من الجلد، والتي شكلتها الأدمة والبشرة. تحت الجلد هناك طبقة من الأنسجة الدهنية اسمه السبلة شحمية. تحت هذه الطبقة هناك طبقة أخرى من العضلات المخططة المعروفة باسم السبلة العضلية 18،28،29. تحت عضلية السبلة هناك الأنسجة الهالي فضفاضة وهي سطحية إلى اللفافة العميقة التي تغطي عضلات البطن أكبر. وبالتالي، فإن رفرف هو كتلة مركب من الأنسجة، والتي تحتوي على جميع هذه الطبقات، باستثناء اللفافة العضلية العميقة (الشكل 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.
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
تلقى واحد من المؤلفين (ديوغو كاسال) على منحة من برنامج للتعليم الطبي المتقدم، الذي ترعاه فونداساو كالوست كولبنكيان، فونداساو Champalimaud، MINISTERIO دا Saúde ه فونداساو الفقرة على Ciência الإلكترونية بحوث والتكنولوجيا، البرتغال.
فإن الكتاب أود أن أشكر مساعدة تقنية من السيد ألبرتو سيفيرينو في تصوير وتحرير الفيديو. الكتاب ممتنون أيضا للسيد اوكتافيو Chaveiro، السيد ماركو كوستا والسيد كارلوس لوبيز لمساعدتهم في إعداد العينات الحيوانية الواردة في هذه الورقة.
وأخيرا، فإن الكتاب أود أن أشكر السيدة Gracinda مينيزيس لمساعدتها في جميع الجوانب اللوجستية المتعلقة اقتناء الحيوانات والصيانة.
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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