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ORIGINAL ARTICLES
NUMBER 3 YEAR 2005
Limb Replantation in Rats - Experimental Model
1 Department of Plastic and Reconstructive Surgery
2 Division of Microsurgery, Victor Babes University of Medicine and Pharmacy Timisoara, Romania

Correspondence to:
Z. Crainiceanu, MD, Department of Plastic and Reconstructive Surgery, Clinical Emergency Hospital No. 1, 156 Dr. I. Bulbuca Blvd, Timisoara
Email: zcrainiceanu@yahoo.com
ABSTRACT
The purpose of this study is to establish an experimental model for individual training and clinical replantation. The standardized model can be used for advanced microsurgical training. The study was done on 7 Sprague Dawely adult rats weighing between 250 and 300g. Under general anesthesia thigh amputation was performed at the limit between middle and cranial 1/3. The replantation was performed following the standard steps. The viability of the replanted segment was verified by clinical observation: color (pink), consistency (full, elastic) and by pulsoximetry for 7 days. All the animals had a good postoperative evolution, with survival of the replanted segment, minimal edema and no trophic or infectious complications.
INTRODUCTION

The idea of replantation is not a new one. The replantation (reattaching all structures important to the function of a complete amputated body part, providing arterial inflow and venous outflow) was a challenge for surgeons for a long time, but better results started to appear after using the surgical microscope.1-3 First experimental replantation was performed by Höpfner in 1903 on dog. Ronald Malt performed the first replantation on May 23, 1962 at Massachusetts General Hospital on a 12-year-old boy who had his right arm amputated in a train accident (The Journal of the American Medical Association, 1964). Since Malt’s first replantation, technological advances and the use of the microscope have made possible the replantation of other body parts, including thumbs, fingers, ears, scalps, facial parts, and genitalia.4,5
Replantation in limb amputations (especially upper limb) is mandatory in the actual technical developmental conditions. Social, economical and psychological implications of limb amputation determine medical responsibilities in emerging conservative surgery instead amputation. The procedure is very complex with critical microvascular stage and difficult to teach with conventional surgical instructive methods.6,7 We describe a teaching model of replantation in rat hind limb.

MATERIAL AND METHODS

Preoperative preparation
We have used 7 Sprague Dawley adult rats, weighing between 250-300g of weight. Under i.m. anesthesia (xylazine and ketamine) the medial and lateral thigh sides were shaved. The rat was positioned in decubitus with the posterior limbs immobilized in extension with elastic bands. The anterior limbs were free in order to permit respiratory movements. Wet swabs were used to protect the subcutaneous tissue and the exposed organs during surgery.
Together with the usual microsurgical instruments, for replantation we used a few osteosynthesis tools: portable motor drill, an 18G needle and a steel 3-0 suture for cerclage.

Anatomy
Fig. 1. Surgical anatomy.
The proximal segment of the hind limb in rat includes:8
- skeleton, represented by the femoral bone;
- the muscles of the thigh, grouped into anterior femoral muscles, medial femoral muscles, muscles of the gluteal region and posterior femoral muscles;
- the vessels: femoral artery, femoral vein and their branches;
- the peripheral nerves: femoral, obturator and sciatic nerves and their branches. (Fig 1)

Surface landmarks
The limit between the middle and the proximal third of the thigh were marked, using as surface landmarks the inguinal flexion fold and the knee joint. We drew a transverse curve line with its concavity towards the cranial extremity. This incision line creates a cranial skin and subcutaneous flap based on the inferior epigastric artery that will protect the anastomosis.

Amputation
The skin of the medial and lateral aspect of the thigh was incised, on the previously marked line. The dissection began by preparing the elements of the vasculo-nervous femoral bundle. The muscular branches of the femoral artery and vein were ligated, to obtain a greater mobility of the vessels to be anastomosed. Once prepared, they can be easily protected while cutting the muscle and performing osteotomy. We cut the gluteus maximus muscle on the lateral aspect of the thigh, dissected and cut the sciatic nerve. Hind muscles were sectioned, carefully protecting, protecting carefully the vasculo-nervous pedicle; the bleeding was controlled with the bipolar. (Fig 2)
Preparing of the femoral vessels for the future anastomosis started before cutting them. First microclamp was put on the vein in a proximal position, thus causing its dilatation, and performed adventicectomy in good conditions. Then we clamped the distal artery, obtaining a partially dilated artery, and perform also the adventitial stripping. Now we cut the vasculo-nervous femoral bundle.
The distal segment is now in ischemia, and the time until revascularisation should not exceed 60 minutes from this moment. We cleaned the femur for about 1cm and cut it with the circular saw, shortening it by 0.5 cm. Bone shortening will allow performing vascular anastomosis without any tension. (Fig 3)
Fig. 2. Vessels and nerves disection.
Fig. 3. The aspect of the amputated segment and stump.

Bone fixation
We washed the femoral medullar channel with saline. We adjusted a fragment of suitable length from the 18G needle and use it for centro-medullar osteosynthesis. We performed a hole in frontal plane into the distal and proximal femoral segments and put the cerclage wire. We put the needle into the medullar channel and made a knot of the wire. A stable bone coaptation was obtained, without permitting bone fragments movements to put the anastomosis at risk. (Fig 4)
Fig. 4. Bone fixation.

Muscle and nerve suture
The muscles were sutured with absorbable 4-0 sutures, starting on the medial aspect of the thigh: vastus medialis, adductors, semimembranosus, semitendinosus, gracilis and than on the lateral aspect: biceps femoris, caudofemoris and partially the gluteus medius.
This is the best moment for sciatic nerve coaptation. Epiperineural suture with a 10-0 stitch is good enough in this exercise. Then we continued suturing on the lateral aspect with gluteus medius, gluteus maximus and tensor fasciae latae. Finish the muscle suture on the internal aspect: rectus femoris, vastus lateralis and intermedius. All muscles are now sutured. We performed coaptation of the femoral nerve, also using a 10-0 stitch. If the time runs up, we first performed the suture of the femoral vessels.

Vascular suture
The vascular step of the surgery started with the end to end artery anastomosis, which is situated in the distal plane, then suture the vein by separate stitches.9 10-0 nylon suture was used. The clamps were removed in the following order: first the proximally clamp of the vein, second the distally clamp of the vein, third the distally clamp of the artery and last the proximally clamp of the artery. (Fig 5)
After performing the patency tests, we covered the anastomosis with the inguinal fat pad irrigated by the inferior epigastric vessels and suture the skin.
Fig. 5. Microsurgical sutures.

Skin suture
The skin suture started on the internal aspect of the thigh, with separate non-absorbable 4-0 stitches. We checked the viability of the replanted segment by inspecting the color (pink), the turgor (full, elastic) and by pulsoxymetry. (Fig 6)
Fig. 6. Pulsoxymetry monitorisation.

Postoperative care
No immobilization of the hind leg or other special treatment is necessary. For a longer term follow-up, the rat should be protected from hurting his anesthetized leg by using a plastic collar.

RESULTS

All the animals had a good postoperative evolution, with the survival of the replanted segment, minimal edema and no trofic or infectious complications.

DISCUSSIONS

Since the first successful digit replantation with microvascular repair (reported by Komatsu and Tamai, 1968), the laboratory microsurgical training importance was obvious, because of surgical procedure complexity and critical microvascular stage being difficult to teach using conventional surgical instructive methods.7,10
Most models from literature use similar technique. Some authors describe techniques very similar with the clinical approach, first detaching the limb by clear cut amputation and than debridement, dissection of the vessels, nerves, muscle and bone, bone fixation, muscle suture, vessels and nerve repair skin suture.6,8 We consider that nerve and vessels dissection and clamping before amputation is mandatory for limited bleeding and ischemic time shortening. Also, the bone fixation by pine and intraosseous wiring will provide better bone alignment and stability than using pine only.
The rat is very convenient animal for teaching microsurgery. They are relatively easy to obtain, manipulate and anesthetize and the size of their vessels is equivalent to digital vessels. They also provide real limb structures handling (bone, muscles, etc.), different from other models.11,12,14 Our model can be used for junior surgeons training to improve their instruction and practice and avoid many possible pitfalls. This mode teaches also microsurgical technique and limb structure handling but also surgical steps in replantation and postoperative manage of the replanted segment.

CONCLUSIONS

Considering limb replantation a very important surgery, with high risk level, the laboratory training is a primordial step to avoid pitfalls, achieve good survival rate and functional results. Our model of rat hind replantation insures all surgical steps of the replantation. It can be considered the ideal experimental model for training in replantation to improve clinical proficiency.

REFERENCES

1. Hopfner E. Ueber Gefassnaht, Gefasstransplantationen und Replantation von Amputierten Extremitaten. Arch Klin Chir 1903;70:417-71.
2. Malt R, Mc Khann C. Replantation of severed arms. J Amer Med Assoc 1964;189:716-22.
3. Biemer E. Definitions and classifications in replantation surgery. Brit J Plast Surg 1980;33:164-8.
4. Biemer E, Duspiva W. Rec Micro Surg 1982;56-58.
5. Jones NF. Replantation in the Upper Extremity, Ch. 82, Grabb and Smith’s Plastic Sugery. 5th Ed., Lippincott-Raven, 1997
6. Ashur H, Owen ED. Reimplantation of completely amputated rat limb.Inter Surg 1979;64(3):45-50.
7. Ad-El DD, Harper A, Hoffman LA. Digital replantation teaching model in rats. Microsurg 2000;20:42-44.
8. Greene EC. Anatomy of the rat. New York and London, Hafner Pub, 1963.
9. Lee S. Manual of Microsurgery. CRC Press Inc, 2000, p. 94-7.
10. Kmatsu S, Tamai S. Successful replantation of a completely cut of thumb. Plast Rec Surg 1968;42:374.
11. Wang H, Gu Y, Dong Z. Rat -tail replantation model. J Rec Microsurg 1999;15(3):203-6.
12. Amara B, Fernandez JJ, Newlin L, et al. Transfer of the rat tail: an experimental model for free osteocutaneous transfer. J Rec Microsurg 1998;14(5):359-62.
13. Zhang F, Chin BT, Ho PR, et al. Rat tail replantation as a training model for microvascular procedures of digit replantation, Microsurg 1998;18:364-7.
14. Nicher HP, Morgan LS, Horowitz GH, et al. A digit repantation model. Microsurg 1985;6:70-2.



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