limb replantation in rats - experimental model - Timisoara Medical ...

LIMB REPLANTATION IN RATS - EXPERIMENTAL
MODEL
Zorin Crainiceanu 1 , Bogdan Bratu 1 , Mihai Ionac 2
REZUMAT
Scopul studiului este de a realiza un model experimental pentru de antrenament n replantare. Modelul standardizat poate fi de asemenea folosit n trainingul
avansat n microchirurgie. Studiul a fost efectuat pe un num\r de 7 [obolani Sprague-Dawely adul]i, cu greutate ntre 250 [i 300g. Sub anestezie general\
s-a practicat amputa]ia de coaps\ la limita dintre 1/3 proximal\ [i mijlocie. Replantarea s-a realizat urmnd timpii standard ai acestiu procedeu. Viabilitatea
segmentului replantat a fost monitorizat\ prin mijloace clinice: culoare (roz), consisten]\ (plin, elastic), dar [i prin pulsoximetrie pe o perioad\ de 7 zile. Toate
animalele au avut o evolu]ie postoperatorie bun\, cu supravie]uirea segmentului replantat, cu edem minim, f\r\ complica]ii trofice sau infec]ioase.
Cuvinte cheie: replantare, microchirurgie, [obolan, membru caudal
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.
Key words: replantation, microsurgery, rat, hind limb
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
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
Received for publication: Sep. 25, 2005. Revised: Oct 28, 2005.
0RIGINAL ARTICLES
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
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Zorin Crainiceanu et al 243

(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
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.
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244 TMJ 2005, Vol. 55, No. 3
Figure 1. Surgical anatomy
Figure 2. Vessels and nerves disection
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)
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)

Figure 3. The aspect of the amputated segment and stump
Figure 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.
Figure 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)
Figure 6. Pulsoxymetry monitorisation
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Zorin Crainiceanu et al 245

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
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246 TMJ 2005, Vol. 55, No. 3
model 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.