Chapter 5. Orthotopic vascularized groin flap transplantation
model in rats
Nicolae Ghetu
Codrin-Nicolae Dobreanu
Victor George Ilie
Vlad Ionut Ilie
Alain-Ali Mojallal
Dragos Pieptu
1. Rationale of model development
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
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Experimental models in rodents 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.
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Experimental models in rodents 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).
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Experimental models in rodents 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
Experimental models in rodents 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).
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Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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).
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Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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
Experimental models in rodents 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-2second 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,
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Experimental models in rodents 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.
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