ORIGINAL ARTICLE
Rat Dorsal Paired Island Skin Flaps: A Precise Model for
Flap Survival Evaluation
Hirotoshi Ohara, Kazuo Kishi and Tatsuo Nakajima

Department of Plastic and Reconstructive Surgery, Keio University, Tokyo, Japan(Received for publication on August 11, 2008)(Revised for publication on September 30, 2008)(Accepted for publication on October 9, 2008)

Abstract. Rectangular flaps on the rat or mouse dorsum are frequently used for flap survival elongation research. However, since rectangular flaps are purely random, the survival
rate varies among individuals. A reliable animal flap model with low individual variation isdesirable for flap survival research, especially in the angiogenic field. We investigated thesurvival rate of paired flaps in the rat dorsum containing 3 vascular territories in each flap,
and determined their usefulness for flap elongation research.
Two symmetrical adjoining rectangular flaps (11×3 cm each) were drawn on the rat dorsum. Two days after material injection, flaps were elevated with only the deep circumflex iliac vessels as the vascular pedicle. Flaps were immediately sutured back and the flap survival area was measured 7 days after the operation. The control group (n = 9) had saline solution injected in both flaps. The bone marrow group (n = 8) had bone marrow injected in
the right flap, and saline solution injected in the left flap.
In the control group, the survival rate of the paired flaps was not statistically different(right flap, 89.0±5.6%; left flap, 89.3±4.5%). In the bone marrow group, the survival rate
between the bone marrow injected flap (89.9±3.7%), and the saline flap (84.8±4.3%) was
statistically different.
The rat dorsal paired island skin flap model shows low difference in flap survival rate anduses an internal control. This is a suitable model for flap survival elongation research. (KeioJ Med 57 (4) : 211－216, December 2008)
Key words: flap model, vascular territory, axial pattern flap, bone marrow, flap survival evaluation

Introduction

flaps require a large number of animals to obtain statistically significant result. Recently, with the advancements
in vascular anatomy and surgical techniques, there has
been greater demand clinically for axial pattern flaps
over random pattern flaps, thus making the axial pattern
flap model more suitable for the study of flap survival.5
Previously, Taylor and Minabe demonstrated that rat
dorsal skin is supplied by 3 vessels; namely, the lateralthoracic (LT), posterior intercostal (IC), and deep circumflex iliac (DCI) vessels.6 A rat dorsal axial pattern
flap model utilizing these vessels was first described by
Syed et al. in 1992.7 Since that time, the model has undergone further developments.4, 5, 8, 9 These previous reports all presented variations of paired rectangular flaps
on the dorsal skin, but did not investigate the creation of

Much time and effort is spent on procedures to elongate flap survival areas. However, it remains difficult tocompletely prevent flap necrosis.1 Although the delay
procedure is a widely used method of improving flap
survival, it requires multiple procedures,1, 2 and a moreconvenient procedure is necessary. In order to study flap
survival, a flap model that can accurately and sensitively
reflect the effects of drugs or cells is necessary. Manyflap models have been developed, and MacFarlane’s dorsal flap model is the one most frequently used.3 However, this model utilizes a purely random flap, resulting inunstable blood circulation, unpredictable necrosis, andvariations in skin vasculature.4 Moreover, the single

Corresponding Author: Kazuo Kishi, M.D., Ph.D., Department of Plastic and Reconstructive Surgery, Keio University, Shinjuku-ku Shinanomachi 35 Tokyo,
160-8582, Japan, Tel: +81-3-5363-3814 , Fax: +81-3-3352-1054, E-mail: kkishi@sc.itc.keio.ac.jp211

Ohara H, et al.: Rat Dorsal Paired Island Skin Flap Model212

with the skin margin, but the soft tissue was gently dissected in order to preserve the DCI vessel. Finally, theflap was elevated, retaining only the attachment to the
DCI vessel as the vascular pedicle (Fig. 2). Flaps werethen immediately sutured back to the donor sites with
5-0 nylon sutures, or stapled with a skin stapler. Sevendays after flap elevation, rats were sacrificed by an overdose of pentobarbital, and the survival areas of the skinflaps were observed. The experimental protocol was approved by the Animal Research and Care Committee of
the School of Medicine, Keio University.
Bone marrow group and control group

Fig. 1 Design of the rat dorsal paired island skin flap model.
Two rectangular flaps (11×3 cm) were marked on the dorsal
area. The flaps included three vascular territories. LT, lateralthoracic vessels; IC, posterior intercostal vessels; DCI, deep
circumflex iliac vessels.

paired axial pattern flaps. In this study, we investigated a
rat dorsal paired island skin (DPIS) flap model covering
the 3 vascular territories, and compared survival rates
between the paired flaps.
Materials and Methods
DPIS flap model design and elevation of flaps
Male Sprague-Dawley (SD) rats, weighing 350-400 g,
were used in this study. Rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital, and thedorsal skin hair was shaved. Next, a flap design, which
was modified from previous reports,5, 9 was drawn on
the skin. The margins of the flaps were as follows: thelateral borders were 3 cm to either side of midline; themedial border was dorsal to midline; the distal border
was a line perpendicular to midline over the apex of thescapula; and the proximal border was a line perpendicular to midline and 11 cm distal to the distal border. Twosymmetrical adjoining rectangular flaps (11 × 3 cmeach) were drawn on each rat (Fig. 1). Two days after
injection of solution, the flaps were elevated under thepanniculus carnosus. The LT and IC vessels were incised

To study elongation of flap survival, we used bonemarrow, which has already been recognized to produce
an angiogenesis effect.10, 11 To harvest bone marrow, SD
rats were sacrificed and their femurs removed. Next,
both edges of the epiphyses were cut and the marrow
was flushed out repeatedly with heparinized saline. The
bone marrow from 2 rats (4 femurs) was collected andmixed with saline for a total volume of 0.3 mL. For rats
in the bone marrow group, 0.1 mL of the bone marrow/saline mixture was injected into the right flap of each rat
intradermally at 3 points on the midline along the longaxis 1, 2, and 3 cm proximal to the distal end. The left
flap was injected with saline at the equivalent 3 points
as an internal control. Injections were performed in thesame manner for the control group, but saline was used
for both the right and left sides. In a preliminary experiment, the flap with the DCI pedicle always became necrotic about 2 cm proximal from the distal end (data notshown). Therefore, we chose this site as the injection
point, along with additional injection sites proximal anddistal to this point, i.e., at 1, 2, and 3 cm proximal to the
distal end. A total of 20 rats were used for this study.
The rats were randomly divided into two groups, with
10 rats in the bone marrow group and 10 rats in the control group.
Measurement of survival rates of flaps
Seven days after flap elevation, the rats were sacrificed and the flap survival areas were observed. Distal
areas of the skin flap that were hard or covered withcrust were judged to be necrotic. The total area of theflaps and the survival area were traced on clear acetate
sheets and scanned as digital images. The digital images
were analyzed using Adobe Photoshop CS3 Extended
software (Adobe Systems, Inc., San Jose, CA) to calculate the percentage of the survival area. Measurements
were performed twice, and mean values were used forstatistical analysis. The survival rate was expressed as
the percentage of the surviving area to the total skin flap
area. The difference in survival rates was expressed asKeio J Med 2008; 57 (4): 211－216

213

Fig. 2 (A) Angiogram showing normal angiographic anatomy of the rat dorsum. The broken lines indicate the vascular territories. The
dotted line indicates the midline of the dorsum. (B) The flap elevated under the panniculus carnosus. Three vessels supply the dorsum
skin. (C) The elevated left flap after dissection of the pedicle. LT, lateral thoracic vessels; IC, posterior intercostal vessels; DCI, deep
circumflex iliac vessels.

the right flap survival rate minus the left flap survival
rate.
Statistical analysis
All values were expressed as the mean ± standard deviation. Statistical significance was determined using thepaired t-test to compare paired flaps, and the MannWhitney U
U-test to examine differences between thegroups. Statistical correlations between paired flaps
were analyzed by Searman’s correlation test. A p-valueless than 0.05 was considered statistically significant.
No correction was made for multiple testing.
Angiographic evaluation
We performed angiography to investigate the relationship between survival area and vascular territory, and tocompare changes in the vessels between paired flaps.
Two rats were randomly chosen from each group. The
rats were anesthetized, the carotid artery was cannulated,
and the rats were euthanized by an overdose of pentobarbital. Next, 150 mL/kg of a lead oxide/gelatin mixture was injected into the cannula and the rats were incubated at 4ºC. After 24 hours, the dorsal skin flap was
harvested and placed on IX Envelopak FR film (Fuji

Photo Film Co., Ltd., Tokyo, Japan) and radiographed(60 kVp, 5 mA, 15-second exposure) using a soft x-ray
machine (ESM; Softex Co., Tokyo, Japan).
Results
We began with 10 rats in each group, but one rat ineach group was excluded from the study because of theabsence of intercostal (IC) vessels, and one rat in thebone marrow group died of unknown causes. All of theflaps showed congestion on the distal border on postoperative day 3, and demarcation was clearly observed atday 7. In the control group, the necrotic area was almost
equal in size between the paired flaps in each individual,
and interestingly, the shape of the necrotic areas was alsonearly symmetrical (Fig. 3A). Angiography revealed flap
necrosis in the LT vessel territory, the third territory (Fig.
3B). In many of the rats in the bone marrow group, theright flaps that received bone marrow showed longer
survival than the contralateral flaps (Fig. 3C). In the angiogram, the necrotic area of the bone marrow injected
flap was localized to the distal end, and vascularity wasmoderately increased in the third territory; however, thesurvival area could not be perfectly determined based on
angiography because some contrast medium flowed
backward into the vessel and the necrotic area (Fig. 3D).

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Ohara H, et al.: Rat Dorsal Paired Island Skin Flap Model

Fig. 3 (A) A representative rat from the control group. The survival areas appear equal on both sides and the areas of necrosis arenearly symmetrical. (B) Angiogram of the same rat from a control group rat. Two vascular territories, posterior intercostal vessels anddeep circumflex iliac vessels, survived equally on both side. (C) A representative rat from the bone marrow group. The survival area inthe right bone marrow flap is larger than in the control flap. (D) Angiogram of the same rat from a bone marrow group rat. The necrotic
area of the right bone marrow flap was localized to the distal end, and vascularity was moderately increased in the third territory.
Arrowheads indicate back flow of the contrast medium. The broken lines indicate the boundary of the necrotic area. IC, posterior
intercostal vessels; DCI, deep circumflex iliac vessels.

There was no significant difference in the right flap survival rate (89.0 ± 5.6%) and the left flap survival rate
(89.3 ± 4.5%) in the control group ((p=0.79) (Fig. 4A).
The flap survival rate ranged from 80.5% to 98.9%, andthe difference in survival rate ranged from 0.2% to 4.8%.
In the bone marrow group, there was a significant difference in the right flap (bone marrow injected) survival
rate (89.9 ± 3.7%) and the left flap survival rate (84.8 ±
4.3%) ((p=0.005) (Fig. 4A). The flap survival rate ranged
from 78.9% to 92.6%, and the difference in survival rate
ranged from 1.5% to 10.8%. The difference in survival
rate was 0.3 ± 3.3% for the control group and 5.1 ± 3.3%
for the bone marrow group, which was a significant difference ((p=0.03) (Fig. 4B). There was also a positive
correlation in survival rate between paired flaps in thecontrol group (rrs=0.67, p=0.02), but no such correlationin the bone marrow group (rrs=0.36, p=0.19) (Fig. 4C).
This correlation suggests that a change in survival rate islinked between paired flaps in the control group, i.e., if
one of the DPIS flaps has a long survival length, then theother side will also have a long survival length.

Discussion
The DPIS flap model consists of paired symmetrical
axial pattern flaps on the rat dorsum. The flaps include 3
territories and are attached only by the DCI vessel. There
were no research about equality of the survival rate between paired axial pattern flaps in previous reports.4, 5, 8, 9
In this study, the difference in survival rates between thepaired flaps was 0.3 ± 3.3% in the control group, which
was contrary to that seen in the bone marrow group at
5.1 ± 3.4%. Thus, in the DPIS flap model one flap can beused as an internal control. The flap survival rate in ourstudy ranged from 80.5% to 98.9% in the control group.
There was an 18.4% difference in the range of each flap,
even when using the same species, sex, body weight,
technique, etc. We speculate that differences in the survival rate of each flap were due to individual differences
between animals, especially with regard to vascularity,
which can contribute to the extent of necrosis in the third
territory. Moreover, subtle differences in technique, suchas those that occur in the preparation around the pedicle
and the position of the flap, will also make a difference.
However, the difference in survival rate between paired
flaps in each individual ranged from 0.2% to 4.8%, andthe individual difference was reduced to as low as 3.6%.

Keio J Med 2008; 57 (4): 211－216

215

Fig. 4 (A) Comparison of the survival rates. There was no significant difference between paired flaps in the control group. In the bonemarrow group, there was a significant difference between paired flaps. (B) Box plot comparing differences in survival rates between
the control group and bone marrow group. There was a significant difference between the groups. (C) Survival rate correlation between
paired flaps. The correlation in survival areas of the paired flaps in the control group suggests that the survival rate between paired flaps
is linked; however, no correlation is seen in the bone marrow group.

The internal control also canceled out differences due tothe influence of the experimental environment. Thus, theDPIS flap model can easily detect changes due to the injected materials by using an internal control.
One reason for the equality in the areas of necrosis in
this model is the 3 territory system axial pattern, whichuses the linking phenomenon.12, 1 3 The linking phenomenon occurs when the adjoining territory can be reliably
captured by the dominant territory, and flap necrosis usually occurs in the third territory. In the DPIS flap model,
DCI is the dominant territory, IC is the adjoining territory, and LT is the third territory. The extent of necrosis in
the third territory varied between animals in the control
group due to individual differences of the animals; however, between paired flaps, the extent of necrosis was
similar, and symmetrical necrosis was seen in the control
group.
We previously created a model with LT dominant double symmetrical flaps, but the dissection of the LT vessel
was difficult since it arises from muscle, contrary to the

DCI vessel, which arises from adipose tissue (Fig. 5).
The DPIS flap model has the same advantages of previous studies.5, 9 It is cost effective since extra animals
are not needed for controls, easy to observe, prevents auto-cannibalism, and it is easy to create the flap. However,a problem with this model, although very rare, is thecongenital absence of IC vessels. In this case, when elevating the flap, it will survive because the DCI and LT
vessels expand into neighboring territories from the beginning (Fig. 6). The only way to prevent this problem is
to directly observe the flap elevation and rule out cases
where the IC vessel is absent.
The DPIS flap model uses two flaps on each rat, whichhave equal areas of necrosis. When introducing target
drugs or cells to a flap, the contralateral flap will be theinternal control used to detect the small change inducedby the drugs or cells. In this study, we used bone marrow
to demonstrate that the paired flaps can produce equal
necroses in the control group. Bone marrow contains
several kinds of stem cells, growth factors, and cytokines

Ohara H, et al.: Rat Dorsal Paired Island Skin Flap Model

216

Fig. 5 (A) Magnification of the lateral thoracic vessels. The
lateral thoracic vessel runs intramuscularly under the flap margin.
Dissection of the lateral thoracic vessels is difficult for creating
a lateral thoracic vessel dominant flap. (B) Magnification of the
deep circumflex iliac vessels. The deep circumflex iliac vessel
arising from adipose tissue. LT, lateral thoracic vessels; DCI,
deep circumflex iliac vessels.

that induce angiogenesis. Although the details of themechanism of action and contribution to angiogenesis
require further exploration, bone marrow transplantation
to the ischemic limb is already in clinical use.10, 11, 14
This model will be especially useful for detecting theeffect of angiogenic agents such as pharmacologic drugs,
cells, and cytokines.
Conclusion
We created a rat dorsal paired island skin flap model.
The paired flaps have equal areas of necrosis, and thedifference in the survival area was 0.3 ± 3.3%. Due tothis low variability in flap survival area, one flap can be
successfully used as an internal control. This flap model
is suitable for investigating flap survival elongation.
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