ARTICLE
Auteur(s) : Berna
Aksoy1,2, Hasan Mete Aksoy3, Ekrem
Civaş4, Hüseyin Üstün5, Nilgün
Atakan6
1TDV 29 Mayis Private Ankara Hospital,
Dermatology Clinic, Ankara, Turkey
2Private Konak Hospital, Dermatology Clinic, Kocaeli,
Turkey
3Private Konak Hospital, Plastic and Reconstructive
Surgery Clinic, Kocaeli, Turkey
4Civaş Private Clinic, Ankara, Turkey
5Ankara Research and Training Hospital, Pathology
Clinic, Ankara, Turkey
6Hacettepe University Faculty of Medicine,
Dermatology Department, Ankara, Turkey
accepté le 13 Juillet 2009
Chronic wounds pose important problems in clinical practice.
Their treatment is difficult, costly and is associated with a high
recurrence rate [1]. The healing of chronic wounds can be of vital
importance and treatment of these wounds has many economic and
social implications [2, 3]. Many different endogenous and exogenous
factors are capable of retardation of wound healing [4].
Retardation of healing is known to be associated with immunological
disorders, disturbance of metabolic processes such as protein
deficiency, postoperative complications, wound infection, hypoxia,
chronic venous insufficiency and hormonal disorders like Cushing’s
syndrome and diabetes mellitus [3-5]. Any of the factors mentioned
above can impair wound healing partially by affecting angiogenesis
[4, 6].
A clinically relevant and easily reproducible chronic wound
animal model has not been described until now [1]. Wounds that are
created in healthy animals heal quickly. A delayed wound
healing animal model simulating slow and complicated healing of
chronic human wounds would be very useful for testing different
treatment modalities used in the management of chronic wounds. Most
of the currently available animal wound healing models are
representative of acute wound healing. Many animal models of
impaired wound healing have been developed for studying the biology
of chronic wounds and testing potentially beneficial treatments
[7]. Surgical wounds have been created in steroid-treated rats [4,
8], diabetic mice [9], radiation damaged rats [10] and pigs [7],
and wounds were produced chemically (sodium dodecyl sulphate) in
mice [4]. Surgically manipulated wounds in rats [1, 11], in rabbits
[12] and in mice [13] are also examples of chronic animal wound
healing models. All these current delayed animal wound healing
models, based on diabetes induced or genetically diabetic animals,
steroid or anticancer treated animals, and irradiated animals, are
difficult to reproduce [1, 13]. They are not widely utilized
because the wounds in these models are not the same as chronic
human wounds where local factors play major roles [13, 14].
Here we describe a new, practical and cheap animal model of
delayed wound healing that can be used to study the biology of
chronic/delayed wound healing and test potential therapeutic agents
that can be used for the treatment of such wounds.
Materials and methods
All animal experiments were conducted under a protocol approved by
the institutional ethics committee and complied with all
requirements of the Animal Welfare Act. Fifteen, male, New Zealand,
young adult white rabbits weighing between 2000-3000 grams were
used in this study. Rabbits were anesthetized by intramuscular
injection of ketamine (60 mg/kg) and xylazine (5 mg/kg)
and their backs were shaved with razors. Skin flaps were outlined
with a surgical marking pen (figure 1A). The dorsal
area skin of each rabbit was then cleansed with an iodine solution
and all the surgery was performed under aseptic conditions.
A 4 cm horizontal incision was made on the skin of dorsal
part of the torso behind shoulders (figure 1B). Pure skin
flaps measuring 2 × 4 cm in size were elevated in front of and
behind this incision (figure 1C). This
dissection exposed the panniculus carnosus layer (figure 1C). Part of the
exposed panniculus carnosus layer, 4 × 4 cm in size, was
resected. During this resection meticulous hemostasis with
electrocautery was important to prevent postoperative hematoma
formation and total skin flap necrosis. Skin flaps were returned to
their places and sutured using 4/0 nylon sutures with cutting tip
needles (figure
1D). Skin flaps were also attached to underlying muscle
layer with one suture placed in the middle of the incision to fix
the skin flaps in place. Following a 3 week healing period (in
consideration of partial skin flap necrosis in some cases), third
degree burn injuries were inflicted by using hot metal plates on
the healed skin flaps and on normal skin at the same location on
the opposite side, under anesthesia (figure 2A). A brass
plate with a handle weighing 500 g was used for inflicting
burn injury. The metal plate had a circular contact surface with a
diameter of 3.5 cm. The metal plate was left in boiling water
at 100 °C for 15 minutes and then applied to the skin of the
animal for 15 seconds to create a full thickness
3rd degree burn injury [15]. Bleaching of the skin
was observed as a sign of burn injury (figure 2B). Topical
antimicrobial treatment was not applied.
The healing rate, healing time and scar quality were evaluated
clinically. We accepted a wound as clinically healed when there was
no de-epithelized open wound area left. Scar samples were obtained
from control and panniculectomy wounds when panniculectomy wounds
healed clinically and these samples were sent for histopathological
examination.
Results
None of the animals were lost during the study. We did not observe
any wound infection or dehiscence. We observed partial flap
necrosis in 4 out of 15 cases before inflicting burn injury.
The wounds on the panniculectomy side healed clinically in an
average of 43.20 (SD ± 3.76) days but on the control side they
healed clinically in an average of 32.80 (SD ± 3.85) days. There
were 10.40 days (SD ± 1.12) between the control and panniculectomy
sides as far as the wound healing period was concerned. Paired
samples t-test was conducted to find out if the difference between
panniculectomy and control sides was statistically significant.
Before the test, the normality of the two data sets was checked and
both seemed to have approximately normal distributions. The result
of the paired samples t-test showed that the difference between the
panniculectomy side and the control side was statistically highly
significant (p-value: < 0.001). In other words, there was a
31.7% delay in healing of the wounds on the panniculectomy side.
Thus, wound healing and contraction were slower on the
panniculectomy side. Moreover, scars were broader and more
irregular on the panniculectomy side (figures 3A and B).
Histopathologically, the panniculus carnosus layer was composed
of loose connective tissue and striated muscle. Histopathological
examination of scar tissue specimens from clinically completely
healed control wounds revealed a thin epidermis including a keratin
layer and granulation tissue including fibroblasts and a mild
mononuclear infiltration. Scar tissue contained regular, coarse and
homogenous collagen fibers which were oriented parallel to the
surface. A panniculus carnosus layer was present in the
deepest parts (figures
4A and B). Histopathological examination of scar tissue
specimens from clinically completely healed panniculectomy wounds
revealed a thin epidermis including a keratin layer and more
prominent granulation tissue, including fibroblasts and a mild
mononuclear infiltration. Scar tissue was composed of irregular
collagen fibers which were oriented longitudinally to the surface
and towards the periphery. A panniculus carnosus layer was not
present in the deepest parts (figures 4C and D).
Discussion
Use of rodents in animal models is practical, due to low cost and
ease of care and handling [11]. However, wounds in these mammals
heal primarily through wound contraction rather than by
re-epithelization. Rodents have a subcutaneous panniculus carnosus
muscle. The panniculus carnosus has been shown to significantly
contribute to wound healing by helping wound contraction and this
layer also contributes collagen to healing wound [11, 16]. Excision
of the panniculus carnosus muscle has been shown to delay wound
healing through limitation of wound contraction [17].
Several chronic animal models based on rodents and the
panniculus carnosus layer have been described. Zhou et al.
[13] developed an animal chronic skin wound model in mice. They
elevated a flap including the panniculus carnosus layer on the back
of mouse. Seven days after the first surgery, they created a soft
tissue defect in the center of this flap. Control wounds closed
completely on day 21, and chronic wound healing model wounds
clinically failed to heal completely on the 21st day.
However, on histopathological examination, epithelization was
almost completely established in both control and model wounds
within 21 days following injury [13]. In this model, although
healing was delayed initially, the wounds on elevated flaps healed
completely almost at the same time as the control wounds. Chen
et al. [1] elevated a longitudinal bipedicled skin flap on rat
dorsum, to the depth of the panniculus carnosus layer, and created
6 wounds. They observed an initial delay in healing of these wounds
but the wounds healed at a similar rate as the controls after day 9
[1]. Gould et al. [11] raised flaps including the panniculus
carnosus layer and created full thickness excisional wounds down to
the panniculus carnosus layer in the center of these flaps. They
placed a silicone sheet underneath the flap to create tissue
ischemia. They showed delayed healing of the wounds located in
2 cm bipedicle flaps with silicone sheet intervention [11].
This is an ischemic delayed wound healing animal model.
Wounds are categorized as chronic when healing with adequate
therapy is delayed beyond 8 weeks in humans [3, 18]. But if one
compares the life span of laboratory animals to that of humans,
healing beyond 8 weeks is not logical for naming any wound as
chronic in animals. Our values may well be considered as chronic
when considering a rabbit’s life span.
In our delayed wound healing animal model, pure skin flaps were
raised on the backs of young rabbits followed by the removal of the
panniculus carnosus layer. The skin flaps were returned to their
places and sutured. We propose that partial flap necrosis before
inflicting burn injury may be related to a reduction of blood
supply to the skin flaps secondary to excision of panniculus
carnosus layer. Three weeks following this operation, full
thickness third degree burn wounds were created in the center of
healed skin flaps. It is known from clinical observations in humans
that wound healing following 3rd degree burn injury is
significantly prolonged. In this model, wound contraction and
closure were definitely prolonged to 43 days in average and
healing quality decreased, due to the excision of the panniculus
carnosus layer. Healing was also delayed in the control wounds as a
result of 3rd degree burn injury, despite the
presence of panniculus carnosus layer. So, a combination of
panniculectomy and full thickness burn injury delayed wound healing
significantly, with a decrease in scar quality. Histopathological
examination revealed that the scars obtained from healed
panniculectomy wounds had characteristics of earlier phases of
wound healing processes. However, the scars from healed control
wounds were more mature and organized, so they showed
characteristics of later phases of the wound healing process.
The delayed wound healing animal model presented in this study
has been developed in rabbits and has some advantages over other
animal models reported in the literature. Rabbits are inexpensive
to purchase and easy to care for. In this new model of delayed
wound healing, the wounds are easy to create and readily
reproducible. There are similarities to chronic or delayed human
third degree burn wounds. The delayed wound healing model presented
is an effective animal model to study the biology of delayed wound
healing and test therapeutic agents.
Acknowledgement
The authors thank gratefully to Baris Surucu for statistical
analysis and to Oner Tuysuz for laboratory technical support.
Financial support: none. Conflict of interest: none
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