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Angiostatic effects
of corticosteroid on wound healing of the rabbit ear
Ichiro Hashimoto*, Hideki Nakanishi*,
Yoshitaka Shono*, Maki Toda*,
Hidetaka Tsuda**, and Seiji Arase**
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*Department of Plastic and Reconstructive Surgery,
and **Department of Dermatology, The University of Tokushima
School of Medicine, Tokushima, Japan
Abstract: Wound healing is a complex biologic process with
initial inflammation, granulation tissue formation, and matrix
remodeling. We observed the relation between angiostatic effects
and corticosteroid administration time in the rabbit ear chamber.
Angiogenesis in the chamber was studied using a microscope-television
system. Two experiments were undertaken to represent the systemic
and the topical administration of steroids. In experiment
1, 10mg of triamcinolone acetonide was injected three times
intramuscularly (on the day of implantation of the chamber,
and the 7th and 14th day after implantation). Vascularization
in this group was significantly delayed at the 7th, 14th,
and 21st days but no difference from controls was observed
in the size and density of vessels after its completion. In
experiment 2, 3 mg of triamcinolone acetonide was injected
once into the skin adjacent to the chamber on the 10th day
after installment of chambers or on the day of installment.
In the former group, new vascular growth was delayed until
the 21st day after installment. The hemorrhagic zone had narrowed
and vascular dilation was observed. In the latter group, endothelial
budding was delayed and vascular constriction occurred. New
vascular growth was severely delayed and granulation filling
of the chamber was not completed. These results suggest not
only that the topical administration had the stronger inhibitory
effect on neovascularization than the systemic administration
but that the effect differed depending on the stage of wound
healing. In view of this effect of this steroid, we should
pay careful attention to the time when steroids are administered
to patients. J. Med. Invest. 49:61-66, 2002
Keywords:angiogenesis, corticosteroid, wound healing, rabbit
ear chamber
INTRODUCTION
Wound healing of the skin is an important clinical factor
for skin surgery, burn injury, and skin ulcers. It is a complex
biologic process with initial inflammation, repair (granulation
tissue formation), and matrix remodeling (1). Since the repair
process comprises angiogenesis and fibroplasia, angiogenesis
is considered to be an indicator of wound healing.
Although suppressive effects on the wound healing by corticosteroids
have been recognized (2), details about the relation between
these effects and administration time remain unclear. Steroid
injection into wounds is performed for treatment of keloids
and hypertrophic scars both intra-or post-operatively (3).
Sometimes, surgery must be performed on patients undergoing
systemic steroid therapy. We undertook two experiments to
represent the systemic and the topical administration of steroids.
The purpose of this study was to clarify the angiostatic effect
of corticosteroids during wound healing and to analyze the
relation between the effect of this agent and its administration
time in the rabbit ear chamber.
MATERIALS AND METHODS
Animals/Rabbit Ear Chamber
Japanese white rabbits weighing between 2.5 and 3.0 kg were
used. Three holes in the edge and one in the center were made
on the distal side of the earlobe after removal of the fur
with a laser. The rabbit ear chamber was implanted after the
epidermis around the central perforation was carefully removed
without damaging the cartilage and the subdermal blood vessels.
The translucent disk and glass cover of the chamber formed
a tissue regeneration space (50 µm thick) (2, 4).
Granulation from the surrounding intact tissue started to
grow into this space. The length of the new vascular growth
was measured on the monitor TV of the microscope-televison
system on the days mentioned below. After the round chamber
was divided into four parts each of 90 degrees, the vessel
growth was rated in the center of each part. The average of
the four points was regarded as the growth for that day. The
average growth on the observation days was calculated based
on the basis of these findings. We observed not only the growth
of vessels but also the size of vessels and the condition
of flow.
Experimental Groups
Triamcinolone acetonide (Sankyo Co., Ltd, Tokyo, Japan) was
used as the corticosteroid and saline as the control. In experiment
1, nine animals each in group, A and B, were injected intramuscularly
three times (on the day of implantation of the chamber, and
the 7th and 14th day after implantation) with saline (1 ml)
or triamcinolone acetonide (10mg, 1 ml), respectively (Fig.
1). The growth of the vessels was assessed on the 5th, 7th,
14th, and 21st day after implantation. In experiment 2, 15
rabbits were divided into three groups (group C, D, and E)
and each received two administrations of intradermal injection
around the chambers (on the day of implantation and on the
10th day after implantation) (Fig. 1). Similar volumes (0.05
ml at one place and total 0.3 ml) of saline or triamcinolone
acetonide were injected intradermally into six places adjacent
to the chamber. The total amount of triamcinolone acetonide
was 3 mg in each administration. In group C, saline was injected
two times, and in group D, saline was injected first and triamcinolone
acetonide second. In group E, the triamcinolone acetonide
was injected first and saline second. The growth of the vessels
was assessed on the 7th, 10th, 14th, 17th, 21st, 24th, and
28th day after implantation.
Statistical Analysis
For all steroid-adiministered groups and the control group
in each experiment, statistical analyses of the between-group
differences in average growth of the vessels were performed
with the Mann-Whitney U test. P values less than 0.05 were
regarded as statistically significant.
RESULTS
Experiment 1
In group A (control group), the neovascularization process
began with the chamber filled with debris. The progression
of the newly formed vessels across the chamber was characterized
by a hemorrhagic zone, which is known as a `fibrin net', prior
to distention of the capillary tips. The fibrin net was apparently
formed by the extrusion of blood cells through the distended
and fragile walls of the blind capillary sprouts as a result
of the constant pulsation transmitted from the general circulation
through the feeding vessels. The diameter of the dilated new
capillaries then became smaller and formed pre- and post-capillary
vessels and eventually arterioles.
The sprouts in group A were observed from the 5th to 7th day
(average:6th day), but in group B from the 7th to 10th day
(average:8th day). The sprouting was delayed significantly
(p<0.05) in the steroid-administered group. In the steroid-treated
animals, the forcible ebb and flow seen in the newly formed
capillaries of the control animals were conspicuously absent,
and the hemorrhagic zone prior to budding of the capillaries
did not develop. Moreover, a very large proportion of the
capillaries were filled with plasma alone, in which a few
almost motionless red cells were suspended.
The neovascularization in group B was significantly delayed
at the 7th, 14th, and 21st days (Fig. 2). At the completion
of the vascularization, however, there was no difference in
the size and density of vessels and the condition of flow
between the control group and the systemic administration
model.
Experiment 2
In group C (control group), endothelial budding, fibrin net,
and vascular new growth were observed in the same manner as
in group A (Figs. 3, 4). The fibrin net prior to all new vessels
was seen on the 17th day. All vessels had started to grow
towards the center of the chamber and the fibrin net to diminish
on the 21st day. The growth of the new vessels was completed
from the 23rd to 25th day.
In group D, sprouts formation and vascular new growth were
not delayed until triamcinolone acetonide was injected (Figs.
3, 5). The normal fibrin net was seen on the 17th day, but
vascular dilation began to be observed from this time. Delay
in vascular new growth was observed and the fibrin net started
to become thinner from the 21st day. The granulation filling
of the chamber was delayed compared with that of control group.
After completion of the vascular growth, however, no differences
between groups C and D in size and density of vessels and
condition of flow were observed.
The debris of group E was less and moved more slowly than
that of groups C and D. In group E, endothelial budding was
significantly delayed (p<0.0001) (Figs. 3, 6). The capillary
network behind the advancing endothelial sprouts was markedly
empty and no hemorrhagic zone prior to capillary budding developed
(Fig. 7). Vascular constriction occurred in both arterioles
and venules, that is, the blood flow was reduced and intermittent
in the area of new vascular growth, which was delayed, while
granular filling of the chamber was not completed in group
E.
DISCUSSION
There are many models for observing angiogenesis in vivo,
including the chorioallantoic membrane of the chick embryo,
the rabbit or rat cornea, and the hamster cheek pouch (5-7).
These models, however, are useful for assessment of angiogenesis
from noninjured existing vessels. The rabbit ear chamber model,
in which we can observe angiogenesis from injured skin, was
suggested to be one of the best models for wound healing in
the skin.
Inhibition of wound healing by corticosteroids is a well-known
phenomenon that was suggested to be caused by the inhibition
of the inflammatory phase of healing as well as of matrix
synthesis (2, 8-10). Corticosteroids reduce vascular permeability
and induce a transient monocytopenia, which results in a decrease
in extravasation and migration of inflammatory cells to the
sites of injury (11, 12). Corticosteroids inhibit the production
of other chemotactic stimuli such as the complement system
and lymphocyte-derived chemotactic factor, and also inhibit
macrophage activation (13).
The aim of experiment 1 was to confirm the anti-angiogenic
activity of corticosteroids upon systemic administration.
Despite the administration of 30 mg of triamcinolone acetonide,
however, we found no differences between the administered
group and the control group after completion of vascularization.
In the comparison between two methods of administration, it
was suggested that the topical administration had the stronger
inhibitory effect on neovascularization than the systemic
administration. In experiment 2, we found that the inhibitory
effect of corticosteroids on neovascularization differed according
to the stage of wound healing. When the steroid was administered
immediately after injury, granulation was not completed. It
is suggested that the delay in vascular new growth was caused
by the inhibition of vascular permeability and matrix synthesis
and that this inhibition was due to suppression of some growth
factors which are normally expressed during the early stage
of wound healing.
Many kinds of growth factors related to angiogenesis have
been identified recently. Endothelial growth factor, transforming
growth factor-α, β, vascular endothelial
growth factor (VEGF), and platelet derived growth factor have
been found to accelerate endothelial growth in vivo and in
vitro (14, 15). Due to the finding that VEGF affects mainly
endothelial cells and can also increase vascular permeability,
it was suggested to be the most effective factor for endothelial
growth. Its expression becomes prominent within 1 to 3 days
after injury (16). This suggests that VEGF is an important
cytokine for vascular hyperpermeability and angiogenesis in
the early stages of wound healing. The findings of the present
study demonstrated that suppression of angiogenesis by corticosteroids
was more effective before rather than during wound healing.
Therefore, corticosteroids may also suppress VEGF or other
growth factors active at the beginning of wound healing.
For surgery in steroid-treated patients, the effects of steroids
on wound healing is very important, so that careful consideration
should be given to the time and period of their administration.
ACKNOWLEDGMENTS
This work was supported in part by Grant-in-Aids for Encouragement
of Young Scientist and for Scientific Research (C) from the
Japan Society for the Promotion of Science.
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Received for publication December 3, 2001 ; accepted January
28, 2002.
Address correspondence and reprint requests to Ichiro Hashimoto,
M.D., Ph.D., Department of Plastic and Reconstructive Surgery,
The University of Tokushima School of Medicine, Kuramoto-cho,
Tokushima 770-8503, Japan and Fax:+81-88-633-7297.
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