Arch Dermatol Res (2001) 293 : 515–521© Springer-Verlag 2001

O R I G I N A L PA P E R

Yukie Kitano · Kotaro Yoshimura · Gentaro Uchida ·Katsujiro Sato · Kiyonori Harii

Pretreatment with topical all-trans-retinoic acidis beneficial for wound healing in genetically diabetic mice

Received: 9 March 2001 / Revised: 3 July 2001 / Accepted: 16 September 2001

Abstract Objective: Topical pretreatment with all-transretinoic acid (atRA) is known to improve healing of cutaneous wounds. We tested the effect of atRA on woundhealing of genetically diabetic db/db mice. It is known
that cutaneous wounds of db/db mice show delayed
wound healing due to impaired wound contraction, delayed granulation tissue formation and underexpressionof keratinocyte growth factor (KGF). Methods: 0.1%
atRA in 100 mg aqueous gel was applied to the backskin of db/db mice as well as to their normal heterozygous littermates, db/+ mice, for five consecutive days,and 2 days after completion of the atRA treatment, tworound excisional wounds were created down the panniculus carnosus with a 6-mm punch biopsy on the backskin of each mouse. Results: After 5 days treatment with
0.1% atRA, significant hypertrophy of the epidermis
and dermis, neovascularization, and inflammatory cell
invasion were seen in the skin of the db/db mice, butthese effects were seen only weakly in db/+ mice.
Wounds in atRA-treated db/db mice closed more rapidly than those in vehicle-treated db/db mice. KGFmRNA expression, which is usually significantly lower
in db/db mice than in normal mice, in wounds of atRAtreated db/db mice on day 1 of treatment was as strong
as in db/+ mice. Conclusion: Pretreatment with atRA
reversed the impaired wound healing in db/db mice.
Keywords All-trans retinoic acid · Diabetic mouse ·Wound healing

Y. Kitano () · K. Yoshimura · G. Uchida · K. Sato · K. Harii
Department Plastic and Reconstructive Surgery,
School of Medicine, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo, 113-8655
e-mail: kitano-pla@h.u-tokyo.ac.jp,
Tel.: +81-3-58008670, Fax: +81-3-58006929

Introduction
Retinoic acid (RA) improves the clinical symptoms of acne
vulgaris, psoriasis and skin photoaging [1]. Histologically,
topical treatment with RA induces hypertrophy in the epidermis and dermis via an increase in dermal vasculatureand invasion of inflammatory cells. RA has several contradictory effects on wound healing. For example, systemic
application of RA has deleterious effect on healing of fullthickness skin wounds and corneal wounds of diabetic animals [2, 3]. On the other hand, systemic RA reverses delayed wound healing in steroid-treated animals [4–7]. RA
is also known to improve wound healing of abraded skin byacceleration of epidermal turnover when it is applied topically prior to wounding [8]. Topical pretreatment with RA
also enhances healing of full-thickness excisional woundsof photoaged and normal skin [9, 10]. These results suggest that wound healing may be affected by the route orschedule of retinoid administration, and that, at least insome skin conditions, topical pretreatment may be the mosteffective method of administration of all-trans-RA (atRA).
We hypothesized that impaired wound healing of genetically diabetic db/db mice may be reversed by topical pretreatment with atRA. Db/db mice show clinical symptoms
similar to insulin-resistant diabetes mellitus patients, suchas severe obesity, hyperglycemia, hyperinsulinemia, andinsulin resistance [11–14]. These symptoms were derived
from genomic mutation in the leptin receptor. Db/db mice
are also known to show impaired cutaneous wound healing,
as is universally observed in diabetic patients [11]. Woundhealing of db/db mice is characterized by weak contraction,
slow inflammatory cell invasion and delayed granulation
formation. The characteristic anatomy of the skin of db/db
mice, including thick subdermal fat and an extremely thindermis, may also contribute to impairment of wound healing.
Many growth factors and their receptors, such as FGF,
FGF receptors, KGF, IGF and VEGF, have been found to
be aberrantly expressed in wounds of db/db mice [11, 12].
Of these, the factor that is considered to be the most important for delayed wound healing in db/db mice is KGF

516[15]. In normal mice, the mRNA levels of KGF are acutely
upregulated early in wound healing [16, 17]. However,
KGF mRNA levels in db/db mice are unchanged throughout the healing process [15]. Since upregulation of KGF
mRNA is one of the earliest events of wound healing, KGF
is considered to be an important factor that might regulate
downstream events during wound healing.

Materials and methods
Animals
Female C57BLK/ksj db/db mice at 8–12 weeks of age (Saitama Experimental Animals Supply Company, Saitama, Japan) and theirnormal littermates (db/+ mice) were used throughout this study.
Animal experiments were performed according to the Laboratory
Animal Guidelines of Tokyo University. All animal procedures were
performed under general anesthesia with diethyl ether. Three independent experiments were performed, and 12 to 15 mice were usedin each experiment. The back skin of each mouse was shaved andtopically treated for five consecutive days with 100 mg 0.1% atRA
(Sigma Aldrich, Tokyo, Japan) in aqueous gel vehicle or 100 mg
aqueous gel without atRA. Scaling was graded by three investigators according to the visual scoring method of Effendy et al. [18]:0, no scaling; 1, weak scaling; 2, moderate scaling; 3, large flakes,
intense peeling.
On the 2nd day after completion of atRA treatment, two 6-mm
round excisional wounds, down the panniculus carnosus and its associated fascia, were created in each mouse. The wound area wascalculated as the product of the long and short diameters of eachwound. Mice were killed on days 1, 3, 7 and 14 by cervical dislocation under general anesthesia with diethyl ether. Wound tissue
was processed for histology and RT-PCR.
Histology
The mice were injected subcutaneously with 30 µg/g 5-bromo-2′deoxyuridine (BrdU labeling reagent; Amersham Pharmacia, ShinFig. 1 Scaling score of db/db
mice treated with 0.1% atRA,
and of db/+ mice treated with
0.1% or 0.4% atRA. Three
mice in each group were evaluated. The values are means ±SD of duplicate measurementsfrom three independent experiments

juku, Tokyo) 3 and 24 h before they were killed. Skin samples
were fixed in 10% formalin, dehydrated and embedded in paraffin.
Paraffin sections were cut at a thickness of 5 µm. Sections were rehydrated and stained with hematoxylin and eosin, and with Gomori’s
trichrome stain. Immunohistochemistry to determine the level of
BrdU incorporation into cells was performed as follows. Sections
were incubated in 4 N HCl for 30 min at 37 °C followed by 0.1 M
borax buffer, pH 9.0, for 10 min at room temperature. The specimens were then treated with anti-BrdU monoclonal antibody(Amersham Pharmacia) followed by biotinylated anti-mouse IgG.
The specimens were then incubated in ABC reagent (Vector,
Burlingame, Calif.), and developed with 3,3′-diaminobenzidine.
RT-PCRmRNA was extracted from a 2-mm wound margin using ISOGENLS (Nippon Gene, Shinjuku, Tokyo). The mRNA (1 µg in a total
volume of 20 µl) was transcribed to cDNA by AMV reverse transcriptase (Promega Japan, Higashinihonnbashi, Tokyo) at 37 °C
for 1 h. For semiquantitative detection of KGF and β-actin cDNA,
1 µl cDNA in a total volume of 50 µl (50 mM KCl, 15 mM TrisHCl, pH 8.0, 4 mM MgCl2) was amplified with Ampli-Taq Gold(PE Biosystems, Roppongi, Tokyo) in a PC-960G thermal cycler(Cosmo Bio, Toyo, Tokyo). The sequences of the oligonucleotide
primers used for amplification of KGF were TTGCAATGAACAAGGAAGGA and GAATTCTATCTTGCAATGAA, and those forβ-actin were TGAGGAGCACCCTATGCTGC and TAGCCCTCGTAGATGGG. Amplification was carried out as follows: pre-PCR
step of 10 min at 94 °C; 32 PCR cycles of 30 s at 94 °C, 30 s at
58 °C and 1 min at 72 °C; and a final step of 10 min at 72 °C. We
had already confirmed in a preliminary study that the PCR products would be within the linear log phase under these conditions.

Results
The effects of topical application of atRA
Scaling was more intense in db/db mice than in db/+ micefollowing treatment with 0.1% atRA (Fig. 1). Although517

Fig. 2 a–d H&E staining of the back skin of db/+ mice (a vehicletreated mouse, b 0.1% atRA-treated mouse) and db/db mice (c vehicle-treated mouse, d 0.1% atRA-treated mouse). Arrows indicate
sebaceous glands

0.1% atRA did not induce significant scaling in db/+ mice,higher concentrations of atRA did induce significant scaling
in these mice, so the effect of atRA appears to be dose-dependent.
Histologically, the skin of db/+ mouse showed only
slight dermal and epidermal hypertrophy after treatment
with 0.1% atRA. On the other hand, the skin of atRAtreated db/db mice showed significant hypertrophy of epidermis and dermis, scaling of stratum corneum, an increase in cellularity and proliferation of subepidermal
vessels (Fig. 2). Elongation of the rete ridges and atrophyof the sebaceous glands were also characteristic of atRAtreated db/db mice. In vehicle-treated db/db mice, BrdU incorporation was observed throughout the basal layer ofthe epidermis. However, in atRA-treated db/db mice, BrdU
incorporation was greater in the follicular epidermis, but
not in the interfollicular epidermis (Fig. 3).

Wound healing of atRA- and vehicle-treated db/db mice
Day 0 after wounding
The wounds in vehicle-treated db/db mice were 20–30%
larger than the original wounds, but the wounds in atRAtreated db/db mice did not show enlargement just after
wounding (Fig. 4).
Day 1 after wounding
Vehicle-treated db/db mice on day 1 after wounding
showed only a small number of inflammatory cells in thewound cleft. Only a small amount of extracellular matrix
had formed at this time. However, atRA-treated db/db miceon day 1 showed massive invasion of inflammatory cells
in the wound cleft. In addition, a large amount of extracellular matrix staining bluish with trichrome reagent hadformed within the granulation tissue, which connected the
granulation tissue with the wound margin (Fig. 5a, b).

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Fig. 3 a–d BrdU staining of back skin of db/db mice treated witheither of vehicle (a) or 0.1% atRA (b) for 5 days. c, d High-power
magnification of a and b (a, b ×100; c, d ×200)

Day 14 after wounding
Macroscopically, 78% of wounds in vehicle-treated db/db
mice had not closed on day 14. Histologically there was alarge amount of granulation tissue within the wide woundcleft. Epidermal regeneration had started, but due to the
distance between the wound margins, there still was an
uncovered area within the cleft. On the other hand, 89% of
the wounds in atRA-treated db/db mice had already closedby this time. Histologically the wound margins had comeclose to each other, and reepithelialization was almost complete (Fig. 5c, d).
Expression of KGF mRNA
mRNA expression of KGF at the time of wounding did notshow significant differences between atRA- and vehicletreated db/db mice (data not shown). However, KGF

mRNA in the wounds of atRA-treated db/db mice on day 1
after wounding was significantly higher than in vehicleor sodium lauryl sulfate-treated db/db mice (Fig. 6).

Discussion
In the study reported here we demonstrated that (1) topical treatment with atRA had stronger effects in db/db micethan in db/+ mice, and that (2) atRA pretreatment reversed
delayed wound healing in db/db mice.
It is noteworthy that atRA induced cutaneous changes
in db/db mice much more strongly than in db/+ mice. This
does not mean that db/+ mice are totally refractory to theeffects of atRA, but that db/db mice are more sensitive toatRA than db/+ mice, because the latter showed effects similar to those seen in db/db mice when they were treated withhigher concentrations of atRA. It is not clear whether thisincreased sensitivity was due to downstream events resulting from the leptin receptor deficiency, or due to lossof barrier function. Similar model-specific sensitivity has
been observed in other experiments. For example, atRA
increases the synthesis of collagen and hyaluronic acid in

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Fig. 4 Wound area of db/+ anddb/db mice. Round wounds ofdiameter 6 mm were made on
the back skin of mice pretreated with either 0.1% atRA or
vehicle. Wounds of db/+ mice
had almost closed by day 7
whether they had been treatedwith 0.1% atRA or vehicle. On
the other hand, vehicle-treateddb/db mice showed enlargement of the wound area on day
1, which was followed by delayed wound contraction. Pretreatment with atRA prevented
the wounds of db/db mice fromexpanding, which may havecontributed to the earlier
wound closure. Data representmeans ± SD of duplicate measurements from three independent experiments

Fig. 5 a–c Wound sections of db/db mice pretreated with vehicle(a, c) or atRA (b, d) were stained with Gomori’s trichrome. Arrows

indicate the wound margin (a, b 1 day after wounding; c, d 14 days
after wounding). a, b ×200; c, d ×50

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Fig. 6 Expression of KGF mRNA in day 1 wounds of db/db anddb/+ mice treated with atRA (RA), sodium lauryl sulphate (SLS), or
vehicle (V). A representative result of RT-PCR from three independent experiments is shown (KGF KGF mRNA, BA β-actin mRNA)UVB-irradiated mice, but not in non-irradiated hairlessmice [19, 20]. It has also been reported that atRA effectively reverses steroid-retarded repair, which may be a result of a mutual interaction between glucocorticoids andretinoids in the process of inflammation, immunity andconnective tissue production [4–7]. It is interesting thatthe sebaceous glands of db/db mice showed extremely
strong changes. Sebaceous glands almost completely disappeared in db/db mice after treatment with 0.1% atRA,
but those in db/+ mice did not show significant changes
after the same treatment. Even with 0.4% atRA, about 30%of sebaceous glands remained intact in db/+ mice, althoughsignificant dermal hypertrophy was observed with this concentration of atRA.
Pretreatment with 0.1% atRA had little effect on thewound healing in normal mice, but it significantly accelerated wound healing in db/db mice. This may reflect the biological effects of atRA pretreatment in these animals. Delayed wound healing in db/db mice seemed to be enhanced
by treatment with atRA. First, full-thickness wounds in
db/db mice had enlarged 20–40% just after wounding due
to their extremely thin dermis with a small amount of extracellular matrix, which was the primary cause of delayed
wound healing in db/db mice during the early stages ofwound healing. Treatment with atRA made the dermis thickand stiff preventing enlargement of excisional wounds in
db/db mice. Second, granulation tissue and extracellularmatrix formation induced by atRA may also accelerate
wound healing in db/db mice. This granulation tissue contains huge numbers of inflammatory cells that release cytokines and growth factors that enhance wound healing.
In addition, smooth muscle actin fiber involved in woundgranulation may exert a contractile force.
The mechanism of action of atRA in wound healing iscomplicated. A study using a dominant-negative retinoic
acid receptor mutant strongly indicates that epidermal proliferation and regeneration is initiated by heparin-binding
epidermal growth factor (HB-EGF) induced by atRA [21,22]. Since atRA induction of HB-EGF occurs in suprabasal
cells prior to the onset of basal cell hyperproliferation, thismechanism may well explain the effectiveness of atRA
pretreatment. On the other hand, atRA may also enhancemacrophage function, and thus finally may enhance macrophage-derived growth factors such as FGF, TGF and IGF-I[6]. This is also plausible since macrophage dysfunction isseen in db/db mice [23]. A more sensitive real-time RT-PCR
could detect quantitative differences in these macrophagederived growth factors.
Although suppression of KGF is characteristic of woundhealing in db/db mice, KGF mRNA levels in unwoundedskin are almost the same between db/db and db/+ mice [20],which was also confirmed by our RT-PCR (data not shown).
In addition, atRA-induced epidermal proliferation in db/db
mouse was not associated with KGF upregulation before
wounding. Taken together, atRA does not directly induce
KGF in unwounded skin but it causes the skin of db/db
mice to induce KGF.
Previous studies of atRA application to already existing
wounds have revealed conflicting results. Some investigators have shown enhanced healing [7, 14, 24], but others
have shown retardation of healing [10, 25, 26]. On the other
hand, topical pretreatment with atRA has consistently
shown improvement in wound healing. In addition, our experimental and clinical experience has revealed that atRA
worsens wound healing of full-thickness open wounds (unpublished data). For these reasons we do not recommend
atRA application to diabetic ulcers. In practice, atRA may
enhance wound healing when it is applied before surgery
in diabetic patients. In the present study, we only evaluated the effects of atRA pretreatment in diabetic wounds,
but it may be possible to apply this strategy to other healing-impaired wounds, such as those in the skin of the elderly or malnourished, or ischemic skin. Pretreatment with
atRA may prevent delayed healing, infection and dehiscence in these potentially healing-impaired wounds.

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