CellBio, 2013, 2, 248-256
Published Online December 2013 (http://www.scirp.org/journal/cellbio)
Open Access CellBio
Wound Closure on the Neonatal Rat Skin I. The
Modulation of the Thickness of Epidermis at
the Closing Incisional Wounds
Mary Arai, Takashi Matsuzaki, Setsunosuke Ihara*
Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan
Email: *ihara@life.shimane-u.ac.jp
Received September 24, 2013; revised October 24, 2013; accepted November 1, 2013
Copyright © 2013 Mary Arai et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Full-thickness incisional wounds were made on the dorsal skin of 1-day-old rats to elucidate the mechanism of the fluc-
tuation of the epidermal thickness after the wound closure. The thickness of the epidermis covering the wound reached
a peak around 96 h post-wounding (PW), and became thinner thereafter. The analyses of the cell proliferation and
apoptosis at the epidermal wound regions revealed that the rate of TUNEL-positive cells that displays the cells under-
going apoptosis increased as the epidermis became thinner around 120 h PW. Next, immunohistochemical analyses
using antibodies against keratinocyte differentiation marker proteins indicated that the delay or interruption of the
spinous to granular transition from 96 to 120 h PW might result in the epidermal thickening in the wound region. Third,
the region undyed with anti-caspase-14 antibody extended downward in the thickened epidermis by 96 h PW, and in
turn, it became intensely and widely stained with this antibody in the thinning epidermis by 120 h PW. Taken together,
it is likely that the delay and acceleration of the terminal differentiation, including cornification of the epidermal kerati-
nocytes may coordinately cause the fluctuation of the thickness of the epidermis at the wound site in rat neonates.
Keywords: Wound Healing; Reepithelialization; Rat; Neonate; Epidermis; Terminal Differentiation; Cornification
1. Introduction
Reepithelialization, a part of the process of the wound
healing, is essential for organisms to survive. The pat-
terns of reepithelialization are known to differ during the
developmental stages [1-6]. First, the wounds in mam-
malian adult skin are sealed with fibrin clot and then the
wound closure is driven by epidermis crawling from the
wound margin [7]. Meanwhile, the wounds in the neona-
tal rat skin are covered rapidly with the extended wound-
surrounding epidermis, and the abnormal expression pat-
terns of keratins and cadherins at the wound site have
been revealed by immunohistochemical analyses [6,8].
However, the late stages of reepithelialization after the
wound closure have not yet been well investigated. In
this study, we focused on the phenomenon that, once full-
thickness incisional wounds made on the skin of neonatal
rats were closed, the thickness of the covering epidermis
increased and temporarily overran and, in turn, restored
its normal level.
First, we examined the possible fluctuation of the epi-
dermal cell number at the wound site, and calculated the
rate of the cell proliferation as well as that of the cell
death detected by anti-BrdU immunostaining and
TUNEL, respectively. Second, we analyzed the terminal
differentiation of the epidermal keratinocytes. In order to
identify the differentiation stage of the wound epidermis
after the wound closure, the immunohistochemistry was
performed using antibodies of involucrin, profilaggrin/
filaggrin, and loricrin as the epidermal differentiation
markers [9-12]. Finally, we examined the distribution of
CCAAT/enhancer-binding protein-α (C/EBP-α) and cas-
pase-14. These two proteins are known to be endogenous
factors regulating the terminal differentiation of the epi-
dermal keratinocytes at the early or late stages, respec-
tively [12-20].
The findings in the present study are summarized as
follows. The delay or derangement in the terminal dif-
ferentiation of keratinocyte occurs within the areas from
the upper spinous to the granular layer in the thickening
epidermis at the wound region. The thinning of the
*Corresponding author.
M. ARAI ET AL. 249
wound epidermis may also be associated with the corni-
fication probably accompanied by an enhancement of the
cell death and the action of caspase-14.
2. Materials and Methods
2.1. Wounding
Sprague-Dawley rats were anesthetized at 1 day after
birth. Two incisional wounds 3 mm in length were made
in the back skin on either side of the dorsal midline with
a disposable scalpel (FEATHER). The wound sites were
immediately covered with the cover agent Opsite (Smith
& Nephew), and the wounded individuals were returned
to the mother rats. The Opsite treatment had no effects on
the wound healing except that it avoided unnecessary
retardation of healing due to the infection that otherwise
occasionally occurred (Koizumi et al., 2004).
2.2. BrdU Injection
Two hours before sampling, 10 μl/gram body-weight 10
mM 5-bromo-2’-deoxyuridine (BrdU) were injected into
the abdominal cavity of rats.
2.3. Histology and Immunohistochemistry
The skin surrounding the wound was cut out at 0, 48, 72,
96, 120, or 144 h PW. Pieces of the skin were fixed with
4% paraformaldehyde in Ca2+, Mg2+-free phosphate-buff-
ered saline [PBS()] for 1 h at room temperature, and
embedded in paraffin. Sections (4 μm thickness) were
stained with hematoxylin-eosin (HE) or analyzed immu-
nohistochemically. For immunohistochemistry, deparaf-
finized sections were pretreated with citric acid buffer
(pH 6.0) at 95˚C, and rinsed with PBS(). Alternatively,
the sections were pretreated with 2N HCl, followed by
digestion with 0.05% trypsin at 37˚C. The pretreated
sections were incubated with 1% normal horse serum in
PBS() for 20 min, and then reacted with primary anti-
bodies at 4˚C overnight. They were washed with PBS(),
then incubated with secondary antibodies for 2 h. Sam-
ples were washed with PBS(), counterstained with 4’,6-
diamino-2-phenylindole dihydrochloride (DAPI, Poly-
sciences) at 0.1 μg/ml, and mounted with Fluoromount
(Japan Tanner Corporation). The following primary anti-
bodies were used: mouse anti-cytokeratin 14 monoclonal
antibody (mAb) (Chemicon, 1:100); mouse anti-cy-
tokeretin 10/13 mAb (Lab Vision, 1:100); mouse anti-
cytokeratin 6 mAb (Progen, 1:50); rabbit anti-mouse
involucrin poriclonal antibody (pAb) (Berkley antibody
company, 1:500); rabbit anti-mouse loricrin pAb (abcam,
1:500); rabbit anti-rat profilaggrin/filaggrin pAb (466)
(generously supplied by Dr. R. B. Presland, 1:250); and
mouse anti-BrdU mAb (Dako Cytomation, 1:100). Alexa
fluor 594 goat anti-mouse IgG1 (Molecular Probes,
1:1500); Alexa fluor 488 goat anti-mouse IgG2a (Mo-
lecular Probes, 1:500); Alexa fluor 488 goat anti-mouse
IgG1 (Molecular Probes, 1:500); and Alexa fluor 488
goat anti-rabbit IgG (Molecular Probes, 1:500) were used
as secondary antibodies.
The double staining of loricrin with involucrin and in-
volucrin with filaggrin, and the staining of C/EBP-α and
caspase 14 were performed using immunoenzyme tech-
nique. Deparaffinized sections were treated with citric
acid buffer (pH 6.0) at 95˚C or 10% TritonX-100 in
PBS(), and endogenous peroxidase was inactivated by
H2O2 before blocking process. After incubation with
HRP-conjugated anti-rabbit immunogrobulins (DakoCy-
tomation, 1:100) as the secondary antibody, the samples
were reacted with DAB for color reaction. The samples
for the double staining were subjected to staining with
the next primary antibodies, followed by staining the
alkaline phosphatase-conjugated anti-rabbit immunogro-
bulins (sigma-aldrich, 1:100) as the next secondary anti-
body. The immunostained sections were finally counter-
stained with hematoxylin. The following primary anti-
bodies were used: anti-C/EBP-α rabbit pAb (Santa Cruz,
1:100); and anti-caspase 14 rabbit pAb (IMGENEX,
2.4. TUNEL
The TUNEL reaction was performed using in situ cell
death detection kit and TMR red (Roche Diagnotics)
according to the manufacture’s instructions.
2.5. Numerical Applications of the Rate of BrdU
and TUNEL Positive Cells
The ratios of the numbers of BrdU- or TUNEL-positive
cells to those of DAPI positive cells in the wound areas
or the intact areas of epidermis were calculated. The
epidermal regions between both wound edges and the
normal interfollicular epidermal regions more than 5 mm
away from the wound edges were defined as the wound
areas and the intact areas of epidermis, respectively, in
this study.
3. Results
3.1. Morphological and Immunohistochemical
Changes in the Incisional Wound
The standard technique using a scalpel No. 11 (FEATHER)
in this study usually generated the full-thickness inci-
sional wounds 1 mm in depth, as shown in Figure 1(A)
(0 h PW). By 48 h PW, wound closure was completed by
the epidermal sheets that crawled from the wound edge
and contacted with each other at the wound center (Fig-
ure 1(B)). From 72 to 120 h PW, the epidermis covering
the wound was thickened downward the dermis as com-
pared with the normal area of epidermis, and the thick-
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HE K 14K 10K 6
Figure 1. Morphological and immunohistochemical changes
in neonatal rat skin wounds during reepithelialization. Se-
rial cross sections were prepared and stained with hema-
toxylin-eosin ((A)-(F)), and anti-K14 ((G)-(L)), K10 ((M)-
(R)), or K6 ((S)-(X)) antibodies. At 0 h PW, K14 and K10
keratins were localized at the basal layer (G), and the su-
prabasal layer (M), respectively. In contrast, K6 keratin
was undetectable at 0 h PW (S). Around 48 h PW, wound
closure was completed (B). By that time, the distribution of
K14-positive cells were expanded vertically throughout the
suprabasal layers and stretched horizontally from the
wound edge (H); K10-positive cells were absent from the
wound-covering areas (N); and K6 keratin became detect-
able in the suprabasal layers through the wound-covering
and the wound-surrounding areas (T). By 72 h PW when
the wound had been closed, the epidermis was thickened
downward the dermis (C). The thickening lasted up to 96 h
PW (D). These extending patterns of K 14 and K 6 were
gradually shifted to the normal patterns from the distal
region of the wounds from 72 to 120 h PW ((I)-(K), (U)-
(W)). Conversely K10-positive cells were gradually in-
creased with time in the wounded areas ((O)-(Q)). At 120 h
PW, the thickened epidermis was beginning to be thinner
((E), (F)). The normal localization of these keratins was
almost recovered in the wounded areas at 144 h PW ((L),
(R), (X)). Scale bars, 100 μm; and Counterstained, with
ening peaked around 96 h PW (Figures 1(C)-(E)). At
144 h PW, the thickened epidermis tended to become
thinner (Figure 1(F)), and finally, the wound-covering
epidermis almost restored the normal thickness 192 h PW
(data not shown).
In addition, the immunohistochemical analyses using
the epidermal differentiation markers showed that K14
keratin was localized at the basal layer, and the localiza-
tion of K10 keratin was observed at the suprabasal layer,
whereas K6 keratin was not detectable, at 0 h PW (Fig-
ures 1(G), (M), (S)). The localizations of these keratins-
positive cells in the wound-covering and the wound-
surrounding epidermis fell into disorder within 48 hours
after wounding (Figures 1(H), (N), (T)), and gradually
recovered the normal patterns from the distal region of
wound during reepithelialization (Figures 1(I)-(L), (O)-
(R), (U)-(X)).
3.2. The Analyses of the Process in Which the
Temporarily Thickened Epidermis Becomes
From the above-mentioned histological observations, we
noticed that the epidermis was thickened just after wound
closure, and thereafter became thinner. Then, we became
interested in what was the cause of the fluctuation of the
epidermal thickness at the wound areas. To begin with,
we speculated that the number of the epidermal cells
would change at the wound. Exactly the living cells in
the thickened epidermis outnumbered those in the control
epidermis, as long as the total numbers of DAPI-positive
nuclei were compared (Figure 2(Q)). Therefore, we fur-
ther analyzed cell proliferation and apoptosis in the
wound and intact areas of epidermis to find the answer to
the fluctuation of the epidermal cell number.
BrdU-positive cells as the proliferating cells were de-
tected in the basal layer at 0 h PW and in the wound-
covering epidermis from 48 h PW onward, too (Figures
2(A)-(F)). Here, the absolute number of BrdU-positive
cells in the epidermal wound regions suggested that the
cell proliferation took place during the thickening of the
epidermis in the wound region. However, when the ratios
of the number of the proliferating cells to the total num-
ber of living cells were estimated, the rate in the wound
areas of epidermis were almost constant throughout the
period tested and, moreover, there was no great differ-
ence between these rates in the wound and the intact
epidermis (Figures 2(G) and (H)). Consequently, it was
likely that the down-regulation of cell proliferation, if
any, did not so much contribute to the thinning of the
wound-covering epidermis.
Next, we did TUNEL staining to test the possibility
that apoptosis is induced in the thickened epidermis as
the epidermis became thinner and reduced in cell num-
ber. From 0 h to 96 h PW, the rates of TUNEL-positive
cells in both the wound and intact epidermis were gener-
ally similar, though more or less variable (Figures
2(I)-(L), (O), (P)). At 120 h PW, however, the rate of
TUNEL-positive cells in the epidermal wound site sig-
nificantly increased, as compared with that in the intact
or 0 h PW (Figures 2(M), (O), (P)). The wound region at
144 h PW had still a high rate of TUNEL-positive cells
(Figures 2(N), (O), (P)). The epidermis in the wound
area reached a rate equivalent to that of the normal epi-
dermis at 192 h PW (data not shown). It should be noted
that TUNEL-positive cells were observed only in the
vicinity of the nucleated upper granular layer in both
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Figure 2. Cell proliferation and apoptosis in the wound and intact areas of epidermis. The rate of the number of
“BrdU-positive cells (stained red)”, which represents the proliferating cells, to the total number of living cells in the wound
areas of epidermis were almost constant throughout the period tested (0 - 144 h PW, (A)-(G)). In contrast, the rate of
“TUNEL-positive cells (stained red)” that displays the cells undergoing apoptosis increased as the epidermis became thinner
(around 120 h PW), ((I)-(O)). The total numbers of DAPI-positive nuclei in the wound areas of epidermis at 48 - 144 h PW
were counted as the living cells (Q). Scale bar, 100 μm; counterstain, DAPI; dashed lines, the border between the epidermis
and dermis; *, p < 0.05; **, p < 0.01; and Red asterisks, comparisons of intact values.
We examined the localizations of these markers. First,
the double immunostaining of loricrin and involucrin was
executed during the process of wound healing. In both
the wound and normal areas of the neonatal skin at 0 h
PW, loricrin (brown) was expressed at and above the
granular layer, whereas involucrin (blue) at and above the
upper spinous layer (Figure 3(A)): i.e., there was a clear
spatial (and possibly temporal) difference in the start of
expression between loricrin and involucrin. At 48 h PW
when epidermis from the wound margin completed
wound closure, such normal expression pattern remained
wound and intact epidermis.
These results suggested that cornification accompany-
ing cell death is significantly elevated during the thinning
of the epidermis at the wound region.
3.3. The Rates of the Terminal Differentiation
May Be Perturbed in the Epidermal Wound
The epidermal keratinocytes generate diverse differentia-
tion marker proteins during the terminal differentiation.
wintactwoundint int
Lor Inv
0hPW 48 72
96120 144
Lor Inv LorInv
Lor InvLor InvLorInv
Figure 3. The loricrin-positive lowermost epidermal layer
became identical to the involucrin-positive one from the
middle of reepithelialization. The upper and middle panels
(the magnifications of wound (left) and intact (right) areas)
show the expression of anti-loricrin (brown) and involucrin
(blue) detected by double staining using the enzyme-labeled
antibodies, and the lower panels show these proteins sin-
gle-stained with fluorescence-labeled antibodies (green) in
the adjacent sections. Loricrin (brown) was expressed at
and above the granular layer, and involucrin (blue) ap-
peared from the upper spinous layer in the neonatal skin at
0 h PW (A). It should be noted that these two proteins were
apt to be stained from the same layer at 96 and 120 h PW in
the epidermal thickening regions ((D) and (E) wound). Scale
bars, 100 μm, 10 μm, and 100 μm from above; counterstain,
hematoxylin and DAPI; and dashed lines, the border be-
tween the epidermis and dermis.
at the wound-covering epidermis (Figure 3(B)). On the
contrary, at 96 and 120 h PW when the epidermis at the
wound areas was thickened, these two proteins were ex-
pressed from the identical cell layer in the epidermal
thickening regions (Figures 3(D) and (E)), although it
remained unclear which protein sifted. Later, the epider-
mal wound area getting thinner inchmeal recovered the
normal expression pattern (144 h PW, Figure 3(F)).
Next, we carried out the double immunostaining of the
epidermis of the neonates using the anti-involucrin and
anti-profilaggrin/filaggrin antibodies. In both the wound
and normal areas at 0 h PW, the expression of profilag-
grin/filaggrin (blue) was observed at the zone lower than
involucrin (brown), concretely, at and above the middle
spinous cell layer (Figure 4(A)). In contrast, unlike the
relationship of expression of loricrin and involucrin, the
spatial difference in the expression of involucrin and pro-
filaggrin/filaggrin were maintained in the same way as in
the normal areas in the same thickening epidermis spe-
cimens. Such an expression pattern remained unchanged
by the thinning phase of the wound-covering epidermis
(Figures 4(B)-(F)).
wintactwoundint int
Inv Fil
0hPW 48 72
96120 144
InvFil Inv Fil
Inv FilInvFilInv Fil
Figure 4. The expression patterns of filaggrin remained
unchanged during reepithelialization. The results of double
staining of involucrin (brown) and filaggrin (blue) and sin-
gle staining in the adjacent sections (green) are arranged in
the same way as in Figure 3. Unlike the expression of in-
volucrin (brown) that started from upper spinous layer,
filaggrin (blue) was expressed at and above the middle
spinous layer in the 0 h PW (A), and maintained this hier-
archical relation until 144 h PW ((B)-(F)). Scale bars, 100
μm, 10 μm, and 100 μm from above; counterstain, hema-
toxylin and DAPI; and dashed lines, the border between the
epidermis and dermis.
3.4. The Localizations of Expression of
Molecules Participating in the Epidermal
Terminal Differentiation Fluctuated during
We immunohistochemically examined the expression of
C/EBP-α and caspase-14, known to participate in the
terminal differentiation of the epidermal keratinocytes at
the early and late stages, respectively. At 0 h PW, C/EBP-
α was intensely stained at both the nuclei and the cyto-
plasm of the cells in the suprabasal layer, while caspase-
14 at the cytoplasm in the granular layer (Figures 5(A)
and (H)). The staining intensities of both molecules at the
leading edge of the epidermis were obviously fallen by
24 h PW (Figures 5(B) and (I)). At 48 h PW, these im-
munostaining intensities were restored to the normal lev-
els at the wound-covering epidermis. The epidermis at 72
h PW showed a similar tendency (Figures 5(C), (D), (J ),
(K)). By 96 h PW when the thickness of the wound-
covering epidermis reached the peak, the region intensely
stained with anti-caspase-14 was confined to the upper
layer at the thickening epidermis, and the unstained re-
gion remarkably extended (Figure 5(L)). Subsequently,
however, the region intensely stained with anti-caspase-
14 in turn enlarged downward by 120 h PW in the midst
of the thinning phase (Figure 5(M)). From 96 to 120 h
PW, the localization of C/EBP-α-positive cells was
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M. ARAI ET AL. 253
Figure 5. The expression of C/EBP-α and caspase-14 in the
wound areas. The localization of C/EBP-α and caspase-14
was immunohistochemically examined. In the neonatal epi-
dermis at 0 h PW, C/EBP-α and caspase-14 were distrib-
uted in the suprabasal layer (A) and granular layer (H),
respectively. At 24 h PW, both of these molecules were di-
minished at the wound edge ((B), (I)). After wound closure
was completed, these immunostaining intensities were re-
stored to the normal level at the wound-covering epidermis
by 48 h PW ((C), (J)). After that, the localization of C/EBP-
α-positive cells was hardly changed, although the thickness
of wound epidermis was varied ((D)-(G)). In contrast, the
region unstained with anti-caspase-14 extensively extended
downward in the thickened epidermal region by 96 h PW
(L). Subsequently, the region intensely stained with anti-
caspase-14, in turn, became enlarged more downward in
the thinning epidermis at 120 h PW (M). Scale bar, 100 μm;
and counterstained, hematoxylin.
hardly changed (Figures 5(E) and (F)). The epidermis at
144 h PW showed that both of these two molecules
tended to restore the normal level of expression (Figures
5(G) and (N)).
These results suggested that the modulation of the ex-
pression of caspase-14 might be responsible for the epi-
dermal thickness.
4. Discussion
HE and immunostaining experiments in this study re-
vealed the healing process of the incisional wounds on
neonatal rat skin to be outlined as follows (Figure 1).
The opened wounds were first closed by the epidermal
cells that, keeping the cell-to-cell contact, rapidly ex-
tended from the wound margin. Simultaneously, the wound-
covering epidermis became thickened. Subsequently, the
epidermis at the wound area became thinner up to the
normal thickness, accompanying an elevation of the lev-
els of the basal cells and the basement membrane. So, we
have further continued morphological analyses of the
incisional wounds, with the aim of pursuing the cause of
above-stated fluctuation in the epidermal thickness.
4.1. The Phase of Thinning of Epidermis at the
Wound Area and That of the Cell Death in
the Upper Epidermis Overlap Each Other
We estimated the rates of cell proliferation and cell death
using anti-BrdU immunostaining and TUNEL staining,
respectively, to verify the possible causal relationship
between the epidermal cell numbers and the fluctuation
of the epidermal thickness in the wound area. The analy-
ses of cell proliferation revealed that there is no clear
correlation between the fluctuation of thickness at the
wound-covering epidermis and the rate of the proliferat-
ing cells (Figures 2(A)-(H)). By contrast, the rate of
TUNEL-positive cells rose shortly after peak (96 h PW)
of the thickness (and the cell number) of the epidermis in
the wound area, and reached the peak at 120 h PW, at
which the thinning phase of epidermis was considered to
start (Figures 2(M) and (O)). The peak value was 1.4
times as much as that at 96 h PW. It should be empha-
sized here that the rates of cell death in the wound areas
at 120 and 144 h PW were significantly higher than those
in the intact areas at 120 and 144 h PW, respectively
(Figures 2(O) and (P)). Thus, the fluctuation of the rate
of cell death and that of the thickness of epidermis
seemed to coincide with each other. In other words, an
elevation of the apoptosis in some cell populations in the
upper epidermis may partially contribute to the reduction
in the epidermal thickness.
A previous study showed that TUNEL-positive cells
were often observed at the granular layer just below the
horny layer of epidermis [21]. However, it has recently
been reported that apoptosis and the terminal differentia-
tion of keratinocytes are two different independent path-
ways [22,23]. Meanwhile, there is an article claiming that
keratinocytes in the granular layer that express active
caspase-14 display TUNEL-positive, being indicative of
the physiological role of caspase-14 in relation to the
DNA degradation [24]. Namely, an elevation of cell
death (TUNEL-positive cells) at the upper part of the
granular layer in the midst of thinning of the epidermis
may result in an augmentation of cornification.
4.2. The Derangement Is Generated in the
Terminal Differentiation on the
Thickness-Fluctuating Epidermis at the
Wound Area
One of the two daughter cells that generates from an
epidermal stem cell via the asymmetric cell division is
known to have the developmental fate toward the termi-
nal differentiation through which, following several cy-
cles of proliferation, the cell differentiates into spinous,
granular and horny cells in order [25]. Such a terminal
differentiation process involves not only the shift of
keratin expression from K5 and K14 (in the basal layer)
to K1 and K10 (in the suprabasal layers) but also a se-
quential switch-on of various differentiation markers
such as involucrin, filaggrin, and loricrin in the supraba-
sal zone [26]. We made the simultaneous comparison of
the intraepidermal localization of these differentiation
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marker proteins, using a series of immunostaining ex-
periments, by which we could detect a one-cell-layer
difference, if any, between the lowermost localizations of
these proteins in the normal neonatal rat skin (Figure 3).
Our experimental results showed that, at 96 and 120 h
PW, loricrin and involucrin were expressed above the
same cell layer in the thickened epidermis (Figures 3(D)
and (E)). On the other hand, profilaggrin/filaggrin was
found to be distributed from a cell layer lower than in-
volucrin. The difference in staining regions lasted until
144 h PW (Figure 4). The results obtained from the two
sets of analyses of the differentiation markers support the
possibility that the stasis or the alteration of the rates in
the epidermal terminal differentiation, which occurs proba-
bly at the zone from the upper spinous layer to the
granular layer, may contribute to the fluctuation of the
epidermal thickness at the wound areas.
Taken together, we speculated the post-wound-closure
process of the epidermal wound healing, as follows
(Figure 6): First, the epidermis at the wound site after
the wound closure is thickened by an increase in the
number of keratinocytes by an elevation of their prolif-
eration and/or by a delay of differentiation within the
pert urb the order
of terminal differentiation
prolifer ation?
mi grat i o n?
accel e ra te d
the cornification
compl et ion
0h PW
Figure 6. The scheme of re-epithelialization process in the
wound of neonatal rat skin. The epidermal stratification at
0 h PW (A) is arrayed in the same order as that of the nor-
mal skin. The granular layer is colored red throughout this
figure. The epidermis of the wound edge, which possibly
comprises the basal cells and “immature spinous cells”
(K14- and K6-positive, but the other differentiation mak-
ers-negative; blue) (Koizumi et al. 2004, 2005), migrates into
the wound bed by 24 h PW (B). Wound closure is completed
around 48 h PW (C). The wound-covering epidermis begins
to thicken by 72 h PW, though the cause is unclear (blue
arrows in (D)). Around 96 h PW, the epidermal thickening
region in the wound area becomes thicker, and the terminal
differentiation in the suprabasal layers is deranged ((E);
purple). At 120 h PW, wound-induced thickened epidermis
starts to be thinner by some mechanisms possibly involving
the accelerated cornification ((F); increase granular cells,
red; TUNEL-positive cells, green nuclei). Then, the wound-
covering epidermis restores the normal thickness from 144
h PW onward ((G), (H)). b, stratum basale; s, stratum
spinosum; g, stratum granulosum; c, stratum corneum; w,
wound; and black arrows, the initial position of the wound
spinous and granular layers. Next, the epidermal kerati-
nocytes are rapidly cornified by a sudden acceleration of
the differentiation to restore the steady state of epidermis.
Although more evidence is necessary to establish this
line of scheme, another noteworthy fact is the presence
of the thick-multilayered horny layers on the thickening
epidermis at the wound. This is an indication of the oc-
currence of transient and active cornification that may
couple with the thinning of epidermis at the wound re-
4.3. A Spatial Expansion of Caspase-14 May
Correlates with the Thinning of the
Epidermis at the Wound Area
As the last analysis in this study, we examined the ex-
pression of C/EBP-α and caspase-14 that are known to
closely participate in the terminal differentiation of
keratinocytes. C/EBP-α is a transcription factor, acting
on the early phase of the terminal differentiation [14,15],
whereas caspase-14 a proteolytic enzyme involved in the
cornification step [12,17]. During the early stage when
the epidermal sheets were migrating for the wound clo-
sure, C/EBP-α and caspase-14 were absent from the
leading edges (Figure 5), fairly consistent with the de-
crease of K10 expression and the spatial expansion of
K14 staining (Figure 1). Following that, at 96 h PW, the
pachychromatic region for caspase-14 was confined to
the upper layers at the wound epidermis, and the region
undyed was widely observed in the lower layers than that
(Figure 5(L)). We inferred from these results that, at the
thickening epidermis in the wound site, the cornification
might be delayed by some down-regulation of the ex-
pression of caspase-14. The proportion of the granular
layer to the total number of the epidermal cells at wounds
might otherwise be reduced by a delay in the terminal
differentiation process or activation of cell proliferation.
Around the middle of the thinning phase at 120 h PW, the
areas intensely stained with anti-caspase-14 at the wound
epidermis were extended more downward (Figure 5(M)).
Accordingly, the breakthrough of the terminal differen-
tiation that had been arrested until then may be related to
the expansion of expression of caspase-14 or the rapid in-
crease of granular cells there.
5. Conclusion
In conclusion, one of the most plausible causes for the
fluctuation of the epidermal thickness at the wound areas
is considered to be a delay and acceleration of the termi-
nal differentiation, including cornification, in the epi-
dermal keratinocytes. Further analyses from the other
viewpoints of cellular migration, intercellular and intra-
cellular signaling, and regeneration of some specific cells,
etc. are needed for thorough elucidation of how and why
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M. ARAI ET AL. 255
the thickness of the wound-covering epidermis fluctu-
6. Acknowledgements
We thank the members of our Morphogenesis Laborato-
ries for their support and helpful discussions in weekly
seminars for furthering this study.
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