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Citation: Lin, M.; Liu, X.; Wang, X.;
Chen, Y.; Zhang, Y.; Xu, J.; Bu, L.;
Zhang, Y.; Liang, F.; Zhang, X.; et al.
A Comparative Study of Skin
Changes in Different Species of Mice
in Chronic Photoaging Models. Int. J.
Mol. Sci. 2023,24, 10812. https://
doi.org/10.3390/ijms241310812
Academic Editors: Sigrun Lange
and Jameel M. Inal
Received: 28 May 2023
Revised: 19 June 2023
Accepted: 26 June 2023
Published: 28 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
A Comparative Study of Skin Changes in Different Species of
Mice in Chronic Photoaging Models
Meifen Lin 1, †, Xiaoran Liu 2 ,† , Xueer Wang 1, Yinyan Chen 1, Yijia Zhang 1, Jinfu Xu 1, Lingwei Bu 1, Yarui Zhang 1,
Fengting Liang 1, Xinyue Zhang 1, Bingli Huang 3, Min Zhang 1, * and Lin Zhang 1,*
1GDMPA Key Laboratory of Key Technologies for Cosmetics Safety and Efficacy Evaluation, NMPA Key
Laboratory for Safety Evaluation of Cosmetics, Department of Histology and Embryology, School of Basic
Medical Sciences, Southern Medical University, Guangzhou 510515, China; linmf8995@163.com (M.L.);
wangxueer123@smu.edu.cn (X.W.); xujinfu@126.com (J.X.); zhangyruia@163.com (Y.Z.)
2Guangzhou Dublin International College of Life Sciences and Technology, South China Agricultural
University, Guangzhou 510642, China; Liuxiaoran05@126.com
3
School of Public Health, Southern Medical University, Guangzhou 510515, China; Huangbingli1998@163.com
*Correspondence: zhangying@smu.edu.cn (M.Z.); zlilyzh@smu.edu.cn (L.Z.); Tel.: +86-02061648204 (M.Z.);
+86-02061648205 (L.Z.)
† These authors contributed equally to this work.
Abstract:
This study aimed to design a novel mouse model of chronic photoaging. We used three
different species of mice (C57BL/6J, ICR, and KM) to create a chronic photoaging model of the
skin. The irradiation time was gradually increased for 40 consecutive days. The skins of the mice
were removed on day 41 and subjected to staining to observe them for morphological changes.
Immunohistochemistry was used to detect tumor necrosis factor-
α
(TNF-
α
) and p53 expression;
superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as well. Compared with
C57BL/J mice, which showed hyperpigmentation, the irradiated skin of ICR and KM mice showed
more obvious skin thickening and photoaging changes of the collagen and elastic fibers. KM mice had
higher levels of inflammation, oxidative stress, and senescent cells. Compared with the 5-month-old
KM mice, the photoaging changes of the 9-month-old KM mice were more pronounced, the SOD
values were lower, and the MDA values were higher. In summary, KM mice have higher levels of
abnormal elastic fibers, inflammation, cellular senescence, and oxidative stress than ICR mice, and
are more suitable for studies related to chronic skin photoaging. C57BL/6J mice were found to be
suitable for studies related to skin pigmentation due to photoaging.
Keywords:
chronic skin photoaging model; C57BL/6J mice; superoxide dismutase; malondialdehyde;
ultraviolet
1. Introduction
Skin aging can be classified into endogenous and exogenous aging [
1
]. Endogenous
aging is the process of decline and degradation of the whole organism, and is influenced
by both endocrine and genetic factors. Exogenous aging occurs gradually from childhood,
and is caused by long-term exposure to sunlight, pollution, ionizing radiation, and toxins.
Among these, ultraviolet (UV) light is the most important factor in exogenous skin aging [
1
],
which is called skin photoaging when caused by UV from sunlight. Acute UV irradiation
can cause skin damage, which manifests as sunburns, peeling, and inflammation. Chronic
UV irradiation can cause aging in skin cells and tissue structure. UV can be classified by
wavelength as UVA (400–315 nm), UVB (315–280 nm), and UVC (280–100 nm). UVB is
mostly absorbed by the epidermis, and causes damage to cellular DNA [
2
–
6
]. UVA can
penetrate deep into the epidermis and induce various types of DNA lesions through direct and
indirect mechanisms, and is now considered to be the main factor in skin photoaging [
7
–
9
].
Both UVA and UVB are important factors in photoaging, and the use of combined UVA
and UVB irradiation is significant for photoaging models.
Int. J. Mol. Sci. 2023,24, 10812. https://doi.org/10.3390/ijms241310812 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 10812 2 of 12
The classical mechanisms of photoaging include cellular senescence, inflammatory
responses, and oxidative stress [
1
]. Cellular senescence is an important driver of pho-
toaging [
7
]; p53 signaling is involved in the systemic regulation of the aging process
and plays an important regulatory role [
10
]. UV light induces the production of cy-
tokines such as tumor necrosis factor-
α
(TNF-
α
) in epidermal keratinocytes and dermal
fibroblasts. TNF-
α
stimulates the release of other cytokines, chemokines, and adhesion
molecules, which trigger skin inflammation [
11
], making TNF-
α
an important indicator
of inflammation. There is a close relationship between oxidative stress and inflamma-
tory response [
12
,
13
], and oxidative stress is considered an important factor contributing
to aging [14].
Recent studies have found that skin aging and various chronic diseases are related [
15
,
16
].
Animal models of chronic skin photoaging have core value in the study of photoaging.
The selection of an appropriate skin photoaging model can help reveal the molecular
mechanism of skin photoaging, and can be used to conduct effective prevention and
treatment intervention research. ICR and KM mice have been utilized in chronic photoaging
models [
17
–
20
], while C57BL/6J mice have been utilized in acute photoaging models [
21
].
At present, there are no comparative studies on chronic skin photoaging models using
different strains of mice. Therefore, in this study 5-month-old (5M) C57BL/6J, 5M ICR, and
5M KM mice and 9-month-old (9M) KM mice were used as chronic photoaging models.
First, we aimed to investigate the effect of species on chronic photoaging in mice and
compare the changes in skin appearance, morphology, inflammatory response, senescent
cells, oxidative stress levels, and other aspects of the three groups of mice of the same age
were compared to determine which mice are most suitable as mouse models for chronic
photoaging. Then, we aimed to investigate the effect of age on chronic photoaging in mice
and to provide a suitable mouse model for the study and treatment of skin photoaging.
2. Results
2.1. Chronic Photoaging Model of Mouse Skin
The groupings of the mice are shown in Figure 1a. The design is shown in Figure 1b,
where the upper back skin of each mouse was the self-control (unirradiated site) and
the lower back was the UV irradiation site. For the self-control, a mouse fixator was
designed for this study (Figure 1c), and the mice were fixed without anesthesia. During the
photoaging molding process of the mouse skin, the amount of UVA and UVB radiation was
detected using UVA and UVB detection instruments (Figure 1d), and the effect of the tin
foil covering the UV light was detected. Mouse skin photoaging modeling was performed
daily (Figure 1e).
2.2. General Observations in Chronic Photoaged Skin of Mice
The back skin of the mice at days 0, 5, 10, 20, 30, and 40 was recorded using a camera
and dermoscope to observe the skin changes during the process of chronic photoaging.
The unirradiated part of the three different species of mouse was smooth, with no obvious
redness or desquamation (Figure 2a–c). The irradiated skin of the 5-month-old ICR mice
showed typical characteristics of chronic photoaged skin, such as obvious thickening,
leathery skin, redness, and desquamation. The irradiated skin of the 5-month-old KM mice
showed more obvious photoaging features, such as thickening, leathery skin, swelling,
and desquamation. The irradiated skin of the C57BL/6J mice showed desquamation on
day 5, and mainly showed pigmentation on days 10–40; other photoaging characteristics
were not obvious. The results of the general skin observations suggested that 5-month-old
ICR and KM mice are suitable for studies on epidermal morphological changes in chronic
photoaging skin, while C57BL/6J mice are suitable for studies on changes related to chronic
photoaging pigmentation. To investigate whether the skin of KM mice of different ages
showed differences in the process of chronic photoaging, the skin of 9-month-old KM
mice was included (Figure 2d). It was found that skin photoaging in the irradiation site of
9-month-old KM mice was more obvious than that of 5-month-old KM mice.
Int. J. Mol. Sci. 2023,24, 10812 3 of 12
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 14
Figure 1. The method of chronic photoaging of the skin in mice. (a) Animal grouping: a 5-month-
old C57BL/6J group, 5-month-old ICR group, and 5-month-old KM group were used to compare the
skin changes of different strains of mice. The 5- and 9-month-old KM groups were used to compare
the skin changes of mice at different ages. (b) UV irradiation of mice. (c) Mouse fixator: (i) the mouse
fixator consists of the main body of the fixator and the piston. The hollowed part of the main body
is used to expose the skin of the irradiated part of the mouse; (ii) overall image of the fixator; (iii)
the mouse was fixed in the fixator, and its head and upper back were wrapped with silver paper.
(d) UV irradiation box: (i) overall image of the UV irradiation box; (ii). under working conditions,
the UV intensity detected by the measuring instrument is UVA 1 mW/cm2 and UVB 0.16 mW/cm2;
(iii) after covering the probe with silver paper, the UV intensity detected by the measuring instru-
ment was 0, showing that the silver paper-wrapped fixator can isolate UV and protect the head and
upper back of the mice. (e) Time-flow chart of the chronic skin photoaging model. UV, ultraviolet;
5 M, 5-month-old; 9 M, 9-month-old.
2.2. General Observations in Chronic Photoaged Skin of Mice
The back skin of the mice at days 0, 5, 10, 20, 30, and 40 was recorded using a camera
and dermoscope to observe the skin changes during the process of chronic photoaging.
The unirradiated part of the three different species of mouse was smooth, with no obvious
redness or desquamation (Figure 2a–c). The irradiated skin of the 5-month-old ICR mice
showed typical characteristics of chronic photoaged skin, such as obvious thickening,
leathery skin, redness, and desquamation. The irradiated skin of the 5-month-old KM
mice showed more obvious photoaging features, such as thickening, leathery skin, swell-
ing, and desquamation. The irradiated skin of the C57BL/6J mice showed desquamation
on day 5, and mainly showed pigmentation on days 10–40; other photoaging characteris-
tics were not obvious. The results of the general skin observations suggested that 5-month-
old ICR and KM mice are suitable for studies on epidermal morphological changes in
chronic photoaging skin, while C57BL/6J mice are suitable for studies on changes related
Figure 1.
The method of chronic photoaging of the skin in mice. (
a
) Animal grouping: a 5-month-old
C57BL/6J group, 5-month-old ICR group, and 5-month-old KM group were used to compare the
skin changes of different strains of mice. The 5- and 9-month-old KM groups were used to compare
the skin changes of mice at different ages. (
b
) UV irradiation of mice. (
c
) Mouse fixator: (i) the mouse
fixator consists of the main body of the fixator and the piston. The hollowed part of the main body is
used to expose the skin of the irradiated part of the mouse; (ii) overall image of the fixator; (iii) the
mouse was fixed in the fixator, and its head and upper back were wrapped with silver paper. (
d
) UV
irradiation box: (i) overall image of the UV irradiation box; (ii). under working conditions, the UV
intensity detected by the measuring instrument is UVA 1 mW/cm
2
and UVB 0.16 mW/cm
2
; (iii) after
covering the probe with silver paper, the UV intensity detected by the measuring instrument was
0, showing that the silver paper-wrapped fixator can isolate UV and protect the head and upper
back of the mice. (
e
) Time-flow chart of the chronic skin photoaging model. UV, ultraviolet; 5 M,
5-month-old; 9 M, 9-month-old.
Int. J. Mol. Sci. 2023,24, 10812 4 of 12
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 14
to chronic photoaging pigmentation. To investigate whether the skin of KM mice of dif-
ferent ages showed differences in the process of chronic photoaging, the skin of 9-month-
old KM mice was included (Figure 2d). It was found that skin photoaging in the irradia-
tion site of 9-month-old KM mice was more obvious than that of 5-month-old KM mice.
Figure 2.
General observations of the chronic photoaging of skin in different species of mice. Photos
labeled “i” show dermoscopic images of the control skin and those labeled “ii” show dermoscopic
images of the chronic photoaged skin. (
a
) Dermoscopic images of 5 M C57BL/6J, (
b
) dermoscopic
images of 5M ICR, (
c
) dermoscopic images of 5 M KM, and (
d
) dermoscopic images of 9 M KM. 5 M,
5-month-old; 9 M, 9-month-old; Scale bar: 25 mm.
2.3. Skin-Related Physiological Changes in Chronic Photoaged Mice
Compared with the unirradiated skin, the percutaneous water loss (TEWL) of the
irradiated skin of all mice was significantly increased, suggesting significant skin barrier
damage in all chronic photoaged mice (Figure 3a). Among 5-month-old mice, the chronic
Int. J. Mol. Sci. 2023,24, 10812 5 of 12
photoaged skin of ICR mice showed the largest increase in TEWL, followed by KM mice,
with the smallest change observed in C57BL/6J mice. The increase in TEWL in 9-month-old
KM mice was greater than that in 5-month-old KM mice. The measurement of skin epider-
mal moisture content of mice using physiological instruments showed that the moisture
content of all mice exposed to UV irradiation was lower than that of the unirradiated skin
(Figure 3b). Among 5-month-old mice, C57BL/6J mice had the largest decrease in skin
moisture content, followed by KM mice, with the smallest change observed in ICR mice.
The decrease in moisture content in the 9-month-old KM mice was greater than that in the
5-month-old KM mice, indicating that the water retention capacity of the skin epidermis of
mice with chronic photoaging was decreased, leading to skin dryness.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 14
Figure 2. General observations of the chronic photoaging of skin in different species of mice. Photos
labeled “i” show dermoscopic images of the control skin and those labeled “ii” show dermoscopic
images of the chronic photoaged skin. (a) Dermoscopic images of 5M C57BL/6J, (b) dermoscopic
images of 5M ICR, (c) dermoscopic images of 5M KM, and (d) dermoscopic images of 9M KM. 5 M,
5-month-old; 9 M, 9-month-old; Scale bar: 25 mm
2.3. Skin-Related Physiological Changes in Chronic Photoaged Mice
Compared with the unirradiated skin, the percutaneous water loss (TEWL) of the
irradiated skin of all mice was significantly increased, suggesting significant skin barrier
damage in all chronic photoaged mice (Figure 3a). Among 5-month-old mice, the chronic
photoaged skin of ICR mice showed the largest increase in TEWL, followed by KM mice,
with the smallest change observed in C57BL/6J mice. The increase in TEWL in 9-month-
old KM mice was greater than that in 5-month-old KM mice. The measurement of skin
epidermal moisture content of mice using physiological instruments showed that the
moisture content of all mice exposed to UV irradiation was lower than that of the unirra-
diated skin (Figure 3b). Among 5-month-old mice, C57BL/6J mice had the largest decrease
in skin moisture content, followed by KM mice, with the smallest change observed in ICR
mice. The decrease in moisture content in the 9-month-old KM mice was greater than that
in the 5-month-old KM mice, indicating that the water retention capacity of the skin epi-
dermis of mice with chronic photoaging was decreased, leading to skin dryness.
Figure 3. Physiological changes of the skin in chronic photoaged mice. On the 40th day, the changes
in the skin physiology of the control and chronic photoaged skin of the mice were observed. (a) Skin
barrier function was detected by percutaneous water loss (TEWL). (b) Epidermal hydration, (c) skin
elasticity, and (d) dermal density were detected using skin ultrasound. (e) Quantitative statistics of
dermal density were detected using skin ultrasound. The numbers above the bars indicate the spe-
cific difference between the UV irradiated sites compared to the control group. * p < 0.05, ** p < 0.01,
*** p < 0.001. TEWL, skin percutaneous water loss; Der, dermis; Sub, subcutaneous tissue; 5 M, 5-
month-old; 9 M, 9-month-old, Scale bar: 1 mm.
Skin elasticity measurements showed that the skin elasticity at the UV irradiation site
of all mice was lower than that of the unirradiated skin (Figure 3c). Among 5-month-old
mice, KM mice showed the largest reduction in skin elasticity, followed by ICR mice, with
Figure 3.
Physiological changes of the skin in chronic photoaged mice. On the 40th day, the changes
in the skin physiology of the control and chronic photoaged skin of the mice were observed. (
a
) Skin
barrier function was detected by percutaneous water loss (TEWL). (
b
) Epidermal hydration, (
c
) skin
elasticity, and (
d
) dermal density were detected using skin ultrasound. (
e
) Quantitative statistics of
dermal density were detected using skin ultrasound. The numbers above the bars indicate the specific
difference between the UV irradiated sites compared to the control group. * p< 0.05,
** p< 0.01
,
*** p< 0.001
. TEWL, skin percutaneous water loss; Der, dermis; Sub, subcutaneous tissue; 5 M,
5-month-old; 9 M, 9-month-old, Scale bar: 1 mm.
Skin elasticity measurements showed that the skin elasticity at the UV irradiation
site of all mice was lower than that of the unirradiated skin (Figure 3c). Among 5-month-
old mice, KM mice showed the largest reduction in skin elasticity, followed by ICR mice,
with the smallest change observed in C57BL/6J mice. The decrease in skin elasticity
observed in the 9-month-old KM mice was less than that in the 5-month-old KM mice.
Further skin ultrasound results showed a decrease in dermal density of all irradiated
skin (Figure 3d,e). Among 5-month-old mice, KM mice showed the largest decrease in
dermal density, followed by ICR mice, with the smallest change observed in C57BL/6J
mice. There was no difference in density reduction of KM mice at different months.
2.4. Histological Changes in the Skin of Chronic Photoaged Mice
Hematoxylin and eosin (H&E) staining (Figure 4a–d) revealed that the unirradiated
skin of all mice showed normal skin thickness, complete structure, orderly distribution, and
normal arrangement of dermal fibers. Irregularly thickened skin epidermis was observed
at all irradiated sites in the mice. Among all mice, 5-month-old ICR and KM mice showed
significant epidermal thickening, whereas the epidermis thickened slightly in 9-month-old
Int. J. Mol. Sci. 2023,24, 10812 6 of 12
C57BL/6J mice and 5-month-old KM mice. Masson staining further showed that dermal
collagen fibers were disordered at all irradiation sites and that collagen fiber bundles
became thinner, curled, and broken. Among 5-month-old mice, ICR and KM mice showed
the most obvious changes. Compared to 5-month-old KM mice, the changes to the dermal
collagen fibers in 9-month-old KM mice were more obvious. Gomori staining was used
to observe the changes in the elastic fibers of the mouse dermis. The elastic fibers of the
skin at the irradiated sites of all the mice were thickened, broken, twisted, and deformed,
and some of them were clumped up. The changes in dermal elastic fibers in KM mice were
the most obvious in 5-month-old mice, followed by ICR mice. The changes in the dermal
elastic fibers of 9-month-old KM mice were similar to those of 5-month-old KM mice.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 14
Figure 4. Histological changes of the skin in chronic photoaged mice. On the 40th day, the histolog-
ical changes in the control and chronic photoaged skin of the mice were observed. (a) 5-month-old
C57BL/6J mice, (b) 5-month-old ICR mice, (c) 5-month-old KM mice, and (d) 9-month-old KM mice.
The black arrow indicates elastic fibers; HE, Hematoxylin and eosin staining; UV, ultraviolet; Epi,
epidermis; Der, dermis; SG, sebaceous gland; HF, hair follicle; 5 M, 5-month-old; 9 M, 9-month-old;
Scale bar: 50 μm.
2.5. Increased Expression of TNF- α and p53 in the Skin of Chronic Photoaged Mice
We selected TNF-α as an indicator of inflammation and p53 as an indicator of aging
to compare the responses of three different species of mice to inflammation and aging
Figure 4.
Histological changes of the skin in chronic photoaged mice. On the 40th day, the histological
changes in the control and chronic photoaged skin of the mice were observed. (
a
) 5-month-old
C57BL/6J mice, (
b
) 5-month-old ICR mice, (
c
) 5-month-old KM mice, and (
d
) 9-month-old KM mice.
The black arrow indicates elastic fibers; HE, Hematoxylin and eosin staining; UV, ultraviolet; Epi,
epidermis; Der, dermis; SG, sebaceous gland; HF, hair follicle; 5 M, 5-month-old; 9 M, 9-month-old;
Scale bar: 50 µm.
Int. J. Mol. Sci. 2023,24, 10812 7 of 12
2.5. Increased Expression of TNF- αand p53 in the Skin of Chronic Photoaged Mice
We selected TNF-
α
as an indicator of inflammation and p53 as an indicator of aging
to compare the responses of three different species of mice to inflammation and aging
(Figure 5). As shown by immunohistochemical staining of TNF-
α
(Figure 5a,c), compared
with the unirradiated skin, the expression of TNF-
α
and the percentage of positive area
of TNF-
α
expression sites in the epidermis and dermis were significantly increased in
the irradiated skin of all mice. Among the 5-month-old mice, the expression of TNF-
α
was the most significant in KM mice, followed by ICR mice and C57BL/6J mice. TNF-
α
expression was significantly higher in 9-month-old KM mice than in 5-month-old KM mice.
These results indicate that a skin inflammatory response was observed in all strains of mice
during chronic photoaging, most prominently in KM mice.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 14
(Figure 5). As shown by immunohistochemical staining of TNF-α (Figure 5a,c), compared
with the unirradiated skin, the expression of TNF-α and the percentage of positive area of
TNF-α expression sites in the epidermis and dermis were significantly increased in the
irradiated skin of all mice. Among the 5-month-old mice, the expression of TNF-α was the
most significant in KM mice, followed by ICR mice and C57BL/6J mice. TNF-α expression
was significantly higher in 9-month-old KM mice than in 5-month-old KM mice. These
results indicate that a skin inflammatory response was observed in all strains of mice dur-
ing chronic photoaging, most prominently in KM mice.
Figure 5. Increased expression of TNF-α and p53 in the skin of chronic photoaged mice. On the 40th
day, the changes in the expression of TNF-α (a,c) and p53 (b,d) in the control and chronic photoaged
Figure 5.
Increased expression of TNF-
α
and p53 in the skin of chronic photoaged mice. On the 40th
day, the changes in the expression of TNF-
α
(
a
,
c
) and p53 (
b
,
d
) in the control and chronic photoaged
skin of the mice were observed. The numbers above the bars indicate the specific difference between
the UV irradiated sites compared to the control group. UV, ultraviolet; TNF, tumor necrosis factor; Epi,
epidermis; Der, dermis; SG, sebaceous gland; HF, hair follicle; 5 M, 5-month-old; 9 M, 9-month-old.
** p< 0.05, *** p< 0.01; Scale bar: 50 µm.
Int. J. Mol. Sci. 2023,24, 10812 8 of 12
Immunohistochemical staining of p53 showed that, in comparison with the unirradi-
ated skin, the number of p53 positive cells in the irradiated skin of all mice was significantly
increased, and were mainly concentrated in the epidermis, indicating that in the process
of chronic photoaging all strains of mice showed senescence of the skin epidermal cells
(Figure 5b,c). Among the mice, KM mice had the largest number of p53 positive cells
for both 5- and 9-month-old mice, whereas ICR mice had the smallest number of p53
positive cells.
2.6. Increased Levels of Oxidative Stress in the Skin of Chronic Photoaged Mice
Oxidative stress is one of the mechanisms that underlie chronic photoaging [
22
].
Superoxide dismutase (SOD) is an important antioxidant enzyme that removes superoxide
anion free radicals, which can protect cells from damage caused by oxygen free radicals.
Malondialdehyde (MDA) is an end product formed by the reaction between lipids and
oxygen free radicals. UV radiation can induce the production of a large amount of reactive
oxygen species, wherein SOD is greatly consumed and MDA accumulates; therefore, the
oxidative stress process can be evaluated via SOD and MDA. After the chronic photoag-
ing process, the level of SOD was significantly decreased after UV irradiation, with the
most obvious change in
5-month-old
KM mice, followed by
5-month-old
C57BL/6J mice,
while the decrease in SOD levels in 5-month-old ICR mice was not significant (Figure 6a).
The level of MDA (Figure 6b) increased after UV irradiation, with the most obvious change
in
5-month-old
KM mice, followed by 5-month-old ICR mice, and the smallest change
found in C57BL/6J mice. In KM mice of different ages, the oxidative stress level of 5-month-
old KM mice was more obvious. The basal SOD value of 9-month-old mice was lower than
that of 5-month-old mice, while the basal MDA value was higher than that of 5-month-old
mice, indicating that oxidative stress levels are affected by natural aging.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 14
skin of the mice were observed. The numbers above the bars indicate the specific difference between
the UV irradiated sites compared to the control group. UV, ultraviolet; TNF, tumor necrosis factor;
Epi, epidermis; Der, dermis; SG, sebaceous gland; HF, hair follicle; 5 M, 5-month-old; 9 M, 9-month-
old. ** p < 0.05, *** p < 0.01; Scale bar: 50 μm.
Immunohistochemical staining of p53 showed that, in comparison with the unirradi-
ated skin, the number of p53 positive cells in the irradiated skin of all mice was signifi-
cantly increased, and were mainly concentrated in the epidermis, indicating that in the
process of chronic photoaging all strains of mice showed senescence of the skin epidermal
cells (Figure 5b,c). Among the mice, KM mice had the largest number of p53 positive cells
for both 5- and 9-month-old mice, whereas ICR mice had the smallest number of p53 pos-
itive cells.
2.6. Increased Levels of Oxidative Stress in the Skin of Chronic Photoaged Mice
Oxidative stress is one of the mechanisms that underlie chronic photoaging [22]. Su-
peroxide dismutase (SOD) is an important antioxidant enzyme that removes superoxide
anion free radicals, which can protect cells from damage caused by oxygen free radicals.
Malondialdehyde (MDA) is an end product formed by the reaction between lipids and
oxygen free radicals. UV radiation can induce the production of a large amount of reactive
oxygen species, wherein SOD is greatly consumed and MDA accumulates; therefore, the
oxidative stress process can be evaluated via SOD and MDA. After the chronic photoaging
process, the level of SOD was significantly decreased after UV irradiation, with the most
obvious change in 5-month-old KM mice, followed by 5-month-old C57BL/6J mice, while
the decrease in SOD levels in 5-month-old ICR mice was not significant (Figure 6a). The
level of MDA (Figure 6b) increased after UV irradiation, with the most obvious change in
5-month-old KM mice, followed by 5-month-old ICR mice, and the smallest change found
in C57BL/6J mice. In KM mice of different ages, the oxidative stress level of 5-month-old
KM mice was more obvious. The basal SOD value of 9-month-old mice was lower than
that of 5-month-old mice, while the basal MDA value was higher than that of 5-month-
old mice, indicating that oxidative stress levels are affected by natural aging.
Figure 6. The increased skin oxidative stress of chronic photoaged mice. On the 40th day, (a) SOD
and (b) MDA levels in the control skin and chronic photoaged skin were observed using ELISA. The
numbers above the bars indicate the specific difference between the UV irradiated sites compared
to the control group. * p < 0.05, ** p < 0.01. SOD, superoxide dismutase; MDA, malondialdehyde;
ELISA, enzyme-linked immunosorbent assay; UV, ultraviolet; 5 M, 5-month-old; 9 M, 9-month-old.
Figure 6.
The increased skin oxidative stress of chronic photoaged mice. On the 40th day, (
a
) SOD
and (
b
) MDA levels in the control skin and chronic photoaged skin were observed using ELISA.
The numbers
above the bars indicate the specific difference between the UV irradiated sites compared
to the control group. * p< 0.05, ** p< 0.01. SOD, superoxide dismutase; MDA, malondialdehyde;
ELISA, enzyme-linked immunosorbent assay; UV, ultraviolet; 5 M, 5-month-old; 9 M, 9-month-old.
3. Discussion
Changes in human skin occur with age, and repeated exposure to external stimuli such
as the sun accelerates skin photoaging [
1
,
23
], especially with the serious destruction of the
atmospheric ozone layer. The selection of suitable animal models and modeling methods
is helpful for exploring the molecular mechanisms of skin photoaging and prevention of
skin photoaging. This study aimed to identify a rapid and effective modeling method for
Int. J. Mol. Sci. 2023,24, 10812 9 of 12
chronic photoaging modeling and to provide a reference for the selection of mouse species.
At present, there are deficiencies in the photoaging modeling process. First, the irradiation
period for chronic photoaging modeling is long (12 weeks in a study by Li et al. [
19
]), with
the current shortest modeling period proposed by Zheng et al. [
24
] at 8 weeks; moreover,
there remains the problem of non-continuous irradiation. Second, UV irradiation equip-
ment is expensive or lacking, the irradiation equipment used is not specifically designed
for modeling of animal photoaging [
19
,
21
,
24
], and unirradiated parts are not guaranteed
to be protected from UV injury during the irradiation process. Meanwhile, the problem
of animal fixation during irradiation has not been solved [
25
]. To solve these problems,
the present study seeks to improve the modeling method of chronic photoaging mice by
designing a UV irradiation box for photoaging modeling (including adjustable heights
for UVA and UVB light sources) and a matching mouse fixator which can accommodate
multiple mice simultaneously. Mouse fixation can protect the non-irradiated parts from UV
damage and does not require anesthesia, greatly reducing the harm caused by administra-
tion of anesthesia to mice in line with animal ethical requirements. In the UV irradiation
cycle, this study adopted the method of continuous irradiation for 40 days, gradually
increasing the daily irradiation time to 30 min, which shortened the irradiation cycle while
ensuring consistent irradiation intensity. Throughout the experiment, the mice did not
have any serious UV damage such as skin breaking or bleeding. After UV irradiation,
the responses of the three strains of mice in terms of skin appearance, tissue morphology,
inflammatory response, cellular senescence, and oxidative stress responses were consistent
with the characteristics of chronic photoaging reported by Wang et al. [
25
], such as rough
and leathery appearance, peeling, epidermal thickening, dermis thinning, collagen fiber
degradation disorder, elastic fiber degeneration and clumping, skin inflammation, and ox-
idative stress response. The photoaging response was similar to that of human photoaging,
which demonstrates that this experiment successfully established a novel, rapid, operable,
and low-cost method for chronic photoaging in animals that is applicable in experimental
research on photoaging.
This study further compared the morphological and biological changes in C57BL/6J,
ICR, and KM mice aged five months during chronic photoaging. As found in the present
study, C57BL/6J mice showed significant skin pigmentation after UV irradiation, while
compared with ICR mice and KM mice, the skin changes of C57BL/6J mice in the process of
chronic photoaging, such as skin morphology, collagenous fiber, elastic fibers, inflammatory
reaction, apoptosis, and oxidative stress, were the most atypical changes. These results are
consistent with the findings of melanin protecting UV-irradiated skin [
1
,
26
], suggesting that
C57BL/6J mice are suitable for the study of skin pigmentation induced by photoaging but
are not suitable for use in chronic photoaging models, as their black hair interferes with the
observation of gross skin appearance. ICR and KM mice are albino mice with no melanin
in the skin, and their hair color does not affect the observation of skin changes. Hence, it
is more suitable to choose ICR or KM mice to continuously observe changes in gross skin
appearance in the process of chronic photoaging. In this study, ICR mice showed similar
changes in general appearance, tissue morphology, and collagen fibers to KM mice, and both
are similar to the chronic photoaging characteristics in humans [
2
,
23
]. However, KM mice
showed better changes in elastic fibers, inflammation, cellular aging, and oxidative stress
than ICR mice. and had more typical characteristics of chronic photoaging. Therefore, KM
mice are more suitable for use in the study of chronic photoaging.
There is a chronological aging process in which all functions of the skin decline
with age [
2
]. Studies have shown that 5-month-old mice are equivalent to 20- to 30-year-
old humans, while 9-month-old mice are equivalent to 50- to 60-year-old humans [
27
].
Compared to 5-month-old KM mice, the normal skin epidermis of the 9-month-old KM
mice had uneven skin thickness, slender collagen fiber bundles, reduced elastic fibers, and
decreased skin water retention ability, barrier repair ability, and elasticity, which appear
to be characteristic of chronological aging [
28
] and are similar to the chronological aging
characteristics of humans [
2
]. Compared with 5-month-old KM mice, 9-month-old KM
Int. J. Mol. Sci. 2023,24, 10812 10 of 12
mice showed similar changes in collagen degradation and inflammation; however, the
changes in SOD and MDA in the oxidative stress response were not obvious, which is not
characteristic of typical chronic photoaging. Therefore, using 5-month-old mice can reduce
interference from the natural aging process to a certain extent, and 9-month-old mice can
be used in studies of the effects of natural aging in combination with photoaging.
4. Materials and Methods
Specific pathogen free (SPF) mice (three C57BL/6J, three ICR, and three KM mice,
all 5 months old) were used to compare skin changes in a chronic photoaging model.
Three SPF KM mice, (9 months old, female) were used to compare skin changes in KM
mice at different months during the chronic photoaging model. The mice were provided by
the Laboratory Animal Center of Southern Medical University and fed standard animal
feed. All mice were raised according to the Guidelines for Animal Experimentation of
the Laboratory Animal Center of Southern Medical University, and the study was ap-
proved by the Experimentation Animal Ethics Committee of Southern Medical University
(protocol code SMUL2023054, 14 April 2023). The UV irradiation device was a self-made
UV irradiation box equipped with three 60 cm UVA (340 nm, 40 W) and one 60 cm UVB
(313 nm, 40 W) tubes, which could stably provide UVA at 1 mW/cm
2
and UVB at 0.15
mW/cm
2
intensity. A laboratory special mouse fixator, which could isolate the UV light
with a hollowed-out part (
21 ×13 mm
) that exposed the back skin of the mouse, was
used. The UVA and UVB intensity detectors were purchased from Shanghai Sigma High
Technology Co., Ltd., Shanghai, China. The dermatoscope (Trichoscan HD) was purchased
from Dermoscan, Regensburg, Germany. The percutaneous water loss instrument (AF200)
was purchased from Biox, London, UK, and the skin polyguide physiological instrument
and skin composition structural analysis system (DUB-MSTC) were purchased from TPM,
Germany. The paraffin embedding, paraffin slicing machine, and Leica forward microscope
were purchased from Leica, Wetzlar, Germany. The H&E and Masson three-color staining
kits were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou,
China, and the Gomori elastic fiber staining solution was purchased from Beijing Solaibao
Technology Co., Ltd., Beijing, China. Primary antibody p53 was purchased from Thermo
Fisher, Waltham, MA, USA. Primary antibody TNF-
α
was purchased from Proteintech,
Rosemont, IL, USA. Goat anti-rabbit IgG/HRP polymer and DAB Kit were purchased
from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China. Western and PI
cell lysates were purchased from Beyotime Biotech. Inc., Shanghai, China. Total SOD
colorimetric cartridge (WST-1) and MDA colorimetric cartridge (TBA) were purchased
from Wuhan Elabscience Biotechnology Co.,Ltd, Wuhan, China.
The mice were fed adaptively for one week after purchase. After the adaptation
period, three mice from each group were randomly selected and divided into 5-month-old
C57BL/6J, 5-month-old ICR, 5-month-old KM, and 9-month-old KM groups. The day
before UV exposure began, the back hair of the mice was removed using a mild hair
removal cream. In the course of 40 consecutive days of irradiation, hair removal was
performed according to the hair growth of the mice, and the frequency was once every
5–7 days. In order to avoid any influence of hair removal on ultraviolet irradiation, we
would irradiate the mice 12 h after hair removal. The lower back of the mice was the
UV-irradiated part (the exposed skin area was approximately 0.21
×
0.13 cm), the upper
back was the non-irradiated part, and the photographing position was marked with a
marker pen.
The mice were fixed with a special mouse fixator; the front part of the fixator (non-
irradiated skin) was wrapped with aluminum foil to fully block the UV light and was
placed in a self-made UV irradiation box. The chronic photoaging molding period was
40 days, and the irradiation doses were 1 and 0.15 mW/cm
2
for UVA and UVB, respectively,
wherein the duration was 10 min on days 1–5, 20 min on days 6–10, and 30 min on days
11–40 (Figure 1).
Int. J. Mol. Sci. 2023,24, 10812 11 of 12
After 40 consecutive days of irradiation, the mice were anesthetized using intraperi-
toneal injection of 2% pentobarbital sodium and the backs of the mice (6.5
×
3 cm) were
depilated with depilatory cream. Pictures were collected using a camera, and skin TEWL,
elasticity, epidermis water content, and tissue density were measured. After data collection,
the unirradiated and irradiated skin were removed, rinsed in phosphate buffer saline (PBS)
on ice, and the blood was removed. The tissue block was divided into two parts: one for
paraffin embedded sections, H&E staining, Masson staining, Gomori elastic fiber staining,
and immunohistochemistry, and another for tissue homogenate and supernatant prepara-
tion at
−
80
◦
C, which strictly followed the corresponding biochemical kit instructions for
SOD and MDA determination. Masson and Gomori elastic fiber staining were performed
according to the manufacturer’s instructions provided.
After routine dewaxing and rehydration of the paraffin sections, endogenous peroxi-
dase was eliminated with 3% H
2
O
2
using sodium citrate solution to repair thermal antigen
and washed three times with PBS for 5 min each time, enclosed in 10% goat serum for
2 h, and incubated for primary antibody at 4
◦
C overnight and secondary antibody for 1 h.
Then, 3,3’-Diaminobenzidine (DAB) color rendering, hematoxylin counterstaining, dehy-
dration, transparency determination, and sealing with film were performed. Images were
taken with an upright microscope, and automated analysis of sample staining (percentage
of positive area and positive staining cell count) was performed using ImageJ software V1.8.
Statistical analysis was performed using SPSS 20.0 software. Data were expressed
as the mean
±
standard deviation. Global comparisons were performed using repeated
measures analysis of variance, and two independent samples t-tests for the same time
points were considered statistically significant at p< 0.05.
5. Conclusions
In conclusion, this study designed and verified a new method to quickly establish
an animal model of chronic photoaging by comparing the morphological and biologi-
cal changes in C57BL/6J, ICR, and KM mice during the process of chronic photoaging.
This study provides a reference for the establishment and selection of animal models of
chronic photoaging in skin photoaging studies.
Author Contributions:
Conceptualization, M.L. and X.L.; methodology, M.L. and X.L.; software, X.W.
and Y.C.; validation, Y.Z. (Yijia Zhang), J.X., and L.B.; formal analysis, F.L. and Y.Z. (Yarui Zhang);
investigation, M.Z., B.H., and X.Z.; resources, L.Z.; data curation, M.Z.; writing—original draft
preparation, M.L.; writing—review and editing, M.L.; visualization, X.L.; supervision, M.Z.; project
administration, L.Z.; funding acquisition, L.Z. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the National Natural Science Foundation of China, grant
numbers 81402613 and 82073417; the GDMPA Key Laboratory Project of Scientific and Technological
Innovation, grant number 2023ZDZ12; the Guangdong Basic and Applied Basic Research Foundation,
grant number 2019A1515011213, 2020A1515011067, and 2022A1515010768; the Xinjiang Uygur Au-
tonomous Region Natural Science Foundation, grant number 2019D01A83; and the Medical Scientific
Research Foundation of Guangdong Province, grant number A2020136.
Institutional Review Board Statement:
This study was conducted according to the guidelines of the
Declaration of Helsinki, and was approved by the Southern Medical University Experimentation
Animal Ethics Committee (protocol code SMUL2023054, 14 April 2023).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The authors confirm that the data supporting the findings of this study
are available within the article.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2023,24, 10812 12 of 12
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