Naturalistae 28: 1-5 (Feb. 2024)© 2024 by Okayama University of Science, PDF downloadable at http://www1.ous.ac.jp/garden/

Original paper

Ear wound regeneration in the Arabian spiny mouse(Acomys dimidiatus)
Akiko OYAKE1 & Kazuyuki MEKADA1*
Abstract: Skin shedding, skin wound regeneration, and extensive ear punch closure have been reported in three Acomys rodent species (A. cahirinus, A. kempi, and A. percivali) commonly known as
African spiny mice. In this study, we examined the regenerative ability of A. dimidiatus, the Arabianspiny mouse. A 2 mm diameter punch hole was created in the proximal and distal parts of the pinna
and observed for up to 35 days. That in the proximal region almost completely closed, and extensive
elastic cartilage, adipose tissue, dermis, epidermis, and hair follicles appeared in the repaired area.
More rapid skin regeneration was observed closer to the trunk. The punch hole in the distal part did
not reach complete closure during the observation period. However, regeneration was observed in the
repaired area as well as in the proximal part. These results corroborate the presence of regenerative
capabilities of A. dimidiatus and indicate that Acomys species may be useful model animals for regeneration studies.

I. Introduction& Mekada 2017, Mekada et al. 2021). Because
A. dimidiatus is a sister species to A. cahirinus(Kunze et al. 1999, Volobouev et al. 2007, Frynta
et al. 2010, Mochida & Mekada 2017), it is expected to have the same regenerative ability as A.
cahirinus. However, no detailed characterizationof A. dimidiatus has been conducted to date.
Therefore, we investigated the skin regenerative
ability of A. dimidiatus by gross and histological observations of the temporal changes in thewound site of pinna tissue.

Regeneration, the capacity to structurally
and functionally restore damaged tissues andorgans, has been observed in many taxonomic
groups, including amphibians, crustaceans, andfish (Alvarado 2000, Brockes & Kumar 2005,
Slack et al. 2008). However, there are few reports of regeneration in mammals, which show
a greater tendency toward tissue repair withfibrous scarring than regeneration (Clark 1996).
Regeneration has been confirmed in mammalianfetuses and the MRL/MpJ mouse strain, but theseabilities are typically temporary or non-innate(Beare et al. 2006, Larson et al. 2010, Gawriluk
et al. 2016). Notably, high regeneration ability
was recently demonstrated in African spiny mice
(Acomys), including A. cahirinus (Cairo spinymice), A. kempi (Kemps spiny mice), and A.
percivali (Percival’s spiny mice) (Seifert et al.
2012, Matias Santos et al. 2016). These species
were found to regenerate wound-defective skintissue without undergoing the usual scarring orloss of function after fibrosis. Thus, these animals
may be useful models for analyzing the cellularand molecular signals that control regeneration(Gaire et al. 2021).
Acomys cahirinus is the major spiny mouse
species currently maintained and used as anexperimental animal (Haughton et al. 2016).
Acomys dimidiatus (the Arabian spiny mouseor eastern spiny mouse), which exists fromsouthwestern Asia to northeastern Africa, is also
maintained in laboratory colonies in Japan and
Switzerland (Montandon et al. 2014, Mochida

II. Materials and Methods
This study involved male and female (non-pregnant) of A. dimidiatus (Aco strain) (Mochida &Mekada 2017) and Mus musculus (BLAB/cCrSlc
mice strain, purchased from Japan SLC, Inc.,
Hamamatsu, Japan) from the Department of
Zoology, Faculty of Science, Okayama University of Science. The animals were housed at aroom temperature of 24ºC and light/dark periodof 12/12 h. They were fed commercial pellets(Labo MR breeder; Nihon Nosan Kogyo. Co.,
Yokohama, Japan), and both food and water were
provided ad libitum. All animal experiments wereconducted in accordance with the Regulations for
Animal Experiments of the Okayama Universityof Science. All experimental protocols, including
those involving animals (Exp2020-004), wereapproved by Animal Experiments Committee ofthe University.
Under isoflurane inhalation anesthesia, a 2
mm diameter ear punch was used to create punchholes in the proximal part of the right pinna

1.

Department of Zoology, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama-shi, Okayama 700-0005, Japan. 岡山
理科大学理学部動物学科，〒700-0005 岡山県岡山市北区理大町1-1.
*Correspondence: Kazuyuki MEKADA, E-mail: mekada@ous.ac.jp-1-

OYAKE & MEKADA

III. Results(closer to the trunk) and the distal part of the left
pinna (farther from the trunk), avoiding major
ear vessels and resulting in minimal bleeding.
The mice were divided into two groups according
to the starting age of treatment: 3 weeks (W3group) and 9 weeks (W9 group). After treatment,
the animals were euthanized every 7 days until
day 35, at which time the pinna tissues were collected. In total, 40 mice were used (8 [4 females
and 4 males] in each group at each collection
time point). The collected pinna fragments were
subjected to histopathological analysis after the
status of the wound was observed under a stereomicroscope and digital images were acquired.
The area of the wound site was measured using
ImageJ software (National Institutes of Health,Bethesda, MD, USA).
The ear samples were fixed in 4% paraformaldehyde overnight, dehydrated in a graded ethanol
series and embedded in paraffin wax. Transverse
microtome sections with a thickness of 4 µm
were deparaffinized in xylene and rehydrated ina graded ethanol series. A commercial Masson’s
trichrome staining kit (Muto Pure Chemicals Co.,
Ltd., Tokyo, Japan) was used to stain the sections,which were then were rinsed, dehydrated, andmounted.

The A. dimidiatus and M. musculus mice usedin this study included both females and males, but
no statistically significant differences in the areaof the ear punch holes were found between thesexes at each sampling time point (Brunner-Munzel Test, P > 0.05). Because a previous study of A.
cahirinus showed differences between the sexes(Matias Santos et al. 2016), we used a combined
value in this study.
In the proximal pinna of A. dimidiatus, a rapid
decrease in the wound area was observed between
days 7 and 35 after punching treatment in boththe W3 and W9 groups (proximal part in Figs. 1
and 2). The median wound area on day 7 was 3.1
and 3.2 mm2 in the W3 and W9 groups, respectively, indicating the beginning of wound repair
in both groups. On day 14 after treatment, themedian wound area was 1.5 and 1.8 mm2 in theW3 and W9 groups, respectively, and new skinformation was observed proximal to the woundin both groups. On day 21 after treatment, themedian area was much smaller in the W3 group(0.0 mm2), and complete closure of the wound
was observed in five of the eight animals. In
the W9 group, the median wound area was 0.1

Fig. 1. Scatter boxplots of ear punch hole area (by proximal and distal parts) from day 7 (D7) to day 35 (D35) in Acomys dimidiatus
and Mus musculus (BALB/c) of the W3 group versus W9 group.

-2-Ear wound regeneration in Acomys dimidiatus

Fig. 2. Representative images of ear punch closure along timeline from day 7 (D7) to day 35 (D35) in Acomys dimidiatus versus Mus musclus(BALB/c) of W3 group. The distal–proximal axis is shown vertically with distal (D) at the top and proximal (P) at the bottom for all panels. The
arrowhead indicates necrotic skin tissue. Scale bar = 1 mm.

Fig. 3. Representative histological sections of Acomys dimidiatus and Mus musculus (BALB/c) of W3 group, stained with Masson’s trichrome
stain. Acomys dimidiatus on (a) at day 7 (D7), (b) day 14 (D14), (c) day 12 (D12), (d) day 28 (D28), and (e) day 35 (D35). (f) M. musculus on
GD\ ' $UURZKHDGVLQGLFDWHWKLFNHQHGHSLGHUPLV \HOORZ KDLUIROOLFOHV SLQN PXVFOH JUHHQ DQGFDUWLODJH EODFN 9HUWLFDOGRWWHGOLQHVLQGLFDWHWKHRULJLQDOSODQHRIZRXQGLQJ LGHQWL¿HGE\WKHSUHVHQFHRIPDWXUHFDUWLODJH 7KHGLVWDO±SUR[LPDOD[LVLVVKRZQKRUL]RQWDOO\ZLWKGLVWDO(D) to the right and proximal (P) to the left for all panels. Scale bar = 0.3 mm.

-3-OYAKE & MEKADA

mm2, and complete closure of the wound wasobserved in two of the eight animals. On day 35
after treatment, the median wound area was 0.0
mm2 in both the W3 and W9 groups, resulting ina nearly closed wound area in both groups.
In the distal pinna of A. dimidiatus, the woundarea tended to gradually decrease between days 7
and 35 after treatment in both groups (distal partin Figs. 1 and 2). The median wound area on day
7 after treatment was 4.1 and 3.5 mm2 in the W3
and W9 groups, respectively, and necrotic skintissue was observed at the edges of the wounds inall mice of the W9 group. The median wound area
on day 14 after treatment was 4.2 and 4.2 mm2in the W3 and W9 groups, respectively, and the
beginning of wound repair was observed in theW3 group. On day 21 after treatment, the medianwound area was 3.3 and 3.9 mm2 in the W3 andW9 groups, respectively, and new skin formation
was observed on the proximal side of the woundin the W3 group. At day 35 after treatment, themedian wound area was 2.9 and 3.4 mm2 in theW3 and W9 groups, respectively, and closureof the wound from the proximal to distal side
was observed in all A. dimidiatus animals. In M.
musculus animals, both the proximal and distal
pinnae showed a decrease in the wound area in
the W3 and W9 groups, but complete closure of
the area was not observed during the study.
In the W3 group, the wound in the proximal
pinna of A. dimidiatus, showed re-epithelialization by day 7 after ear punch treatment, below
which a mass of loosely packed tissue was observed (Fig. 3a). By day 14 post-treatment, thedistance between the original wound surface andthe wound boundary had increased (Fig. 3b). On
day 21, the formation of elastic cartilage extending from the pinna cartilage beyond the woundsurface and the formation of hair follicles wereobserved (Fig. 3c). On days 28 and 35, morehair follicles as well as muscle formation wereobserved. Extensive growth of elastic cartilage
was present in the regenerated area (Fig. 3d,e). The recovery of the wound area of the distal
pinna of A. dimidiatus in the W3 group and theproximal and distal pinnae in the W9 group exhibited changes similar to these (data not shown).
Although some epidermal regeneration was also
observed at the wound sites of M. musculus by
day 35 (data not shown), no complicated structural change such as those seen in A. dimidiatus
were observed on day 35 (Fig. 3f).

mm in diameter), the punch holes completely
closed within 2 months, and histologic examination showed extensive formation of elastic
cartilage, adipose tissue, dermis, epidermis, andhair follicles at the site of repair (Gawriluk et al.
2016, Matias Santos et al. 2016). In the present
study, complete closure of the proximal pinna
wound was observed in most A. dimidiatus mice(Figs. 1 and 2). The regenerated auricular injury
site exhibited epidermis, hair follicles, elastic
cartilage, and muscle, and the tissue structure
resembled that of the normal skin (Fig. 3). Hence,
A. dimidiatus, like previously reported Acomys
species, was found to have the ability to reconstruct skin wounds structurally and functionally.
Although regeneration occurred in the wounded area of the distal pinna of A. dimidiatus,
complete closure of the wound was not observed
during the study period (35 days). The regenerative process progressed from the proximal
to distal side of the wound hole, suggesting agreater regenerative capacity closer to the trunk.
In experiments involving A. cahirinus, in which
punch procedures were performed in the middleof the auricle without distal–proximal distinction,skin regeneration progressed from the proximal
to the distal side of the wound (Matias Santos etal. 2016). Although no studies to date have performed quantitative measurement of the expression profiles of genes noted during regenerationof Acomys species, studies of laboratory mice
have shown that calpain in neovascular vessels
promotes wound healing and tissue formation(Miyazaki et al. 2018). Because blood vessels are
more abundant closer to the trunk, the presenceof blood vessels may have influenced the large
difference in regenerative capacity between theproximal and distal locations in the present study,
with wounds in more vascularized locations having greater regenerative capacity. The wound area
reduction tended to be faster when the animals
were injured at 3 weeks of age (W3 group) thanat 9 weeks (W9 group), but the difference wasslight. We would not expect a significant difference in regenerative capacity over this age range.
This analysis revealed that A. dimidiatus has
the ability to structurally and functionally reconstruct skin wounds. Acomys cahirinus andthe present A. dimidiatus colonies were raised
as experimental animal strains, facilitating acomparative biological research approach using
both species. We expected that Acomys species
will become an even more useful model animalin the field of regeneration research.

IV. Discussion

Acknowledgements

In previous studies of ear punch regeneration in
A. cahirinus and A. kempi treated with ear punches twice the size of those of the present study (4We would like to thank the members of the-4-Ear wound regeneration in Acomys dimidiatus

Department of Zoology, OUS, for their support
in maintaining the spiny mouse strain.

Mekada, K., Oyake, A., Miyake, S., Yamaguchi, H., Hiraki,E., Yoshida, S., Tokumatsu, E. & Mochida, H. (2021)
Basic reproductive features of captive Arabian spiny
mice (Acomys dimidiatus). The Bulletin of Okayama
University of Science 57A: 7-11.
Miyazaki, T., Haraguchi, S., Kim-Kameyama, J.R. &Miyazaki, A. (2019) Endothelial calpain systems orchestrate myofibroblast differentiation during woundhealing. FASEB Journal 33(2): 2037-2046.
Mochida, H. & Mekada, K. (2017) Taxonomic status of
Aco strain derived from spiny mice. Proceeding of
Okayama Association for Laboratory Animal Science
33: 22-25. [in Japanese]
Montandon, S. A., Tzika, A. C., Martins, A. F., Chopard,
B. & Milinkovitch, M. C. (2014) Two waves of anisotropic growth generate enlarged follicles in the spiny
mouse. EvoDevo 5: 33.
Seifert, A. W., Kiama, S. G., Seifert, M. G., Goheen, J. R.,
Palmer, T. M. & Maden, M. (2012) Skin shedding andtissue regeneration in African spiny mice (Acomys).
Nature 489(7417): 561-565.
Slack, J. M., Lin, G. & Chen, Y. (2008) The Xenopus
tadpole: a new model for regeneration research. Celland Molecular Life Sciences 65(1): 54-63.
Volobouev, V., Auffray, J. C., Debat, V., Denys, C., Gautun,
J. C. & Tranier, M. (2007) Species delimitation in the
Acomys cahirinus–dimidiatus complex (Rodentia,
Muridae) inferred from chromosomal and morphological analyses. Biological Journal of Linnean Society
91(2): 203-214.

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公受暁子・目加田和之：アラビアトゲマウス（Acomys
dimidiatus ）の耳介損傷の再生
要約
アフリカトゲマウスとして一般的に知られるAcomys に属するA. cahirinus, A. kempi およびA. percivali の３種の齧歯類において，皮膚の脱落，皮膚の再生，広範な耳介閉鎖が報告されている．本研究では，第４の種であるA. dimidiatus の再生能力を検証した．耳介の近位部ないし遠位部に直径２mm のパンチ穴を開けたトゲ
マウスを35日間まで経時的に観察をしたところ，近位部のパンチ穴はほぼ完全に塞がり，修復部位には弾性
軟骨，脂肪組織，真皮，表皮，毛包が広範囲に形成された．遠位部に関しては，本研究での観察期間では，完全な閉鎖までは至らなかったが，同様に皮膚の再生が観察された．これらの結果は，A. dimidiatus の再生能力を裏付けるものであり，Acomys 属が再生研究のモデル動物としてさらに注目されるべきものであることを示した．
(Accepted 27 October 2023)-5-