Original Paper

Skin Pharmacol Physiol 2018;31:175–183 Received: December 5, 2017

Accepted after revision: February 2, 2018
DOI: 10.1159/000487404 Published online: April 4, 2018

Topical Treatment of Skin Injury Inflicted

in Mice by X-Ray Irradiation
Evangelia Meimeti a Antonios Kafanas b Panagoula Pavlou a
     

Antonia Evangelatou c Panagiota Tsouparelou c Stelios Kanellopoulos c

     

Panagiotis Kipouros c Nikolaos Koliarakis c Georgios Leonis a

     

Efstathia Ioannou d Vassilios Roussis d Michail Rallis a

     

a Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens,
Athens, Greece; b Pathologist, Parodos Efthalias Adam, Serres, Greece; c Department of Radiation Oncology, Andreas
   

Syggros Hospital of Dermatology and Venereology, Athens, Greece; d Department of Pharmacognosy and Chemistry
 

of Natural Products, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece

Keywords
Radiotherapy · Ceratothoa oestroides · Ionizing radiation ·
Pinus halepensis · Skin injury · Radioprotection
Abstract
Background/Aims: There is no treatment, without side ef-
fects, efficiently preventing or curing skin burns, caused by
radiotherapy. A new experimental topical treatment proto-
col was assessed in mice receiving orthovoltage X-rays at an
equivalent dose to that applied to human breast cancer pa-
tients in conventional radiotherapy. Methods: SKH-HR2 fe-
male hairless mice were irradiated on their dorsum with a
total dose of 4,300 cGy during a 1-month period (20 frac-
tions). The treatment group received a combination of 3 top-
ical products, an oil-in-water cream, a gel containing Pinus
halepensis bark aqueous extract, and an ointment contain-
ing olive oil extract of the marine isopod Ceratothoa oestroi-
des. The positive control group was treated with a conven-
tionally used commercial gel, whereas the negative control
group did not receive any topical treatment. Skin alterations
were evaluated by macroscopic examinations, measure-
ments of transepidermal water loss (TEWL), melanin content,
erythema intensity, hydration, and histopathology assess-
ment. Results: Sixty days after radiation, TEWL and hydration
values were abnormal and elements of acute, chronic, and
granulomatous inflammation were present in all cases. The
severest damage was detected in the deeper dermis. Treat-
ment showed a comparatively beneficial effect on chronic
and granulomatous inflammation while positive control was
beneficial on acute inflammation. Conclusion: Skin anti-in-
flammatory treatment was the most effective but must be
applied for several months. Further preclinical studies should
be conducted, assimilating a human cancer radiation thera-
peutic schema with the aim of optimizing skin inflammation
treatment. © 2018 S. Karger AG, Basel
Introduction
Radiation therapy is applied to women with breast
cancer, before or after surgery, and is usually combined
with chemotherapy. It has been shown that approximate-
ly 90% of women under radiotherapy develop skin in-
flammation. High-energy X- or gamma rays, or beta par-
ticles destroy cancer cells by damaging their DNA either
directly or through generation of free radicals [1–6].
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Univ. of California Santa Barbara

© 2018 S. Karger AG, Basel Michail Rallis

Department of Pharmaceutical Technology
Faculty of Pharmacy, National and Kapodistrian University of Athens
E-Mail karger@karger.com
Panepistimiopolis Zografo, GR–15784 Athens (Greece)
www.karger.com/spp
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E-Mail rallis @ pharm.uoa.gr

 Ionizing radiation can also damage normal cells, espe-
cially those which are rapidly divided [3]. Although it
contributes to the treatment of many cancers, radiation
therapy may also induce serious side effects to the skin,
most commonly radiodermatitis [4].
Radiotherapy-induced skin injury mainly depends on
the dose per fraction and the total one. However, indi-
vidual radiosensitivity plays an important role. In breast
cancer patients subjected to radiotherapy, the side effects
on the skin can be acute (occurring close to the beginning
of radiotherapy) or chronic (lasting for months, even
years after treatment). Acute skin injury is characterized
by erythema, desquamation, edema and pigmentation,
while chronic injury includes dryness, thinning of the
epidermis, decrease in adnexal structures, ulceration, fi-
brosis, pigmentation alterations, progressive decrease in
circulation, and telangiectasia [3, 7–9].
Ionizing and ultraviolet radiations induce skin inju-
ries [7]. Antioxidant enzymes or substances, vasocon-
strictors, and growth factor inhibitors (e.g., imatinib)
can contribute to skin protection [9–14]. Plant products,
such as Aloe vera, sweet almond oil, and olive oil, con-
taining bioactive compounds protect the skin from the
radiation-induced damage and have been used as excip-
ients or ingredients in several pharmaceutical and cos-
metic preparations [8, 15–20]. Pinus halepensis extracts
have shown protective and therapeutic skin properties
[21–23]. Bark extracts from P. halepensis contain poly-
phenols, proanthocyanidins, flavonols, and flavonoids;
therefore, they may exert therapeutic effects against
pain, inflammation, oxidative stress, and cancer. On the
other side of biodiversity, marine benthic organisms rep-
resent a rich reservoir of potentially useful substances
with medicinal properties such as antimicrobial, anti-
inflammatory, anticoagulant, and antineoplastic effects
[24–26].
The isopod Ceratothoa oestroides is a parasite found in
the buccal cavity of fish, causing serious lesions finally
leading to the death of the host, mainly of young fish [27,
28]. Experimental studies in mice have recently demon-
strated a significant wound-healing potential of C. oes-
troides oily extract [29–31].
Even though there are many raw materials with sig-
nificant protective or therapeutic properties, there is no
product safely preventing or curing skin burn caused by
radiotherapy. Topically applied corticosteroids drastical-
ly reduce skin inflammation induced by radiation X;
however, their use is accompanied by serious side effects.
Moreover, there is incomplete knowledge about the tim-
ing and type of topical products which must be used [32].
176 Skin Pharmacol Physiol 2018;31:175–183
DOI: 10.1159/000487404
Based on the aforementioned previous results and ex-
perience, a topical treatment protocol consisting of 3 ex-
perimental products was designed. These products were
applied before, during and after irradiation on the skin of
mice, which received orthovoltage X-ray radiation in a
total dose equivalent to that used for human breast cancer
conventional radiotherapy. These experimental products
had as active ingredients P. halepensis extract, C. oestroi-
des extract, egg white, A. vera gel, and were expected to
exhibit anti-inflammatory, antioxidant, and emollient
properties when applied to the skin. The prophylactic ef-
fect of this protocol against radiation-induced acute skin
injury was evaluated by comparing the treated mice to
those treated with a widely used commercial gel (positive
control) and another set of mice that did not receive any
treatment (negative control).
Materials and Methods
Animals and Radiation Protocol
The experiment was approved by the Prefectural Veterinary
Committee (Protocol No. 719/11.02.2016), and all procedures
were in accordance with the European Communities Council Di-
rective 86/609/EEC. Twenty-one SKH-HR2 female mice 6 months
of age were used in this study. All mice originated from the breed-
ing stock of the School of Pharmacy Small Animal Laboratory (EL
25 BIO 06). The animal room was kept at 23 ± 1 ° C, 25–45% hu-
midity, and was illuminated by yellow fluorescent tubes in a 12-h
light and dark cycle. The mice had unrestricted continuous access
to standard chow diet (Nuevo SA-Farma-Efyra Industrial and
Commercial SA, Greece) and fresh water.
The animals received orthovoltage X-ray radiation on the dor-
sal surface of their trunk (Gilardoni SpA, Mandello del Lario, Ita-
ly). Technical features and information on the radiotherapy sche-
ma used in this study are presented in Table 1. Radiotherapy was
performed at the Radiotherapy Department of the Dermatological
Hospital Andreas Syggros, Athens, Greece. The mice were posi-
tioned in sternal recumbency, and the radiation was administered
with an applicator. The radiation area was 3.14 cm2. Each ani-
mal received 1 fraction of radiation daily, 5 fractions a week, for
1 month (20 fractions). The daily dose of radiation was 180 cGy for
the first 10 fractions and 250 cGy for the remaining 10 fractions,
reaching a total dose of 4,300 cGy. The total radiation dose was
equivalent to that used for conventional breast cancer radiothera-
py in humans [33–35].
The mice were sacrificed by cervical dislocation 2 months after
the end of the irradiations.
Extract Preparation
P. halepensis extract was an aqueous 10% w/w extract prepared
at room temperature under stirring for 24 h.
Ceratothoa oestroides extract was an olive oil 10% w/w extract
prepared at room temperature under stirring for 24 h. It was pre-
served at 4 ° C.
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Table 1. Technical and other information on the radiotherapy
scheme
Radiation area (circular, diameter) 2 cm
Source-skin distance 40 cm
Energy
kV 150
mA 16
Fraction duration 4 min and 18 s
Fraction time 10.30 p.m.
Radiotherapy duration 1 month
Starting radiation dose 180 cGy
Total radiation dose 4,300 cGy
The radiotherapy scheme, equivalent to human breast cancer
conventional radiotherapy, was applied to 21 mice in order to
evaluate the prophylactic effect of a topical treatment protocol on
radiation-induced skin side effects.
Experimental Treatment
Three topical products were applied:
“Product 1” was an oil-in-water cream containing 5% of P. ha-
lepensis bark extract. Due to its phenolic antioxidants, it was ex-
pected to provide a prophylactic effect against radiotherapy-in-
duced skin inflammation.
“Product 2” was an oil-in-water cream containing 5% P. ha-
lepensis extract, egg white, A. vera gel, urea, and sodium pyrrol-
idone carboxylic acid. It was expected to provide anti-inflamma-
tory, hydrating, and emollient skin effects.
“Product 3” was an ointment containing 10% C. oestroides olive
oil extract, bees wax, lipoic acid, and petrolatum. The product was
expected to regenerate the skin and reduce transepidermal water
loss (TEWL) due to its lipid constituents.
Mice (n = 7) received the experimental topical treatment pro-
tocol according to the following schedule: 1 week before the initia-
tion of radiotherapy product 1 was applied on the area of irradia-
tion once a day. On the day of each radiation fraction, product 1
was applied on the skin 1 h before radiotherapy, whereas product
2 was used immediately after irradiation, and product 3 was ap-
plied on the irradiated area at night. The topical treatment protocol
was continued for 6 days after the end of radiotherapy by initially
applying product 1 in the morning, followed by product 2 after
1 h, while product 3 was applied at night. The dose of the product
applied was 5 mg/cm2. The topical treatment protocol was termi-
nated 6 days after the end of radiation treatment.
Positive Control
Mice (n = 7) were treated 1 week before radiotherapy, as well
as 1 h before and immediately after each irradiation, with a gel
(Radiogel; HC Welth, Budapest, Hungary) that is conventionally
used in such cases. The dose of the product applied was 5 mg/cm2.
The gel application was stopped 6 days after the end of radiation
treatment.
Negative Control
Mice (n = 7) were subjected to the radiotherapy protocol as
mentioned above without receiving any topical treatment.
Topical Treatment of Skin Injury Inflicted
in Mice by X-Ray Irradiation
Cutaneous Damage Assessment
Skin changes were evaluated macroscopically and microscopi-
cally.
All skin parameters were evaluated under standard laboratory
conditions. Macroscopic–skin parameter evaluation was per-
formed on day 0 (before radiotherapy) and on days 2, 4, and 15
during the radiotherapy period, as well as 6 days and 2 months
after the last radiation fraction as described below.
(a) Preclinical evaluation and digital photography were carried
out.
(b) Water barrier skin function (TEWL) was evaluated by the
density gradient of the water evaporation from the skin using the
device Tewameter TM 210 (Courage and Khazaka, Cologne, Ger-
many). Reduction in TEWL is indicative of high epidermal barrier
integrity.
(c) Melanin content (pigmentation) and erythema level (red-
ness) measurements were conducted by a Mexameter ΜΧ 18
(Courage and Khazaka Electronic GmbH, Cologne, Germany).
This device is a well-established narrow-band absorbance/reflec-
tance meter used for assessing the quantities of the 2 major com-
ponents (melanin and hemoglobin) responsible for skin color [36].
The measurement is based on the difference in the absorption be-
tween hemoglobin and melanin at specific wavelengths: the green
and the red for hemoglobin (568 and 660 nm), and the red and the
near-infrared for melanin (660 and 870 nm) [34], while the results
are shown in arbitrary units.
(d) Hydration measurement was performed with a Corneom-
eter CM 820 (Courage and Khazaka, Cologne, Germany) by esti-
mating stratum corneum electric capacitance.
(e) Histopathology evaluation was performed on skin speci-
mens obtained from the irradiated area after sacrificing the mice
of the 3 groups 60 days after radiotherapy. The specimens were
pinned on a flat cork surface and fixed in neutral-buffered forma-
lin. They were labeled without the pathologist being aware of
which groups corresponded to the specimens. All specimens were
routinely processed, paraffin embedded, sectioned at 5 μm and
stained with hematoxylin and eosin.
The occurrence of inflammation and its type (acute or chronic)
were examined. In case of acute polymorphonucleated neutrophil
inflammation, the predominant cell type was counted, and inflam-
mation was graded as follows: grade 1 = 1–10 cells, grade 2 = 10–20
cells, and grade 3 = >20 cells. In the case of chronic inflammation,
the predominant cell type that was counted was cells with lympho-
cyte morphology. The granulomatous inflammatory reaction was
also evaluated. The number of granulomas per square millimeter
as well as the largest dimension (length) of the biggest granuloma
in millimeters were also assessed. Furthermore, the status of mast
cells (degranulated, nondegranulated or variable) was evaluated.
In all quantitative measurements, the hot spot method was used,
and a mean value was estimated.
Statistical Analysis
Regarding the evaluated macroscopic variables, the effect of
group and time on each variable separately was studied by using a
linear mixed effects model [37].
The association between group and type of inflammatory reac-
tion (acute, chronic, and granulomatous) or the status of mast cells
(degranulated, nondegranulated, or variable), as revealed from mi-
croscopic evaluation, was studied by the χ2 test for independence
and the Fisher exact test. The effect of group on counts which de-
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Skin Pharmacol Physiol 2018;31:175–183 177
DOI: 10.1159/000487404
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 Color version available online
Negative Positive Treatment Negative Positive Treatment

Mean values of melanin (logarithmic transformation)

Period of X-ray
During After 4.5
20
Mean values of Tewameter, g/n/m2

4.0
15

3.5
10

0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60
a Time, days b Time, days

Negative Positive Treatment Negative Positive Treatment

60
300

Mean value of corneometer

50
Mean value of redness

250
40

30

200
0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60 0 +2 +4 +15 +6 +60
c Time, days d Time, days

Fig. 1. Mean values of transepidermal water loss (a), melanin (b), redness (c), and hydration (d), with 95% con-
fidence intervals for each time period and group (treatment, negative and positive controls); error bars denote
standard deviations.

fined the type of inflammation was studied utilizing the general- TEWL values was observed for all groups during radio-
ized linear model under the Poisson distribution function. Statisti- therapy, indicating a reduced epidermal barrier integrity.
cal analysis was conducted using the statistical language R [38]. It is worth noting that even 60 days after the end of ra-
Finally, the effect of group on granuloma length was studied using
the parametric 1-way analysis of variance. diations, the skin barrier did not come back to normal
In all tests, a difference was considered statistically significant (Fig. 1a).
when the p value was less than 0.05.
Pigmentation
Melanin content measurement revealed a significant
Results effect of time (p = 0.023) on pigmentation. It showed
some fluctuations but not significant changes between
Macroscopic Characterization treatments (Fig. 1b).
No macroscopic skin changes were observed by pre-
clinical evaluation and digital photography during the Erythema
study period. No pain or other significant symptoms were Skin redness evaluation revealed a statistically signifi-
noticed. cant effect of both group (p = 0.043) and time (p = 0.007)
on erythema. The post hoc analysis for the effect of time
Barrier Function revealed a statistically significant increase in erythema
TEWL measurement did not identify any significant from day 4 to 15 during radiotherapy (p < 0.001), while
differences between the treated group, and positive and the post hoc analysis for the effect of group revealed a sig-
negative control groups. However, a statistically signifi- nificant difference only between the treatment group and
cant mean effect of time (p < 0.001) on this variable was the negative control group (p = 0.020) in favor of the lat-
revealed. More specifically, an increase in the mean ter (Fig. 1c).
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 Color version available online
a b c

Fig. 2. Histological patterns of mouse skin on day 60 after radio-
therapy in the treatment group (a), the positive control group (b),
and the negative control group (c). c There is acute, chronic and
granulomatous inflammatory reaction in equal proportions.
b There is also a mixed reaction pattern but the intensity of inflam-
mation and the number of inflammatory cells are lower. a There
are only scattered, small foci of mixed inflammation. Black arrows
indicate collections of inflammatory cells predominantly com-
posed of neutrophils, some of them with karyorrhexis. A blue ar-
row indicates chronic inflammatory round cells. A red arrow
shows a giant cell that engulfed a lipid droplet surrounded by mac-
rophages, thus forming a granuloma. Yellow circles indicate de-
granulated mast cells close to the inflammatory reaction. Hema-
toxylin-eosin. Magnification ×20.

Color version available online

Acute Chronic Granuloma

Treatment
Treatment Treatment
group
group

Negative
Negative control
Negative Positive
Positive control
control control Positive
control
control

a b c

Fig. 3. Pie chart showing the extent of acute (a), chronic (b), and granulomatous (c) inflammation in treatment,
positive control, and negative control groups after histological evaluation on day 60 after radiotherapy.

Hydration Histological Observations

Hydration was evaluated by stratum corneum water
content and was significantly increased for negative and
positive controls (p < 0.05) without returning to normal,
even 60 days after the end of radiotherapy (Fig. 1d). Hy-
dration of the treated group significantly increased 2 days
after administration, followed by a noticeable decrease
(p < 0.05) that reached normal levels, 6 days after termi-
nation of radiation. Sixty days after the end of radiation
treatment, a significant increase in hydration was ob-
served.
To evaluate the type of inflammation, quantitative
measurements were performed by histological sections.
Histological patterns of mouse skin of the treatment
group, the positive control group, and the negative con-
trol group are presented in Figure 2a–c.
The extent of acute inflammation found in each group
is shown in Figure 3a. An association between groups and
acute counts was found (p < 0.001). Statistical analysis
based on polymorphonucleated neutrophil counts re-
vealed that acute inflammation was significantly lower in
the treatment group and in the positive control group
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Topical Treatment of Skin Injury Inflicted Skin Pharmacol Physiol 2018;31:175–183 179
in Mice by X-Ray Irradiation DOI: 10.1159/000487404
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compared to the negative control group (p < 0.001). How-
ever, acute inflammation was lower in the positive control
group than in the treatment group (p = 0.021).
A significant effect of groups on chronic counts was
found (p = 0.011). After statistical analysis of measure-
ments, it was shown that chronic inflammation (counts
of cells with lymphocyte morphology) in the treatment
group was lower than in the positive and negative control
groups, but the difference was significant (p = 0.007) only
between the treatment and the negative control group
(Fig. 3b).
Similar results to chronic inflammation were also
found for granulomatous inflammatory reaction (Fig. 3c).
A statistically significant difference was observed only be-
tween the treatment group and the negative control group
(p = 0.001). In the examined hot spots, lipogranulomas,
defined as giant cells that engulf lipid droplets surround-
ed by macrophages, were identified. Statistical analysis
did not reveal a significant effect of group on granuloma
length.
A statistically significant association (p < 0.001) be-
tween group and mast cell instances was found. Specifi-
cally, in almost all cases, in the negative control group
mast cells were degranulated, whereas in the other 2
groups they were nondegranulated or variable.
Discussion
This is the first time that administration of X-ray ra-
diation to mice using a physiologically relevant method is
attempted. The total dose of 43 Gy was fractionated into
20 doses, beginning with milder doses, following the
model of radiotherapy for cancer patients, and especially
focusing on the sensitive case of breast cancer. Except the
very specific type of athymic nude mice, previous studies
reported the use of different types of hairy mice, such as
C3n/HeN, BALB/c, and C57BLJ6, irradiating their flanks
or hind legs by a single dose, such as 11, 15, 22, 30, 35, 44,
50, or 200 Gy [32, 39–43]. Using hairless mice, there is no
need to shave the irradiated area, a factor which facilitates
the repeated topical applications.
The damaging effect of ionizing radiation on mouse
skin is confirmed by this preclinical study. TEWL, which
is the main indicator of skin barrier function related to
dermatitis phenomena [44], was in all cases enhanced
(Fig. 1a). This phenomenon is the most common side ef-
fect of radiotherapy [7, 45]. The fact that TEWL did not
return to normal levels 60 days after the end of irradiation
showed that skin side effects are severe and remain for
180 Skin Pharmacol Physiol 2018;31:175–183
DOI: 10.1159/000487404
long periods. This is in accordance with human data
showing late side effects even years after radiation [5, 7,
45, 46].
Apparently, melanin content does not play a signifi-
cant role in radiation therapy, as there were no significant
pigmentation changes on the treated skin (Fig. 1b).
Skin erythema measurements confirmed the preclini-
cal evaluation of no remarkable redness in treated and
nontreated mice (Fig. 1c). An increased redness was ob-
served only in the treatment group on the last day of the
treatment (day 15). This enhanced redness could be ex-
plained by the edema, which was apparently less in the
case of the treatment group. It is worth noting that the
presence of edema influences skin color by making it
whiter.
Skin hydration measurements showed a significant in-
crease after irradiation (p < 0.05) for both negative and
positive controls, which did not come back to normal
even 60 days after irradiation. For the treatment group,
an important increase was observed 2 days after the be-
ginning of irradiation (p < 0.05), while skin hydration
dropped to normal values 6 days after irradiation (Fig. 1d).
The fact that hydration returns to abnormal values 60
days after irradiation of the treatment group clearly shows
that inflammation continues for at least 2 months after
the end of irradiation exposure, thus recreating edema. It
was repeatedly observed that hydration measurements
are significantly influenced by epidermis and dermis ede-
ma. These data are in accordance with the abnormal
TEWL measurements 60 days after irradiation (Fig. 1a).
Acute inflammation (Fig.  2a–c, 3a) was significantly
pronounced in the negative control group followed by the
treatment and the positive control groups. Chronic in-
flammation (Fig. 2a–c, 3b) was significantly higher in the
negative control group followed by the positive control
and the treatment group. The presence of active inflam-
mation in the negative control group 60 days after expo-
sure to the last radiation dose was confirmed by the ob-
servation of degranulated mast cells. It is noted that mast
cell degranulation contributes to enhanced vascular per-
meability after irradiation and consequently to the in-
flammatory skin injury [47, 48].
The existence of degranulated mast cells could ratio-
nalize the significant edema creation on the irradiated
site, especially because of the enhanced histamine release
[49].
Acute and chronic inflammation (Fig. 3) was signifi-
cantly higher in the negative control group as compared
to the positive control or treatment groups. Positive con-
trol seems to better act in the case of acute inflammation
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while the contrary happened in the case of chronic in-
flammation. Chronic inflammation is difficult to cure in
comparison to acute inflammation, a fact which shows
that treatment has a meaningful effect on radiation ther-
apy [50].
The granulomatous inflammatory reaction was most-
ly enhanced in the negative control group followed by the
positive control, while a minimal reaction was seen in the
treatment group (Fig. 3c). This reaction is very important,
as it is a specific type of chronic inflammation in which
macrophages, epithelioid cells, and multinucleated giant
cells predominate [51, 52]. To the best of our knowledge,
this is the first time that granulomas have been histologi-
cally identified in skin subjected to X-ray radiation.
No skin injuries were identified by visual observation
or photographic evaluation. On the contrary, studies in
patients who received a total dose of 50 Gy after breast
cancer surgery showed that erythema and dry desquama-
tion developed in 38%, or in another study only 4.6% were
radioresistant, 77% had mild to moderate skin reactions
and 12% severe skin inflammation [6, 46]. The histologi-
cal evaluation showed that the severest damage was in-
flicted to the reticular dermis and even to subcutaneous
tissue (Fig. 2). Concerning the histology of epidermis, in-
significant changes were observed. This could be due to
the high penetration capacity of X-rays as well to the thin
skin of hairless mice (540 μm) [53, 54]. Similarly, UVA
light induces skin ageing through damage of dermis pro-
teins instead of enhancing epidermis photocarcinogene-
sis [55].
This in vivo study was conducted at the Radiotherapy
Department of the Hospital Andreas Syggros, a fact that
did not easily offer the flexibility to perform preliminary
experiments for the optimization of the final assay condi-
tions. The main complication related to the timeline of
treatment. Since no clinical indications of inflammation
were observed after the end of radiotherapy, apparent-
ly due to edema, therapy was prematurely terminated
(6 days after the end of radiotherapy). Also, since intense
inflammation was observed deeply in the skin after
2 months from the termination of radiotherapy, a rede-
sign of the experiment is needed, especially with respect
to the duration of therapy, which should be continued
several months after the end of radiation.
Overall, taking into account the data concerning
chronic inflammation, granulomatous inflammatory re-
action and the probable nonexistence of edema during
treatment, the optimal skin protection against X-rays was
obtained by the treatment protocol. This result is appar-
ently due to the P. halepensis extract being rich in pheno-
Topical Treatment of Skin Injury Inflicted
in Mice by X-Ray Irradiation
lic antioxidants, which decreased oxidative stress and
therefore inflammation [21, 56], as well as to the wound-
healing effect of C. oestroides extract [28–31].
In conclusion, using a similar approach and a total
dose of X-ray radiation equivalent to that used for a con-
ventional radiotherapy of human breast cancer, the treat-
ment protocol proved more effective in the reduction of
chronic inflammation, granulomatous tissue and edema
formation. For the first time, granulomatous tissue for-
mation was examined in mouse skin after X-ray radia-
tion. Skin inflammatory damage (both acute and chronic)
was mostly situated in the deep reticular dermis. It ap-
peared severe and continued to be for a long period of
time, therefore, skin anti-inflammatory treatment should
be continued for several months. The obtained results
provide an indication of the X-ray radiation damage and
its treatment approach. Further preclinical studies based
on a human cancer radiation therapy schema should be
conducted so that skin inflammation treatment may be
optimized.
Acknowledgments
The authors would like to acknowledge Dr. Nikolaos Mittas for
his contribution to data analysis, Dr. Dimitra Psalla for scientific
advice on histological evaluation, Nuevo SA company, especially
Mr. Ioannis Karvelis, for the provision of their excellent mouse
chow diet, and the Experimental Research Center ELPEN for its
significant support.
Statement of Ethics
All animal experiments conform to institutional standards.
The experiment was approved by the Prefectural Veterinary Com-
mittee (protocol No. 719/11.02.2016), and all procedures were in
accordance with the European Communities Council Directive
86/609/EEC.
Disclosure Statement
The authors declare no conflict of interest.
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