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This study was designed to evaluate therapeutic
ultrasound (TUS) in improvement of radiation-induced skin damages and
restriction of joint mobility. Eighteen adult male rats were divided into
control (Con-G), 30-Gy x-ray irradiation (30 Gy-G), and 30-Gy x-ray
irradiation with TUS treatment group (US-G), and then 30 Gy-G
and US-G were 30 Gy irradiated to their right hind limb. US-G underwent pulsed
TUS (3 MHz, 0.5 W/cm2, 10 min/d, 5 d/wk) to irradiated skin. The
main outcome included difference in skin reaction score, ankle joint range of
motion (ROM) and histological examination of skin. In our results,
radiation-induced disorders were significantly severe from 14 days after x-ray
irradiation, and TUS significantly improved the skin damages 28 days
after x-ray irradiation and reduced the early restriction of ROM. Then, the present findings indicate
the possibility of TUS as an adjuvant treatment for radiation-induced skin
disorder, though further examinations for the
morphological and molecular changes in the x-ray irradiated skin mediated by
TUS are required.
Keywords: Therapeutic ultrasound, Radiation injury, Skin, Wound healing, Joint
mobility
Abbreviations: Con-G:
Control Group; H.E:
Hematoxylin-Eosin; IQR: Interquartile Range; ROM: Range of Motion; TUS: Therapeutic
Ultrasound; US-G: 30 Gy
x-ray Irradiation with TUS Treatment Group; 30 Gy-G: 30 Gy x-ray Irradiation
Group
INTRODUCTION
The radiotherapy has been widely accepted as an effective treatment for
various type of cancer, but many patients have also suffered from its side
effects because of additional damage to the surrounding healthy tissues [1,2].
The radiation skin injury, which occurred in approximately 95% of patients
receiving radiotherapy for malignant neoplasms, remains a critical problem in
spite of the advances of medical technologies [3]. The harmful severity ranges
from mild erythema to moist desquamation, ulceration and soft tissue fibrosis,
and those lead to impaired wound healing and joint range of motion (ROM)
restriction in severe cases [4,5]. Therefore, it is important for preservation
of the patients’ quality of life to mitigate radiation damage to skin around
target cancer area.
Therapeutic ultrasound (TUS) has
been clinically used for various disorders such as bone fracture [6,7] and
tendon injury [8,9], and recently reported to be effective in
promoting the healing of refractory wound in Escherichia coli-infected [10] or
diabetic murine skin [11]. Previous studies have also revealed the positive
effect of TUS on the improvement of ROM in rat joint contracture model through
the alteration of collagen fibril alignment within its soft tissues [12,13].
TUS, therefore, may be expected to alleviate radiation-induced wounds and
subsequent reduction of joint mobility, but there are few reports which
investigate the effect of TUS on radiation-induced disorders.
The present study aims to demonstrate the effect of
MATERIALS AND METHODS
All procedures were carried out in accordance
with the Guidelines for Animal Experimentation of our institution.
The radiation-induced skin reaction and ankle joint dorsiflexion ROM of
all rat’s right hind limbs were evaluated the day before
irradiation, and at 3, 7, 14, 21 and 28 days after irradiation. The skin reaction scoring [3,14]
(Table 1) and ROM
measurement [15,16] were performed according to the previous reports. To
briefly explain ROM measurement, the ankle joint was passively dorsiflexed
maximally with the hip and knee joints 90 degrees under anesthesia by blinded
investigator. The angle formed by a line connecting the lateral malleolus and
the centre of the knee joint to a line parallel to the bottom of the heel were
measured from vertical position at 5 degree intervals using an angle meter.
At the end of the
experimental period, all rats were sacrificed under deep anesthesia and their skins of irradiated area
were extracted. Extracted skins were fixed in phosphate buffered 4%
paraformaldehyde and embedded in paraffin. Sections (5 mm thickness) were cut
and stained with hematoxylin-eosin (H.E.).
The data of skin reaction score and ROM were
presented as median
with interquartile range (IQR) and means ± standard deviations, respectively.
Both inter- and intra-group comparisons of skin reaction score and ROM were
performed by Steel-Dwass and Tukey’s test, respectively, at a significance level of 0.05 using
Statcel 3 software (OMS Publishing Inc., Saitama, Japan).
RESULTS
In the 30 Gy-G and US-G, radiation-induced skin reddening slightly
appeared 3 days following x-ray irradiation, and became significantly such severe
conditions as hair loss and effusions of the right hind limb from 14 days
post-irradiation onward (Figure 2). Twenty
eight days after x-ray irradiation, the US-G showed a significant improvement
in skin injury, compared with the 30 Gy-G (Figure
2).
The ankle joint ROM in the 30 Gy-G significantly decreased from 14 days after x-ray irradiation, compared with the other groups, whereas there was no significant difference in ROM between the Con-G and US-G throughout the experiment period (Figure 3).
Twenty eight days after irradiation, many neutrophils were
histologically found to infiltrate both epidermis and dermis in the 30 Gy-G,
showing inflammation, whereas the cell number was lower in the US-G (Figure 4).
DISCUSSION
In this study, we demonstrated the mitigative
effect of TUS on radiation-induced skin damages in rats. Twenty eight days
after irradiation, our results showed a significant improvement in skin
reaction score and lower number of neutrophils infiltrating both epidermis and dermis in the
US-G, compared with the 30 Gy-G, suggesting progression from the inflammatory
phase to the scar formation.
Effects of TUS is known to depend on insonation
frequency, intensity, treatment time and pulsed or continuous wave [17,18]. TUS
in the present insonation condition have been reported to accelerate the wound
healing in normal mammal skin, but its effect has not been found on ischemic
and radiation-induced skin wound healing in rodents [19,20]. This inconsistency with our results may
relate to difference in treatment time: only 3 or 5 minutes per day for 3 or 5
days per week in previous studies [19,20]. This idea is supported by Osumi’s
report that TUS for 10 minutes per day for 4 days per week with similar
conditions to ours improved the healing delay of infected wound in mice [10].
Additionally, low intensity (30 mW/cm2) pulsed TUS for 20 minutes
daily has been reported to reverse impaired wound healing in diabetic and aging
mice [11]. Therefore, pulsed TUS in long time (≥ 10 minutes),
frequent (≥ 4 days
per week) and/or low intensity condition may be suitable for the effective
treatment of refractory wound.
Our results also showed the effect of TUS on
early radiation-induced ROM restriction. In some previous reports,
radiation-induced contracture of mice has been evaluated from more than 2
months after x-ray irradiation, because it is due to the skin fibrosis
following prolonged inflammation [4,21]. Meanwhile, the present
study demonstrated the significant reduction of ankle dorsiflexion ROM in the 30 Gy-G
from only 2 weeks after irradiation and reversing effect of TUS on that, though
the mechanism leading the indications remains unclear so far. Clinically, since
long term immobility lead to severe contracture, TUS may be applicable to
maintain joint mobility from early stage after x-ray irradiation.
To our knowledge, this is the first study
showing the usefulness of TUS for radiation skin damage by use of animal model,
but we evaluated the radiation-induced skin reaction and ROM only for a limited
period. Further examinations for morphological changes and molecular mechanisms
mediated by TUS are required for the valid treatment of radiation-induced skin
damage.
ACKNOWLEDGEMENT
This work was supported by JSPS KAKENHI Grant
Number 15K16346.
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