EXPERIMENTAL

A Novel Mouse Model of Cutaneous

Radiation Injury
Vishal D. Thanik, M.D.
Background: Radiation therapy is a cornerstone of oncologic treatment. Skin
Christopher C. Chang, M.D. tolerance is often the limiting factor in radiotherapy. To study these issues and
Richard A. Zoumalan, M.D. create modalities for intervention, the authors developed a novel murine model
Oren Z. Lerman, M.D. of cutaneous radiation injury.
Robert J. Allen, Jr., M.D. Methods: The dorsal skin was isolated using a low-pressure clamp and irradi-
Phuong D. Nguyen, M.D. ated. Mice were followed for 8 weeks with serial photography and laser Doppler
Stephen M. Warren, M.D. analysis. Sequential skin biopsy specimens were taken and examined histolog-
Sydney R. Coleman, M.D. ically. Tensiometry was performed and Young’s modulus calculated.
Alexes Hazen, M.D. Results: High-dose radiation isolated to dorsal skin causes progressive changes
New York, N.Y. in skin perfusion, resulting in dermal thickening, fibrosis, persistent alopecia,
and sometimes ulceration. There is increased dermal Smad3 expression, and
decreased elasticity and bursting strength.
Conclusions: This model of cutaneous radiation injury delivers reproducible lo-
calized effects, mimicking the injury pattern seen in human subjects. This technique
can be used to study radiation-induced injury to evaluate preventative and thera-
peutic strategies for these clinical issues. (Plast. Reconstr. Surg. 127: 560, 2011.)

R
adiation therapy is a cornerstone of oncologic The combination of perivascular fibrosis from these
treatment for the destruction of neoplastic cells chronically activated myofibroblasts and direct endo-
and their vascular beds. Although delivery of the thelial cell damage can lead to a chronically fibrotic
highest effective dose of ionizing radiation is desirable and hypovascular state. Alopecia occurs because of the
from an oncologic perspective, it must be balanced combination of direct follicular stem cell injury and
against the risk of radiation injury at the target area and overabundant fibrous matrix.6 These factors lead to the
at collateral regions. One of the most common areas long-term consequences of thickened, fibrotic, inelas-
for collateral injury is the skin. Skin tolerance is often tic, and alopecic skin.
the limiting factor in radiotherapy, with its injury lead- The clinical effects seen from ionizing radia-
ing to both short- and long-term sequelae. Despite re- tion are mediated at the cellular level by multiple
finements in the delivery of radiation therapy, cutane- cytokines, most notably transforming growth fac-
ous injury remains a complication of this treatment tor (TGF)-␤.7 TGF-␤ is mediated through Smad3,
modality. These injuries present a challenge in plastic a nuclear transcription factor, regulating inflam-
surgery and have limited therapeutic options. mation and chemotaxis.7 Activation of these path-
Exposure of the skin to ionizing radiation initiates ways is critical in inducing the injury pattern seen
a cascade of cellular events that lead to acute and from ionizing radiation.
chronic effects. In the acute phase, there is an influx of To study these changes and develop modalities
inflammatory cells and macrophages, which can clin- for intervention, we sought to develop a reproduc-
ically result in erythema, desquamation, and ulceration ible animal model of cutaneous radiation injury. A
of the skin and soft tissue. This phase is followed by the murine model was used because of its easy availabil-
proliferation of specific fibroblasts called myof- ity and low cost. It was designed to isolate the skin to
ibroblasts.1–3 These activated fibroblasts contribute to obviate any systemic responses from ionizing radia-
the longer term outcome of radiation fibrosis because tion. A single dose of irradiation was used to allow for
of their deposition of overabundant fibrous matrix.4,5

From the Institute of Reconstructive Plastic Surgery Labo-

ratories, New York University School of Medicine. Disclosure: Dr. Coleman receives royalties from
Received for publication January 26, 2010; accepted July 14, Byron Medical and is an advisor with stock options
2010. with Becon Medical and Cytori Therapeutics. No
Copyright ©2011 by the American Society of Plastic Surgeons other author has a financial interest to declare.
DOI: 10.1097/PRS.0b013e3181fed4f7

560 www.PRSJournal.com
Volume 127, Number 2 • Murine Model of Radiation Injury

ease of delivery while still maintaining the prototyp- male mice (stock no. 1800; Jackson Laboratories,
ical injury pattern seen with radiation injury. We also Bar Harbor, Me.) were anesthetized using a mixture
sought to create a model that did not require com- of ketamine, xylazine, and acepromazine. Hair was
plicated mechanisms or radiation shielding. Most removed from the dorsal skin using an electric clip-
importantly, we aimed to develop a model that mim- per and depilatory agent (Nair; Church & Dwight
icked both grossly and histologically the changes Co., Inc., Princeton, N.J.). Dorsal skin was then
seen in cutaneous radiation injury in humans, me- stretched and positioned between a low-pressure
diated through common cellular pathways. Al- clamp, which was created using bamboo sticks from
though other investigators have studied and devel- cotton-tipped applicators secured with rubber fas-
oped radiation models for hind-limb ischemia, teners. This design allowed for isolation of the dorsal
antitumor effects, or gastrointestinal injury, there is skin without ischemia (Fig. 1).
no well-established model of isolated radiation-in-
duced skin injury and ulceration. In this article, we Radiation
describe a new murine model for isolated radiation
Mice were laid longitudinally and secured with
injury to the dorsal skin resulting in alopecia, dermal
TegaDerm (3M, St. Paul, Minn.). Standard, off-
thickening, fibrosis, and ulceration.
the-shelf lead shields were placed to protect the
head, body, spine, and tail of the animal while
MATERIALS AND METHODS exposing the distracted skin to the radiation field.
Mice Treated animals were exposed (n ⫽ 5) to 30-, 40-,
All experiments were performed in accordance 45-, and 50-Gy external beam radiation therapy
with New York University Animal Care and Use Com- using a Varian 2300 Linear Accelerator (Varian
mittee guidelines. Ten- to 12-week-old wild type FVB Medical Systems, Inc., Palo Alto, Calif.). The dose

Fig. 1. (Above) Skin distraction model. After anesthesia and hair removal, the
dorsal skin was tented and distracted using a low-pressure wooden clamp to
isolate the skin and separate the underlying spinal column and musculature.
(Below) Irradiation apparatus. Once clamped, the subject’s skin was affixed to a
fiberglass plate using TegaDerm while the body was shielded using 0.25-inch-
thickleadsheeting.Theexposedskinwasthencoveredwitha0.5-cmradiation
bolus. XRT, external beam radiation therapy.

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 Plastic and Reconstructive Surgery • February 2011

energy was 6 MeV, with a dose rate of 1000 cGy/ highest dose groups (ⱖ45 Gy) had inadequate
minute. Field size was a 20 ⫻ 20-cm electron cone, lead shielding, resulting in tail exposure with sub-
with the source-to-skin distance being 100 cm to sequent necrosis and infection, and died within
the top of the 1-cm bolus. the first week. Of the 22 remaining animals, those
with isolated irradiation to the dorsal skin survived
Doppler Examination without diminished lifespans. There were no
Skin perfusion was analyzed using a color laser changes in behavior or feeding, and there was no
Doppler probe. The dorsal skin of the mice (n ⫽ incidence of infection.
5 per group) was scanned in situ and while
clamped to ensure the skin was not ischemic be- Skin Distraction and Clamping Do Not Cause
fore irradiation. Serial Doppler assessments of the Tissue Ischemia
dorsal skin were then made to evaluate perfusion Ischemia of the target area during irradiation is
after irradiation. a potentially confounding factor. To ensure that the
dorsal skin being irradiated had normal blood flow
Photography and perfusion, laser Doppler studies were per-
Serial photography of the dorsal skin surface formed before irradiation to measure perfusion to
was performed to evaluate for macroscopic evi- the target zone. Analysis demonstrated no change in
dence of radiation injury. blood flow with use of the low-pressure clamp for
skin distraction (Fig. 2), with equal perfusion be-
Histology tween clamped and nonclamped dorsal skin.
Skin biopsy specimens were taken from the zone
of irradiation at 2, 4, 6, and 8 weeks after external High-Dose Irradiation Causes Progressive
beam radiation therapy and fixed in 10% formalin Changes in Skin Perfusion
(n ⫽ 3 per group). Specimens were paraffin-embed-
Aberrant and overactive myofibroblasts are
ded and sectioned for hematoxylin and eosin stain-
known to cause excessive collagen matrix deposi-
ing. Fibrosis was assessed by picrosirius red stain
tion, resulting in perivascular fibrosis and hypoper-
and Smad3 immunohistochemistry (Cell Signaling
fusion. To evaluate this phenomenon, serial Dopp-
Technology, Inc., Danvers, Mass.). To quantify the
ler examinations of the dorsal skin were performed
amount of fibrosis and collagen content, the scar
in the irradiated versus nonirradiated mice, to ex-
index, a previously validated methodology, was cal-
amine whether changes in perfusion occurred over
culated as described previously.8
time. Laser Doppler monitoring demonstrated pro-
Tensiometry gressively worsening blood flow to the irradiated tis-
sue (Fig. 3) over a 6-week period.
Cutaneous rigidity (Young’s modulus) can be
evaluated by calculating the linear portion of the
stress-strain curve of the skin specimen. Dorsal skin High-Dose Radiation Injury Causes Prolonged
was harvested at 8 weeks after irradiation. Using a Alopecia
Chatillon TCD-200 tensiometer (Commercial Scale Alopecia is a common complication after
Co., Inc., Agawam, Mass.), consistent stress-strain radiotherapy.9,10 Its etiopathogenesis is thought to
curves of control and irradiated skin were calculated be multifactorial, with direct injury to basal cells,11
(n ⫽ 3 per group). A straight line is fit to the linear destruction of follicular stem cells,6,11 and cicatri-
part of the stress-strain curve. The value of the slope cial alopecia secondary to fibrosis.9 Effects are
of this line is the Young’s modulus. thought to be dose, technique, and location
dependent.12 Our results demonstrate this dose
Statistical Analysis dependency, with mice exposed to radiation doses
All data are expressed as the mean ⫾ SEM. Data greater than 40 Gy developing alopecia isolated to
were analyzed with an independent samples two- the dose field (Fig. 4). This alopecia persisted for
tailed t test assuming unequal variances. A value of longer than 12 weeks.
p ⬍ 0.05 was considered to be statistically significant.
High-Dose Irradiation Causes Progressive
RESULTS Dermal Thickening and Fibrosis
High-Dose Irradiation Isolated to Dorsal Skin Mice exposed to radiation doses greater than 40
Does Not Compromise Mouse Viability Gy developed progressive skin thickening. When the
A total of 25 mice were irradiated and allowed animals were examined, the skin in the irradiated
to recover. Of the 25 treated animals, three in the area was thickened and inelastic, with areas of non-

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Fig. 2. To ensure that the model did not cause ischemic damage, Doppler perfusion was
used to measure peripheral blood flow before and after clamping.

Fig. 3. After exposure to 45-Gy irradiation, progressive, decreased blood flow is seen by laser Doppler examination in the
irradiated skin over 21 days.

healing ulceration (Fig. 4). When analyzed histolog- changes were seen past 12 weeks. The scar index was
ically with hematoxylin and eosin and Sirius red calculated to quantify the differences between irra-
staining (Fig. 5, above), there was thickening of the diated and control mice. The irradiated groups had
dermis, with exuberant and progressive collagen scar indices twofold greater than controls (Fig. 5,
deposition over time. Changes became obvious by 2 below).
weeks and continued to progress up to the 6-week
time point. This was accompanied by an inflamma-
tory infiltrate and ulceration. There was also a loss of High-Dose Irradiation Causes Increased Dermal
hair follicles and decreased vascularity. The expan- Smad3 Expression
sion phase appeared to end by 6 weeks, at which Radiation-induced skin injury is mediated
time the fibrosis became chronic. Chronic fibrotic through various cytokines, most notably TGF-␤.

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Fig. 4. Over several weeks after 45-Gy radiation, there was clinical evidence of radiation
damage to the skin, resulting in dermal thickening, erythema, alopecia, and ulceration.
XRT, external beam radiation therapy.

Smad3 is an intracellular mediator of TGF-␤ changes in elasticity and bursting strength in

signaling13–15 and is the primary mediator of wounds placed in irradiated skin.16,17 Young’s
TGF-␤ cytostatic signaling.7 Sections underwent modulus was used to evaluate cutaneous rigidity.
immunohistochemical staining for Smad3. At the The control skin (Fig. 7, above, left and below, left)
2-week time point, we began to see increased has a lower slope, which represented a calculated
Smad3 expression (Fig. 6, above). By 6 weeks, there Young’s modulus of 7.78 MPa (N/m2 ⫻ 106). Ir-
was a distinct thick layer of collagen that stained radiated skin 8 weeks after irradiation had a
densely for Smad3 (Fig. 6, below). Young’s modulus of 19.63 MPa (Fig. 7, right).

Irradiated Skin Has Decreased Elasticity and DISCUSSION

Bursting Strength Radiation injury of healthy skin is a common
In addition to phenotypic changes, irradiated clinical problem that causes both severe cosmetic
skin demonstrated altered biomechanical proper- deficits and complex surgical complications, such
ties. Radiation has profound effects on the elas- as skin ulceration and delayed wound healing. As
ticity of the skin. Previous studies have shown targeted oncologic treatment makes technologi-

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Fig. 5. Localized irradiation induces site-specific fibrosis. (Above) Histologic sections from skin biopsy specimens
taken 2, 4, and 6 weeks after 45-Gy irradiation demonstrate increased dermal thickening and increased picrosirius
red staining, a marker of fibrosis. (Below) Scar index analysis demonstrates a greater than twofold increase in irra-
diated animals compared with controls at 6 weeks (p ⫽ 0.0002). XRT, external beam radiation therapy.

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Fig. 6. Increased Smad3 expression. (Above) Smad3 stain (brown) of skin 2 weeks
after 45-Gy irradiation (magnification, 20⫻). Brown areas indicate that Smad3 pro-
duction is initiating. (Below) At 6 weeks, there is a thick layer of dermal collagen
staining positive for Smad3 (magnification, 10⫻; inset, 20⫻).

cal improvements, damage to surrounding tissue hair follicles, and dermal thickening.19,20 This pro-
decreases, but overlying dermal structures remain gression of findings is seen in our model (Fig. 5).
exposed to radiation. Therefore, it is important to There is an initial influx of inflammatory cells with
establish a reliable animal model to mimic this epidermal ulceration. This is followed by over-
injury and study its pathogenesis and potential abundant collagen deposition by myofibroblasts
treatment and prevention. as demonstrated by the prominent picrosirius red
This model accurately reproduces the changes staining pattern. There is resultant dermal thick-
seen in human subjects from radiotherapy-in- ening, and progressive loss of vascularity caused by
duced injury. The hallmarks of this process involve perivascular fibrosis with parallel loss of hair fol-
progressive skin thickening, with decreased per- licles. When this dermal thickening is examined
fusion, elasticity, and alopecia. All of these find- quantitatively by determining Young’s modulus,
ings are recapitulated in this murine model. After we see the expected decrease in elasticity and
irradiation of the mice, the area of irradiated skin bursting strength.
becomes initially inflamed and erythematous, with These effects have been demonstrated to be
areas of superficial ulceration and desquamation. predominantly attributable to overactive myofi-
This is followed by progressive thickening and in- broblasts depositing excessive collagen through a
elasticity of the skin compared with the nonirra- TGF-␤/Smad3–mediated pathway.4,5 Tissue irra-
diated areas. Concomitant alopecia develops rap- diation activates multiple intracellular pathways,
idly within 2 weeks, consistent with direct follicular including interleukin-1 and interleukin-6, tumor
stem cell injury, and persists because of the effects necrosis factor-␣, TGF-␤, and the chemokines in-
of perivascular fibrosis and cicatricial alopecia. terleukin-8 and eotaxin.21 Of these, TGF-␤ has
This is seen in human patients, where alopecia has been implicated as the most potent mediator of
been noted at similar doses in those subjected to radiation-related skin injury. TGF-␤ has been
cranial irradiation.18 shown to regulate inflammation and fibrosis in
When we examine these areas of radiation both physiologic and pathologic states by acting as
injury at the histologic level, we see again that our a potent chemotactic factor for inflammatory cells
model of injury accurately reproduces the injury and being a key activator of these cells.4,5 Ionizing
pattern seen in human subjects. Radiation injury radiation serves as a potent inducer of TGF-␤
is characterized by excessive collagen deposition, which, in turn, stimulates myofibroblast prolifer-
with stromal encasement of blood vessels, loss of ation and deposition of collagen matrix.4 TGF-␤

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Fig. 7. Decreased elasticity and breaking strength. Stress-strain curves of control (above, left) and irradi-
ated (below, left) skin were calculated and demonstrate the lower slope of Young’s modulus between
control and irradiated tissue in identical mice at the same age. (Right) The average Young’s modulus of the
control tissue was 7.78 MPa, compared with 19.63 MPa in the irradiated tissue at 8 weeks (p ⫽ 0.033).

exerts its cellular actions by binding to trans- ated Smad3 activity in the areas of radiation fi-
membrane serine/threonine kinase receptor brosis, we observed that the activity of Smad3 was
subunits.22 In turn, the activated receptor phos- elevated in the areas of collagen deposition in the
phorylates the receptor-regulated cytoplasmic dermis. This is first noted at 2 weeks and persists
protein Smad3. Smad3, a nuclear transcription past the 6-week time point. This long-term up-
factor, is the primary mediator of TGF-␤ cytostatic regulation of Smad3 activity is expected, as TGF-␤
signaling.7 Irradiated human tissue experiments and Smad3 activity in human subjects with radia-
and several mammalian irradiation models have tion fibrosis has been found to be elevated as long
demonstrated increased levels of TGF-␤ and as 20 years after injury.4
Smad3 in vitro and in vivo.15 Tissue irradiation has The radiation model we describe is unique
both early and late effects that result in tissue and simple to recreate, allowing a single re-
damage. Studies in TGF-␤–null mice and efforts to searcher with few resources to generate single-
neutralize TGF-␤ have identified its role in both.23 dose radiation injury and minimizes animal mor-
This response is combined with increased apopto- tality. Furthermore, our model of skin clamping
sis of epithelial and hematopoietic cells present in and distraction does not crush the underlying tis-
the targeted radiation field.24,25 When we evalu- sue and vascular perfusion is not compromised, as

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 Plastic and Reconstructive Surgery • February 2011

shown by our laser Doppler analysis. The clamping 7. Kim SG, Kim HA, Jong HS, et al. The endogenous ratio of
system is simple and requires no specialized ma- Smad2 and Smad3 influences the cytostatic function of
Smad3. Mol Biol Cell 2005;16:4672–4683.
terials or jigs, using readily available sticks from 8. Flanders KC, Major CD, Arabshahi A, et al. Interference with
ubiquitous cotton-tipped applicators. The use of transforming growth factor-beta/Smad3 signaling results in
nonmetal materials is essential, as diffraction of accelerated healing of wounds in previously irradiated skin.
the electron field can cause unpredictable scatter Am J Pathol. 2003;163:2247–2257.
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model for creating a reproducible cutaneous in- Dowell HP. Surgical interventions for the treatment of ra-
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tom-made jigs,5,16,26 and have typically included Cancer 2007;49:731–736.
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Using a murine model offers many advantages 12. Lawenda BD, Gagne HM, Gierga DP, et al. Permanent alo-
in the study of radiation-induced cutaneous in- pecia after cranial irradiation: Dose-response relationship.
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