Vol. 123 No.

5 May 2017

A domestic porcine model for studying the effects of radiation

on head and neck cancers
Christoph R. Arnold, MD, PhD,a Frank Kloss, MD, DMD, PhD,b Sarvpreet Singh, MD,c Danijela Vasiljevic, MD,a
Robert Stigler, MD, DMD,b Thomas Auberger, MD,d Volker Wenzel, MD, PhD,e Günter Klima, MD, PhD,f
Peter Lukas, MD, PhD,a Günter Lepperdinger, PhD,c,g and Robert Gassner, MD, DMD, PhDb

Background. Radiation therapy (RT) of the head and neck region is often accompanied by serious side effects. Research in this
area is needed to improve treatment outcomes and ameliorate therapy tolerance. Laboratory rodents are barely matching
today’s clinical standards in RT research. Yet domestic swine (Sus scrofa domestica) have previously proved suitable for various
advanced tests in clinical research and training. We therefore investigated whether S. scrofa domestica is also appropriate for
irradiation of the mandible.
Study Design. A common scheme for irradiation treatment of S. scrofa domestica mandibles in a split-mouth design was
acquired by applying computed tomography (CT) scanning under sedation. Basing on close anatomic resemblance, a standard
treatment plan comprising 2 opposed irradiation fields could be accomplished.
Results. RT was carried out in a clinical environment with 2
9 Gy. The resulting operating procedure facilitated
complication-free sedation, transport, positioning, CT scanning, and effective irradiation.
Conclusion. Based on common standards applied for RT in humans, domestic pigs can be employed to progress RT clinical
research. Due to their human-like anatomy, physiology, size, and weight, the swine model is expedient for advancing
experimental RT of the head and neck area. (Oral Surg Oral Med Oral Pathol Oral Radiol 2017;123:536-543)

Most tumors of the head and neck region develop from surgical and chemotherapeutic approaches, RT effec-
within the mucosa and are hence termed head and tively allows for addressing microscopic tumor exten-
neck squamous cell carcinomas (HNSCCs).1 HNSCC sions or draining lymphatic vessels. Commonly, RT is
is the sixth leading cancer by incidence worldwide, applied in an adjuvant or neoadjuvant setting, but is
accounting for approximately 3% of adult malignancies also often employed as a primary treatment with or
in North America and Europe.2 The choice of without simultaneous chemotherapy. Around half of
therapeutic modality depends primarily on localization patients diagnosed with various types of cancer undergo
and disease progression. Early-stage HNSCC, to date, RT, either with or without surgery or chemo-
has most often been surgically treated,3,4 but radiation therapy.11,12 Recent technical innovations, such as
therapy (RT) has become increasingly important. 3-dimensional conformal RT (3-DeCRT) and
Currently, RT is the primary treatment for locally intensity-modulated RT (IMRT), have resulted in
advanced HNSCC.5-7 improved clinical outcomes, in particular regarding
In general, RT plays an important role in locore- locoregional disease control and survival rates. Owing
gional therapy for malignant diseases, not only of the to enhanced precision, these modern techniques yield
head and neck region.8-10 Being positioned between the reduced adverse effects, in particular by leaving sur-
rounding healthy tissue untouched as much as
This work was presented as a poster at the DEGRO-ÖGRO annual
possible.13-18
conference in Mannheim, Germany, in June 2016. This work was
supported by the Jubilee Fund of the Austrian National Bank, 2006 Although RT has been widely applied in treating
(project number 12246) and Synthes Austria. malignant diseases for many years, its outcomes are
a
Department of Therapeutic Radiology and Oncology, Innsbruck considered far from satisfactory, often resulting in
Medical University, Innsbruck, Austria. serious and impairing adverse effects. Especially
b
Department of Maxillofacial Surgery, Innsbruck Medical University,
complicating in the head and neck area are numbers,
Innsbruck, Austria.
c
Institute for Biomedical Aging Research, Innsbruck, Austria. spatial location, radiosensitivity, and individual size of
d
Department of Radiotherapy-Radiooncology, Hospital Traunstein,
Traunstein, Germany.
e
Department of Anaesthesiology and Critical Care Medicine, Inns-
bruck Medical University, Innsbruck, Austria.
f
Statement of Clinical Relevance
Division of Histology and Embryology, Innsbruck Medical Univer-
sity, Innsbruck, Austria.
g Domestic pigs can be easily employed as an exper-
Department of Cell Biology, University Salzburg, Salzburg, Austria.
Received for publication Aug 1, 2016; returned for revision Oct 11, imental animal model to mimic therapeutic irradia-
2016; accepted for publication Nov 25, 2016. tion in the head and neck region to gain information
Ó 2016 Elsevier Inc. All rights reserved. about radiation-induced pathologies and improve
2212-4403/$ - see front matter existing or advance new radiotherapeutic strategies.
http://dx.doi.org/10.1016/j.oooo.2016.11.017

536
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Volume 123, Number 5
tissues. Complications in the oral region after head and
neck RT can be manifold and include mucositis,
hyposalivation, xerostomia, excess mucous and secre-
tions, radiation-induced caries and periodontitis, oral
infections, trismus, taste and smell disorders, pathologic
fractures, radiation-induced stenosis of the carotid ar-
teries with severe neurologic consequences, and
osteoradionecrosis (ORN).19 ORN emerges as a
delayed radiation injury emanating from defective
bone healing for longer than 3 months. This occurs in
approximately 2-8% of cases, most commonly
affecting the body of the mandible, supposedly
because of compromised vascularization.19-23 Typi-
cally, ORN develops 2 to 4 years after RT completion.
In severe cases it can lead to facial deformities, barely
manageable pain, pathologic fractures, sequestration of
devitalized bone, and oral-extraoral or oroantral
fistulae. Suffice it to say, therapeutic strategies need to
be improved considerably in order to reduce side effects
and ameliorate clinical outcomes.
Research concerned with advancing RT is addressing
cellular mechanisms, physical principles, technical in-
novations, and clinical routines. Basic research in
medicine often relies on animal models. They provide
insight into disease mechanisms, allow determination of
efficacy and safety of therapies, and represent unprec-
edented options for testing novel therapies. For obvious
reasons, small rodents are most commonly used. The
advantages are availability, handling and effort in
housing, maintenance costs, low ethical concerns, ease
of genetic modifications, and reproductive traits.24 On
the other hand, limitations of these model organisms
compared to humans are enhanced metabolic rates,
disparate genetic backgrounds due to inbreeding, little
immunologic challenge due to sterile housing, and
relatively short lifespans. Therefore, studies involving
large animals, which are more closely related to
humans, are being pursued. Apparently, nonhuman
primates would be an optimal match. However, these
do not reach sexual maturity before the age of 3 years
and give birth at best once a year, with only few
offspring per litter. Furthermore, they require very
sophisticated maintenance and handling, and, for
political reasons, experimentation on primates is often
banned. In contrast, domestic pigs (Sus scrofa
domestica) can produce up to 30 offspring per year
and are widely available, easily maintained, and
relatively inexpensive. Moreover, many aspects of
their physiology and anatomy greatly resemble the
human situation.25-29 Also, due to technical con-
straints, large animal models are found to be better
suited for testing new RT approaches, in particular
because rodents are too small for defined irradiation of
specific anatomic regions, especially the head and neck
region. Domestic swine have already been applied in
ORIGINAL ARTICLE
Arnold et al. 537
RT research in many aspects. Distinguished protocols
have been established for RT procedures on skin, lung,
and kidneys.30-33 The previously established experi-
mental procedures, employing radiation technology,
date back 2 to 3 decades and have been further devel-
oped by introducing more sophisticated techniques and
powerful machines. We here provide an operation
procedure for irradiation of the mandible employing
domestic pigs, describing all procedures and technical
refinements, which should ease future large animal
studies for the advancement of RT, especially for the
treatment of malignancies of the head and neck region.
MATERIAL AND METHODS
Permission to perform experimentation on S. scrofa
domestica (domestic pigs, weight w60 kg) was granted
by the Austrian Government and National Ethics Com-
mittee (permission no. BMBWK-66.011/0143-BrGT/
2006). All experimental and analytical work concerning
animal experimentation was conducted in accordance to
the European Union directive 86/609/EEC.
CT scanning for planning of the irradiation was
performed on a Philips Brilliance Big Bore CT scanner
(Philips, Hamburg, Germany). The irradiation plan was
created using the PrecisePLAN treatment planning
system, release 2.12-477.08 (Elekta, Stockholm, Swe-
den). Irradiations were exclusively performed with a
6 MV Elekta SLi Precise linear accelerator.
The biologically effective dose (BED) was deter-
mined using the modified linear quadratic model (LQ-
L), which can be used to elicit biological responses to
different irradiation fractionation regimes, also when
employing high single doses.34 The formula for the
BED is as follows:
BEDn ¼ DT þ DT2/(a/b) þ [(g/a)(D  DT)], where g
is the proportion of killed cells per Gy in the final linear
portion of the survival curve, DT is the dose at which the
survival curve becomes linear, and D is the dose applied
per fraction. g/a was calculated according the formula
g/a ¼ 1 þ (2 DT/(a/b)). DT was estimated to be 2 a/b.
The a/b ratio was assumed to be 3.85, a value that is
typically used for late-reacting tissues such as bone.35-37
Electronic portal images were taken via the Elekta
portal imaging system.
For sedation, the following drugs were used: aza-
perone (Eli Lilly, Indianapolis, IN, USA), atropine
(Braun, Melsungen, Germany), ketamine (Intervet,
Vienna, Austria), and propofol (Fresenius, Bad Hom-
burg, Germany).
For histologic analysis, biopsies from 16 irradiated
and 8 nonirradiated animals were taken from equivalent
regions of the mandible 8 weeks after irradiation. After
embedding in poly-methacrylate, blocks were crude
sawed, grounded, polished, and stained with toluidine
blue.
ORAL AND MAXILLOFACIAL SURGERY OOOO
538 Arnold et al. May 2017

Fig. 1. Positioning and safety fixation of the swine on the linear accelerator table. A and B, left-lateral view; C and D, ventrocranial
view. The putative field of irradiation was marked with blue ink. A and B, The sedated animals mounted on a stretcher were placed
in a supine position with a plastic tube between the jaws and mouth slightly open, allowing the animal to breathe. Large support
pads wedged underneath the entire length of the animal laterally on both sides increased positional stability. C and D, Propofol was
administered through a cannulized ear vein.

RESULTS sedation. Sedated animals were then brought to the

Preparing for animal experimentation
The method and experimental regimen presented here
were designed and tested on 16 female pigs of the same
age weighing w60 kg. Eight animals served as nonir-
radiated controls. Prior to and during the time of
experimentation, the animals were in good condition,
without displaying obvious medical indications. The
experimental animals were housed at a registered farm,
where they were accommodated in a specially adapted
facility equipped with a CCT system with uninterrupted
24-hour video observation. Irradiations were performed
at the Department of Radiotherapy-Radiooncology of
Innsbruck Medical University. In order to preclude
impinging on clinical routines, irradiation and accom-
panying treatments were pursued only on weekends.
The animals were transported from the farm to the
animal facility of the university, where sedation was
induced. For this purpose, animals received a simulta-
neous intramuscular injection of azaperone (2 mg/kg),
atropine (0.01 mg/kg), and ketamine S (20 mg/kg) into
the shoulder or buttock muscles. After 5-10 minutes, an
ear vein was cannulized. Propofol (6-8 mg/kg/h) was
administered via an infusion pump in order to maintain
radiation oncology department on standard animal
carriers attached with stretcher-like handle bars. During
sedation, the animals received oxygen via nose cone,
and oxygen saturation was measured at the tail. Seda-
tion in this way actually proved sufficient to pursue
subsequent procedures, thus sparing the animals from
general anesthesia. Working along this line, CT scan-
ning as well as irradiation and backhauling could be
performed exclusively under sedation.
Radiation treatment planning and irradiation
The animals were positioned on a linear accelerator
table in the supine position. A polyamide bite block was
inserted to preclude occlusion of the teeth (Figure 1).
To prevent inadvertent rolling, immobilization was
accomplished by pushing wedge-shaped support pads
underneath the trunk. After irradiation and before
backhauling to their original housing, the animals were
allowed to recover from sedation in a quiet area within
the animal facility.
Planning of the irradiation procedure was pursued
by employing CT imaging of the first animal. Based
on the acquired CT data, a standardized 3-D model
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Volume 123, Number 5
Fig. 2. Compilation of images derived from computer to-
mography scanning yielding a 3-dimensional model of a
swine’s head (x and z directions are highlighted in green
letters, beam paths are depicted by yellow lines).
was compiled using a clinical treatment planning
system (Figure 2). Based on that model, 2 opposed
irradiation fields of 7
3 cm were chosen to
irradiate both rami of the mandible. This way,
comprehensible and reproducible conditions for
homogenous dose distribution of irradiation could
be modeled (Figure 3). Working along this line, a
set of preliminary default specifications was
developed. The procedure was refined by reiterating
the same workflow on the remaining animals. This
first model actually proved sufficient later on,
because clinical measurements using a pelvimeter
revealed stable jaw width and length. Primarily due
to the interanimal anatomic similarities of the head
region, subsequent CT imaging of each individual
animal became obsolete. A standardized treatment
plan was thus developed. Taking an electronic
portal image of the irradiation field shortly before
each irradiation was sufficient to first confirm
anatomic resemblances and eventually place each
animal in a correct position. This greatly
accelerated the individual sequences, in particular
because CT scanning as well as implementing and
finalizing treatment planning is time-consuming. In
conclusion, we were able to establish a standardized
procedure that was generally applicable to preparing
all cases by simply confirming correct positioning
within the irradiation fields by taking an electronic
portal image of the irradiation fields before irradia-
tion. Then the total dose applied was 18 Gy in 2
fractions one week apart with 9 Gy per fraction,
resulting in a BED of 59.2 Gy.
ORIGINAL ARTICLE
Arnold et al. 539
During transportation, sedation, positioning, and
irradiation, no unusual incidents were reported.
Although handling large animals is tedious, all pro-
cedures and treatment of the animals in the clinical
surroundings turned out to be completely uncompli-
cated. Neither sedation-induced incidents, damage to
clinical equipment and installations, nor any unex-
pected incidents were reported during the stay at the
hospital, the latter owing to the fact that irradiation
treatment was conducted on weekends, thus avoiding
any interference with patients.
Following this regimen, 16 pigs were irradiated. For
all cases, no contingent events or critical circumstances
occurred. In stark contrast to our initial experimental
design, we were able to irradiate the pigs without using
general anesthesia. This was advantageous, as endo-
tracheal intubation is very difficult in pigs, considered
much more difficult than intubation in humans. Around
the porcine larynx, deep blind-ending pouches exist that
pose an increased risk of unintentional misplacement of
an endotracheal tube.29 Sedation using azaperone,
atropine, ketamine, and propofol was sufficient to
enable safe transportation, accurate positioning,
reproducible irradiation, and uncomplicated return
transport of the animals. After irradiating a few
animals, preparing an animal, transporting it to the
irradiation room, positioning, and irradiating lasted no
longer than w30 minutes. Staff involved were a
radiation oncologist, a physicist, a radiation therapist,
an animal care attendant, and an anesthesiologist. For
positioning, no special equipment was required. The
animals were positioned on the regular linear
accelerator table and stabilized with standard support
pads. The only extra demand was stringent cleaning
of all facilities in order to meet the hygienic
requirements of a hospital.
Biomedical implication
The biological effectiveness of the treatment was inves-
tigated 8 weeks after irradiation. Animals were sacrificed,
and irradiated mandibular bone was inspected by histo-
logic means. Notably, within irradiated bone, osteocytes
exhibited apparent swelling of the cell bodies, indicative
of intracellular edema (Figure 4). This is a known early
response to irradiation.38 This animal model has been
applied to investigate osseous healing and fracture
closure in the context of potential ORN.39 As published
previously, mesenchymal stem cells (MSCs) have been
isolated from irradiated pig mandibles. Unlike MSCs
irradiated in culture, MSCs explanted from bone
several days and weeks after irradiation provided
experimental evidence of enhanced radioresistance,
since they exhibited no obvious deficiencies regarding
proliferation or differentiation capacity.40
ORAL AND MAXILLOFACIAL SURGERY
540 Arnold et al.
Fig. 3. Treatment plan for the irradiation of the mandible. A and D, transverse section; B, coronal section; C, sagittal section. The
95% isodose areas are presented in magenta. The inlying green lines represent the 100% isodoses. Rectilinear green lines depict
field boundaries.
DISCUSSION
RT is an effective therapeutic approach in the treatment
of HNSCC and other malignant diseases. By now,
standards have been distinguished and widely agreed,
leading to an increasing use of RT in many therapeutic
routines.8-10 Technological advances, accumulated
detailed knowledge regarding cellular and organismic
responses after irradiation, and a sound understanding
of biological repair and regeneration mechanisms are
continuously improving clinical outcomes and reducing
radiation-induced side effects. Still, unresolved adverse
effects persist. Prime topics in this respect are impair-
ment of patients and pain directly related to RT, which
in due course and often in the long run negatively
impact quality of life. The head and neck region is
particularly vulnerable to radiation-induced damage, as
there are many small, sensitive tissues and organs
located in intimate proximity to one another. In lieu of
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May 2017
very precise irradiation routines, particularly due to
technical limitations of the currently available irradia-
tion devices, anatomic structures are unintentionally
harmed, resulting in unwanted side effects. Although
complications can partly be reduced by careful planning
and implementing individualized RT schemes together
with effective post-RT treatment, the prime demand of
research in this field is to minimize damage while
maximizing therapeutic outcome.
In this context, much of the basic scientific research
is commonly performed in cell cultures or with small
rodent models, such as mouse or rat. Together these
approaches have limitations, in particular when
attempting to extrapolate the experimental results in
order to identify new avenues for human therapies.
Also due to the spatial constraints of radiation tech-
nology, rodents and other small animals are simply too
small to allow accurate irradiation of specific anatomic
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Volume 123, Number 5
Fig. 4. Bone histology 8 weeks after irradiation. A and C, Biopsies from nonirradiated controls and B and D, irradiated (2
9 Gy)
animals taken from equivalent regions of the mandible. Cells in irradiated bone display edema (red arrows pointing at enlarged and
brightly stained nuclear regions), indicating an early reaction to the radiation. Scale and respective measures are indicated.
regions, such as distinct increments of the mandible.
While nonhuman primates are apparently the animals
most closely related to humans in terms of size, anat-
omy, and physiology, they have unfavorable repro-
ductive traits, are costly, and require sophisticated
maintenance and handling. In general, studies in
nonhuman primates have become scarce due to rigorous
legislation. Last, and most important, the public fully
refuses to acknowledge experiments on primates. In
contrast, research on pigs has many advantages. They
reproduce frequently and are relatively cheap and easily
housed, which raises less ethical and legal concerns.
Domestic pigs have been employed previously in RT
research to study the effects of radiation, e.g., on skin,
lung, and kidneys.30-33 Seminal work to establish
standardized procedures was done decades ago. To
date, these procedures may only partially suit present
clinical routines. First, irradiation machines and tech-
niques employed in those studies hardly compare to
current devices and methods, particularly concerning
irradiation accuracy and computer-assisted techniques.
Second, as irradiation is becoming increasingly
important in the treatment of various malignancies,
patient numbers have greatly increased in recent years
and radiation oncology departments have had to
establish more efficient therapeutic schemes to handle
ORIGINAL ARTICLE
Arnold et al. 541
the increasing numbers of patients in less time. This
greatly restricts time available for research, regarding
both available staff and technical equipment. In this
context, distinct RT research addressing the head and
neck region employing the domestic pig model has not
been reported. Particularly regarding ORN, which is
considered one of the most devastating late side effects
of HNSCC radiotherapy, experimental studies have not
been performed in extenso. Taking these issues into
consideration and appreciating the fact that many as-
pects of domestic pigs, particularly regarding anatomy,
physiology, and cell biology, are comparable to
humans, we established a swine model for irradiation of
the mandible.
To gain insight into the pathogenesis of ORN, non-
rodent experimental models have been established.
Minipigs have been studied previously, which,
although much smaller, greatly resemble domestic
pigs.41,42 Not only because of obvious factors such as
size and anatomy, but more importantly due to the
rationale of exhibiting comparable physiologic param-
eters, the domestic pig is closer to the human situation.
The model described here was initially developed for
investigating ORN and potential underlying mecha-
nisms. Particularly addressed was the role of mesen-
chymal progenitor cells, which are potentially capable
ORAL AND MAXILLOFACIAL SURGERY
542 Arnold et al.
of obliterating the development of fibrotic lesions, in
wound and fracture healing and their potential to
modulate immune response.39,40 It is conceivable that
the procedure described here can be adapted to other
regions of the head and neck area, initiating a plethora
of different irradiations.
In conclusion, we were able to show that S. scrofa
domestica can be effortlessly employed as an experi-
mental animal model in order to obtain knowledge on
radiation-induced pathologies in the head and neck
area, and subsequently improve the management of
devastating and impairing reactions. Moreover, the
described operation could further popularize the use of
large animal models, particularly because the protocol
can be easily adopted with virtually no need for special
equipment or otherwise trained personnel.
We thank Paul Eichberger and Alexandra Wegmayr, Depart-
ment of Therapeutic Radiology and Oncology, University
Hospital of Innsbruck, for outstanding technical assistance and
Rita Vitolo, School of Dental Medicine, University of Pitts-
burgh, for careful proofreading of the manuscript.
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Reprint requests:
Christoph R. Arnold, MD, PhD
Department of Therapeutic Radiology and Oncology
Innsbruck Medical University
Anichstrasse 35
6020 Innsbruck
Austria
Christoph.arnold@i-med.ac.at