Original Research Proliferative Activity of Myoepithelial Cells in Irradiated Rabbit Parotid and Submandibular Salivary Glands

Abstract

Background:
The behavior of salivary myoepithelial cells (MEC) during chronic irradiation exposure is unknown. This study aimed to investigate the response of MEC to prolonged radiation exposure.

Materials and methods:
16 rabbits and four controls were irradiated with either 10 Gy, 20 Gy, 30 Gy or 40 Gy (Gray units) of direct axial beam radiation. Parotid and submandibular glands were removed and examined using immunohistochemical double staining. Proliferating MEC were semi-quantified using alpha smooth muscle actin antibodies and proliferating cell nuclear antigen (PCNA) antibodies.

Results:
MEC proliferative activity increased after radiation in both submandibular (P = 0.037) and parotid groups (P = 0.006) compared to controls. Hyper-proliferation was seen only in parotid glands which was almost dose-dependent. Mean percentage MEC proliferation did not correlate with the clinical grading or recovery from oral mucositis (P = 0.47).

Conclusions:
Parotid glands are more sensitive to radiation compared to submandibular glands. Further research is needed to determine the role of MEC proliferative activity in response to radiation.

Full text

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Journal of International Oral Health 2015; S(2):1-5
Radiotherapy impact on salivary myoepithelial cells activity … Kujan O et al
Original Research
Received: 10th April 2015 Accepted: 16th July 2015 Confl icts of Interest: None
Source of Support: Nil
Proliferative Activity of Myoepithelial Cells in Irradiated Rabbit Parotid and Submandibular
Salivary Glands
Omar Kujan1,2, Rania Othman3, Mohammed Alshehri4, Fareed Iqbal5, Nabil Kochaji6
Contributors:
1Assistant Professor, Department of Oral and Maxillofacial Sciences,
School of Dentistry, Al-Farabi Colleges, Riyadh, Kingdom of
Saudi Arabia; 2Assistant Professor, Department of Oral Pathology,
School of Dentistry, Hama University, Hama, Syria; 3PhD
Student, Department of Oral Pathology and Histology, School of
Dentistry, Damascus University, Damascus, Syria; 4Consultant,
Department of Dental, King Khalid University Hospital, King Saud
University, Riyadh, Saudi Arabia; 5Senior Registrar, Department
of Gastrointestinal Physiology, St. Mark’s Hospital & Academic
Institute, London, UK; 6Assistant Professor, Department of
Oral Pathology and Histology, Faculty of Dentistry, Damascus
University, Syria.
Correspondence:
Dr. Kujan O. Department of Oral and Maxillofacial Sciences,
Al-Farabi College for Dentistry and Nursing, Riyadh, Kingdom
of Saudi Arabia. Tel.: +966501158867, Fax: +966112324580.
Email: omar.kujan@gmail.com
How to cite the article:
Kujan O, Othman R, Alshehri M, Iqbal F, Kochaji N. Proliferative
activity of myoepithelial cells in irradiated rabbit parotid and
submandibular salivary glands. J Int Oral Health 2015;S(2):1-5.
Abstract:
Background: The behavior of salivary myoepithelial cells (MEC)
during chronic irradiation exposure is unknown. This study aimed
to investigate the response of MEC to prolonged radiation exposure.
Materials and Methods: 16 rabbits and four controls were
irradiated with either 10 Gy, 20 Gy, 30 Gy or 40 Gy (Gray units)
of direct axial beam radiation. Parotid and submandibular glands
were removed and examined using immunohistochemical double
staining. Proliferating MEC were semi-quantifi ed using alpha
smooth muscle actin antibodies and proliferating cell nuclear
antigen (PCNA) antibodies.
Results: MEC proliferative activity increased after radiation in
both submandibular (P = 0.037) and parotid groups (P = 0.006)
compared to controls. Hyper-proliferation was seen only in parotid
glands which was almost dose-dependent. Mean percentage MEC
proliferation did not correlate with the clinical grading or recovery
from oral mucositis (P = 0.47).
Conclusions: Parotid glands are more sensitive to radiation
compared to submandibular glands. Further research is needed
to determine the role of MEC proliferative activity in response to
radiation.
Key Words: Experimental, mucositis, myoepithelial cells,
proliferating cell nuclear antigen, salivary
Introduction
Head and neck cancers are relatively uncommon accounting
globally for only 4% of all cancers.1,2 However, 5 years survival
within developing nations remains disproportionately inferior.3
This may be due to inadequate public awareness and late
presentation. Radiotherapy is an established treatment for
head and neck cancers.4 Radiotherapy causes xerostomia,
which is the subjective feeling of oral dryness. It is caused by
an inadequate or absence of saliva fl ow. It can also be caused
by changes to the saliva composition.5 Prolonged xerostomia
can cause mucositis which is the most commonly reported
complication following radiation for head and neck cancer.6
Severe mucositis causes signifi cant morbidity, reduced quality
of life, and in some cases, interruption to treatment schedules.4-8
Salivary glands are composed mainly of parenchymal
cells (acini) and myoepithelial cells (MECs). MECs
play an important role in supporting the morphogenesis,
di erentiation and polarization of salivary acini.9-12 A reduction
in saliva flow is seen almost immediately after radiation
exposure.8 The long-term exposure to radiation may induce
permanent gland damage, infl ammation, and fi brosis. The
precise pathophysiology of this process is unknown. On a
cellular level, several pathways have been implicated.8 During
infl ammatory conditions like sialadenitis, an increase in MEC
proliferative activity is observed which can be up to 10-fold
higher than that seen in normal salivary tissue.13-15 Conversely, a
decrease in the number of MECs after a single dose of radiation
has also been reported.16 This would suggest that post-radiation
infl ammation induces a di erent MEC behavior than that seen
in general infl ammatory conditions. The behavior of MECs
during chronic radiation exposure is unknown. Understanding
these pathways are important as it may help to develop novel
therapies which inhibit cellular processes which induce salivary
gland damage.
In order to characterize the response of salivary MEC’s to the
full radiation exposure the following study was conducted.
It also aimed to determine MEC proliferative activity post-
radiation correlated with the clinical grading of mucositis in
an animal model.
Materials and Methods
Study design
All experiments were conducted in strict accordance to the
recommendations laid out by National Institutes of Health
and Research guidance on the “Care and Use of Laboratory
Animals” guidelines.17 The study protocol was approved by the
University of Damascus Animals in Science Ethics Committee

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Radiotherapy impact on salivary myoepithelial cells activity … Kujan O et al
(Approval ref: 528/2012). 6-month-old female New Zealand
rabbits (n = 20) with an average weight of 2.5 kg were used in
this study. The animals were bred in the Damascus University
Animal Centre. All experiments were performed, while the
rabbits were under general anesthesia following sodium
pentobarbital infusion through the femoral vein. The rabbits
were exposed to 12 h-12 h light-dark cycles and had free access
to water and standard diet.
A protocol developed and validated by Hakim et al. was
followed.16,18 In brief 16 rabbits were divided into four groups
(n = 4 per group). Rabbits from each group received either
10 Gray units (Gy) (Group A), 20 Gy (Group B), 30 Gy
(Group C) or 40 Gy (Group D) of ionizing radiation using
a treatment schedule of 2 Gy/day over 5 days. Treatment
schedules ranged between 5 days (Group A) and 20 days
(Group D). Control group (n = 4) received no radiation. The
protocol aimed to mimic typical radiation schedules given
to patients with head and neck cancers. Radiation doses are
increased in gradual fractions as described above. An equivalent
dose of 40 Gy confers a 100% risk of mucositis.19 The study
design hence simulated high (Group D), moderate (Group B
and C), and low radiation (Group A) exposure.
Radiation was delivered using the ALCYON II telecobalt
therapy device (Georges Speicher, France). An axial beam
was directed toward the head of the rabbit at a extending
from the retro-auricular region to the tip of the nose after
a bolus delivered from 0.5 cm (Figure 1). A radiation field
size of 5 cm × 10 cm was created, which allowed all salivary
glands to be irradiated. All procedures were performed by
a single researcher (RO). Rabbits were irradiated daily for
5 min. An experienced radiotherapist from the Nuclear
Medicine Hospital in Damascus University Hospital was
used for consultation and advice to ensure correct radiation
dose delivery. The oral mucosa of animals was examined
by an oral medicine specialist (OK) prior to execution and
graded according to the oral mucositis assessment scale
(OMAS).20
Following their respective radiation regimens all animals
were killed immediately. Controls were killed at the end of
20 days. Parotid and submandibular glands from all animals
were removed and fi xed in 10% neutral bu ered formalin for
24 h. Specimens were para n-embedded and sectioned for
hematoxylin and eosin staining. Histopathological analysis was
performed by a blinded histopathologist (NK).
An immunohistochemical double staining technique was used
to quantify proliferating MECs. MECs were double-stained
using antibodies against -smooth muscle actin (-SMA)
while active proliferation was quantified using antibodies
against proliferating cell nuclear antigen (PCNA). 4 m
formalin fi xed sections were placed on poly-lysine-coated
glass slides. Sections were dewaxed and rehydrated in standard
serial dilutions in ethanol. Sections were then incubated with
200 l of dual endogenous enzyme block for 5 min and then
washed three times in 0·1 mol/L phosphate-bu ered saline
(PBS). Slides were incubated for 1 h at room temperature with
PCNA primary mouse monoclonal antibody (Monoclonal
anti-PCNA, clone PC10, DakoCytomation, Denmark) diluted
1:100 in antibody diluent (50 ml/PBS, 0.5 ml goat serum and
0.5 g bovine serum albumin). Slides were then washed in PBS
and incubated in 200 l of polymer/HRP for 5 min. After
further PBS washes, slides were incubated in 200 l DAB for
5 min followed by further PBS washes and incubation in PBS
bu er for 1 h at room temperature. A 200 l Doublestain block
(EnVision™ DuoFLEX Doublestain System, DakoCytomation,
Denmark) was applied for 5 min, then washed in PBS.
Slides were incubated with 1:100 -SMA second antibody
(Monoclonal anti--SMA, Clone 1A4, DakoCytomation,
Denmark) for 1 h at room temperature. The slides were
then washed three times with PBS each for 3 min, followed
by incubation with 200 l Rabbit/Mouse (LINK) for 5 min.
The slides were then washed 3 times with PBS each for 3 min,
followed by incubation with 200 l polymer/AP for 5 min. After
a further three washes in PBS, the sections were incubated with
200 l permanent red working solution (EnVision™ DuoFLEX
Doublestain System, DakoCytomation, Denmark) for 15 min.
Slides were washed with deionized water and placed in a
distilled water bath for 5 min. Slides were counterstained for
2-3 s with hematoxylin. A negative control experiment was
carried out on random sections from each gland, in which the
primary antibody was omitted.
Statistical analysis
The labeling index of PCNA in MECs was calculated. MECs
were counted under a microscope at ×400 magnifi cation by two
pathologists simultaneously (NK, RO) using eight randomly
selected fi elds. The percentage of cycling (PCNA-positive)
MECs was calculated by dividing the number of PCNA positive
Figure 1: The rabbits in set-up position using the ALCYON
II telecobalt therapy device.

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Journal of International Oral Health 2015; S(2):1-5
Radiotherapy impact on salivary myoepithelial cells activity … Kujan O et al
cells with the number of -SMA positive cells from within the
eight randomly selected fi elds. An average was then calculated
Data are presented as mean (standard deviation). To evaluate
statistical di erences between groups, a non-parametric U-test
according to Wilcoxon, and Mann–Whitney was used, with
P < 0.05 considered to be statistically signifi cant. Statistical
analysis was done using Statistical Package for Social Sciences
(SPSS) software version 22.0 (SPSS®: Inc., Chicago, IL, USA).
Results
All animals tolerated radiation and continued to eat and drink.
There were no premature deaths or weight loss.
Oral mucositis was observed in all animals (100%) following
radiation (Table 1). The severity of mucositis directly correlated
with strength of radiation with the greatest severity observed
in Group D and the least in Group A. The severity scores
ranged from 3 to 18 with a mean score of 10 across the groups.
Mucositis was observed on average 2.25 days after the fi rst
dose of radiation and took on average 3 days to heal. Rabbits
in Group D took the longest to recover from the mucositis
(range 3-8 with a mean of 5 days). The OMAS score did not
correlate with mean PCNA activity for any subgroup (P = 0.47).
Histological H and E sections demonstrated mild to moderate
inflammatory cell infiltration in all radiation groups. The
greatest number of infl ammatory cells was seen in Group D.
In this group, parotid glands showed higher infl ammatory
infi ltrates compared to submandibular glands (Figure 2).
Graph 1 illustrates the mean percentage of PCNA-positive
MECs in normal and irradiated salivary glands. Controls
showed low mean MEC proliferative activity in both
submandibular and parotid salivary glands; 9.56% and 8.36%
respectively (P = 0.945).
The highest mean percentage of PCNA-positive MECs was
found in parotid salivary glands in Groups A and D (Figure 3)
which were statistically di erent to other subgroups (P = 0.015)
and normal parotid salivary tissue (P = 0.006).
Parotid Group D (40 Gy) showed higher mean% PCNA-
positive MECs than other parotid subgroups (10, 20, 30 Gy).
In addition, PCNA activity in parotid glands was signifi cantly
higher than submandibular glands, particularly in subgroups A
(10 Gy; P = 0.001), B (20 Gy; P = 0.001), and D (40 Gy;
P = 0.003).
Hyper-proliferation of MECs was graded as a value higher
than mean plus two standard deviations of controls. With this
defi nition, hyper-proliferation of MECs was not observed
in submandibular glands irrespective of radiation dose
(Table 2a and b). Hyper-proliferation of MECs was observed
in all parotid sub-groups except Group C (30 Gy).
Table 1: Correlation of OMAS scale and response to diff erent radiation doses.
Group Duration of
radiation
OMAS
score
Number of days after fi rst
radiation dose mucositis
observed
Number of days taken
for mucositis to resolve
Mean percentage of PCNA
positive cells in parotid and
submandibular glands
A (10 Gy) 5 3 3 2 18.09
B (20 Gy) 10 7 2 2 13.94
C (30 Gy) 15 11 2 3 13.09
D (40 Gy) 20 18 2 5 27.88
Average 12.5 10 2.25 3 18.25*
*P=0.47, OMAS: Oral mucositis assessment scale, PCNA: Proliferating cell nuclear antigen
Figure 3: Double immunohistochemistry for proliferating
cell nuclear antigen (brown) and actin (red) in the irradiated
parotid gland (40 Gy) (×400).
Figure 2: Haematoxylin and eosin sections of control (normal
parotid gland) (a) and irradiated submandibular (b) and
parotid 40 Gy (c) glands (40 Gy), (×200).
c
b
a

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Discussion
Head and neck cancer patients receiving radiotherapy
report poor quality of life. Salivary gland dysfunction and
consequential mucositis hampers speech, causes di culties
with food mastication, swallowing, and taste.5,6,21 These
impaired functions arise from direct acinar and ductal salivary
cell damage. The present study suggests that salivary MECs
play a limited or no role in the pathogenesis of radiation-
induced mucositis but increase their proliferation activity
which may highlight recovery processes. Despite holding
integral roles in salivary gland function, the role of MECs
after ionizing radiation is not well understood. We aimed in
this study to evaluate the proliferative activity of these cells
in a well-known animal model using PCNA antibodies as
markers of proliferation. This approached was justifi ed as the
immunohistochemistry of PCNA has shown to be equivalent
to Western blotting as a semi-quantitative method of assessing
MEC proliferative activity.22 Furthermore, our protocol had
been validated previously by Hakim et al.16
Our findings demonstrate that salivary MECs increase
their proliferative activity after radiation. Moreover, MECs
proliferative activity correlated with an increase in infl ammation
as shown through infl ammatory infi ltrates. This, however,
did not correlate with the degree of mucositis or recovery
from mucositis except at higher (40 Gy) doses. MEC hyper-
proliferation was observed only in parotid glands. Though
the MEC proliferative activity was dose-dependent, the
proliferative activity in parotid glands at 30 Gy was <10 Gy and
20 Gy, with 10 Gy demonstrating higher activity than 20 Gy.
However, at 40 Gy the mean proliferative activity increased
once again. This observation may be explained by suggesting
that the initial response of parotid MECs to ionizing radiation
is to increase activity, however, as the radiation duration
increases, the cells begin to trigger recovery processes. At larger
doses, this recovery is hampered which increases proliferative
activity further. The pro-infl ammatory role of MECs has been
characterized in chronic infl ammatory conditions.23-25 It is
unclear whether MEC proliferative activity was responsible for
inducing a pro-infl ammatory environment. As infl ammatory
infi ltrates were seen in both gland types after ionizing radiation
where only parotid glands signifi cantly increased proliferative
MEC activity; it would seem that proliferating MECs were not
responsible for inducing infl ammation.
We found hyper-proliferation of MECs in parotid glands only
after exposure to radiation doses 10 Gy, 20 Gy, and 40 Gy.
Parotid glands contain a higher number of serous cells which
are more radiation-sensitive.26 Submandibular glands, on the
other hand, contain a higher density of mucous cells.14 The
results suggest that submandibular glands are more resistant
to radiation. As parotid glands are more sensitive to radiation,
increased MEC proliferate may act to compensate for the
gross infl ammation. The role of anti-infl ammatories on MEC
proliferative activity would be an interesting further study.
Mucositis was graded according to the OMAS scale by a
blinded oral medicine specialist. Though the examiner was
experienced in grading mucositis in humans, he lacked full
training in grading this in rabbits. Furthermore, the OMAS
scale has not been validated in animals. Using two blinded
veterinary surgeons with experience in grading mucositis
in rabbits would have added to the strength of the study.
However, as no rabbits lost weight and continued to eat and
drink throughout the study, it is likely that the recording of
mucositis was accurate particularly as the main conclusions
were that all rabbits recovered within 5 days.
Conclusion
Parotid MECs increase their proliferative activity after exposure
to ionizing radiation which is almost dose-dependent. This
observation is absent in submandibular glands. Oral mucositis
is seen after radiation exposure which occurs as a result of
inflammation. Recovery from mucositis is possible after
radiation irrespective of dose. The pathogenesis of mucositis
after radiation is not related to MECs proliferative activity.
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Graph 1: Mean percentage of proliferating cell nuclear antigen
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irradiated salivary glands. MEC proliferative activity calculated
as PCNA +ve cells/ alpha smooth muscle actin antibodies
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Table 2a: Mean proliferative activity of MECs against radiation doses.
Gland type Control 10
Gy
20
Gy
30
Gy
40
Gy
P value
(Wilcoxon)
Submandibular 9.56 6.24 7.78 10.27 21.17 0.037
Parotid 8.36 29.94 20.1 15.91 34.6 0.006
MECs: Myoepithelial cells
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Parotid 17.86 Yes Yes No Yes
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either yes or no. MECs: Myoepithelial cells, SD: Standard deviation

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