International Journal of Pediatric Otorhinolaryngology (2007) 71, 623—628

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Efficacy of vitamin A in experimentally induced

acute otitis media
Ibrahim Aladag *, Mehmet Guven, Ahmet Eyibilen, Semsettin Sahin,
Dogan Köseoglu

Gaziosmanpasa University Medical Faculty, Department of Otorhinolaryngology, 60100 Tokat, Turkey

Received 20 October 2006; received in revised form 22 December 2006; accepted 26 December 2006

KEYWORDS Summary
Vitamin A;
Acute otitis media; Objective: Vitamin A plays a role in the prevention of oxidative tissue damage. In the
Free radicals present study we investigated therapeutic role of this substance on healing of middle
ear mucosa in experimental acute otitis media (AOM).
Materials and methods: Otitis media was induced by inoculating Streptococcus
pneumoniae via transtympanic injection. Thirty rats were divided into two groups.
Group I treated with parenteral ampiciline-sulbactam. Group II received same anti-
biotic regimen and parenteral single dose of 100,000 IU vitamin A in palmitate form.
At tenth day post-inoculation, animals were sacrificed and mucosal samples were
excised from the infected tympanic cavities for histpathological examination and
blood samples were obtained for measurements of activities of superoxide dismutase
(SOD), glutathione peroxidase (GSH-Px) and evaluation of levels of malondialdehyde
(MDA) and nitric oxide (NO).
Results: All the infected middle ear mucosas displayed various degrees of the
inflammation, but there was no meaningful difference between two groups. However,
epithelial integrity was significantly better in group II than group I ( p < 0.01).
While serum NO and MDA levels decreased in the group receiving both antibiotic
and vitamin A, serum SOD and GSH activity were found to increased. All of the
statistical differences are significant.
Conclusions: Pretreatment with vitamin A increases antioxidant enzyme activities
and reduces formation of NO and MDA.
Vitamin A may be considered as an additional medicament for the medical
treatment of AOM.
# 2007 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
* Corresponding author. Tel.: +90 356 212 95 00;
fax: +90 356 213 31 79. Acute otitis media is a common pediatric disease.
E-mail address: ibrahimal@hotmail.com (I. Aladag). Because of its persisting or recurring mature, it has

0165-5876/$ — see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijporl.2006.12.019
624
still been a major cause of morbidity in children in
spite of antimicrobial therapy [1]. Inflammatory
damage to the middle ear mucosa during the infec-
tion may be responsible for delay in recovery and
may predispose the patient to recurrence or persis-
tence. Some studies have documented the impor-
tant role of free radicals (FRs) in mucosal damage
during middle ear infection [2,3]. Although antibio-
tics are mainstays of treatment, they do not speci-
fically address the issue of FRs damage.
Free radicals including hydroxyl radicals, super-
oxide anions, and hydrogen peroxide, which are
produced by activated granulocytes, play an impor-
tant role in many biochemical processes such as
intracellular messaging in the cell differentiation,
apoptosis, immunity and defense against microor-
ganisms [4]. The shift of the delicate balance
between FRs and the cellular antioxidant defense
system in favor of FRs might lead to development of
oxidative stress [5]. Main targets of FRs are the
polyunsaturated fatty acids in cell membranes caus-
ing lipid peroxidation and MDA formation, which
may lead to damage of the cell structures and
function [6]. Aerobic organisms are protected
against free radicals by enzymatic and nonenzy-
matic antioxidant defense systems [4]. The most
important enzymatic antioxidants are superoxide
dismutase (SOD) and glutathione peroxidase
(GSH-Px). Increased activity of radical scavenger
enzymes, such as SOD and GSH-Px to prevent the
detrimental effects of oxidative stress in different
organs, has been declared in recent reports [7]. Free
radicals and lipid peroxides have been implicated in
the pathogenesis of many diseases, including dia-
betes mellitus, cancer, rheumatoid arthritis, sys-
temic lupus erythematosus, Behcet’s disease,
infectious diseases, atherosclerosis, and aging [8].
Vitamin A is a fat-soluble substance and essential
for immunity, cellular differentiation and mainte-
nance of respiratory and gastrointestinal epithelial
surfaces, growth, reproduction and vision. Although
vitamin A is recognized for its ocular benefits, its anti-
infective effect has been known for a century. Recent
clinical studies and appropriately designed experi-
ments in animals demonstrated that vitamin A
enhances immunity, thereby reducing childhood mor-
bidity and mortality from infectious diseases [9]. We
aimed to investigate the effect of vitamin A on heal-
ing of experimentally designed AOM in addition to
standard antibiotic therapy histopathologically.
2. Materials and methods
A total of 30 female Wistar rats weighing 240—260 g
were used. The study protocol was approved by the
I. Aladag et al.
Ethics Committee of Gaziosmanpaşa University and
the animals were handled in accordance with guide-
lines of the National Council for the Care of Labora-
tory Animals. Animals with otoscopic signs of active
or recent infection were discarded.
The animals were anesthetized with a combina-
tion of ketamine (100 mg/kg) and diazepam (0.1 mg/
kg) given intraperitoneally. All procedures were per-
formed under sterile conditions. Under otomicro-
scopic view, 0.04 ml of type 1 Streptococcus
pneumoniae solution, consisting of 107 to 109 pneu-
mococci per milliliter, was inoculated into the left
middle ear cavity via transtymphanic route. The
animals were initially examined at 48 h post-inocu-
lation in order to perform otomicroscopy and confirm
the presence of otitis media. Then animals were
randomly divided into two groups: group I was trea-
ted with intramuscular ampiciline-sulbactam
(50 mg/kg) twice daily for 7 days, group II was treated
with intramuscular single dose of 100,000 unit vita-
min A (in palmitate form, Türk-Roche, Istanbul,
Turkey) in addition to intramuscular ampiciline-sul-
bactam for 7 days. After 10 days, all animals were
sacrificed by using a lethal dose of intracardiac
sodium pentothal. Blood samples were collected
for biochemical analysis. Temporal bones were taken
and middle ear mucosas were prepared for histo-
pathologic evaluations.
3. Histopathological analyses
Specimens were stained with hemotoxylin—eosin and
were examined using light microscopy. Histopatho-
logic analysis of the group I was compared to group II.
The examiner (DK) was unaware of the groups. The
histopathologic analysis was scored for inflammation
and integrity of epithelium. The severity of grade of
inflammation was assessed on a scale of none (0),
minimal (1): rare individual inflammatory cells within
the mucosa and submucosa, mild (2): light infiltrate
Fig. 1 Middle ear epithelium integrity is complete with
minimal inflammation (group II).
Efficacy of vitamin A
of individual and clusters of inflammatory cells, mod-
erately (3): dense infiltrate of inflammatory cells and
severe (4): inflammatory infiltrate so dense as to
obscure the normal architecture of the mucosa and
submucosa. The integrity of middle ear epithelium
was assessed on a scale of complete (0) (Fig. 1), focal
epithelium lost but no vacuolization and metaplasia
(1), focal epithelium lost and vacuolization (2), and
focal epithelium lost and epithelial metaplasia (3)
(Fig. 2).
4. Biochemical analyses
Fasting blood samples were drawn into heparin-free
tubes during routine blood sampling for biochemical
analyses. After immediate centrifugation (2000  g
for 15 min at +4 8C), serum samples were stored
frozen at 70 8C. The following determinations
were made on the samples using commercial
chemicals supplied by Sigma (St. Louis, USA). Total
(Cu—Zn and Mn) SOD (EC 1.15.1.1) activity was
determined according to the method of Sun et al.
[10] and Durak et al. The SOD activity was expressed
as mmol/L. Glutathione peroxidase (GSH-Px) activ-
ity was measured by the method of Paglia and
Valentine [11]. Activity was expressed as mmol/L.
The serum thiobarbituric acid-reactive substance
(TBARS) level was determined by a method [12]
based on reaction with thiobarbituric acid (TBA)
at 90—100 8C. In the TBA test reaction, malondial-
dehyde (MDA) or MDA-like substances and TBA react
to produce a pink pigment with an absorption max-
imum at 532 nm. Results were expressed as mmol/L,
according to the standard graphic prepared from
measurements with a standard solution (1,1,3,3-
tetramethoxypropane). NO measurement is very
difficult in biological specimens, therefore tissue
nitrite (NO2 ) and nitrate (NO3 ) were estimated
Fig. 2 Thick arrows show an area of focal epithelium lost
and thin arrow shows two areas of squamous metaplasia
(group I).
625
as an index of NO production. Samples were initially
deproteinized with Somogyi reagent. Total nitrite
(nitrite + nitrate) was measured after conversion of
nitrate to nitrite by copperized cadmium granules
by a spectrophotometer at 545 nm. Results were
expressed as mmol/L.
5. Statistical methods
All parameters showed a normal distribution; there-
fore parametrical statistical methods were used to
analyze the data. The findings were expressed as the
mean  S.D. Mann—Whitney U-test and two indepen-
dent sample t-test were performed for pathological
parameters and for biochemical parameters, respec-
tively. p-Values <0.05 were regarded as statistically
significant. All calculations were analyzed by using
Statistical Package for Social Sciences (SPSS) 14.0
demo Version. (SPSS Inc., Chicago, IL).
6. Results
In group I, inflammation was found minimal in one
section, mild in five sections, moderate in eight
sections and severe in one section. In group II,
inflammation was found minimal in three sections,
mild in four sections moderate in one section and
severe in seven sections. The inflammatory changes
in middle ear mucosas in both groups were similar.
However, epithelial integrity in animals receiving
antibiotic and vitamin A was significantly better
from than the animals receiving only antibiotic
( p < 0.001). Summarized data for each treatment
group was presented in Table 1.
Therapeutic effects of vitamin A on AOM were
examined by means of histopathologic examina-
tions as well as SOD, GSH-Px enzyme activities,
and blood levels of NO and MDA. The results are
presented in Table 2. Blood NO and MDA levels of
group II receiving antibiotic and vitamin A were
significantly lower than those receiving only anti-
biotic ( p < 0.001). However, SOD, GSH-Px enzyme
activities were significantly increased in group II
compared with group I.
7. Discussion
Vitamin A is a micronutrient, essential for immunity
and maintenance of respiratory epithelium. This
fat-soluble substance is found in foods from animal
sources, including dairy products. Vitamin A serves
as a prohormone for retroretinoids and intersects
with signal transduction at cytoplasmic and
626 I. Aladag et al.

Table 1 The comparison of the histopathological changes of group I and those of group II
Group I Group II z p
Inflammation
None 0 0 0.625 0.532
Minimal 1 3
Mild 5 4
Moderate 8 1
Severe 1 7
Epithelium integrity
Complete 0 3 3.647 <0.001
Focal lost 6 12
Focal lost with vacuolization 0 0
Focal lost and metaplasia 9 0

membrane sites. Although, several retinoids have
been used for therapy of several diseases, the
applicability of this class of compounds is limited
by their teratogenic activity. Hence, great atten-
tion should be paid for the risk assessment of
embryotoxicity resulting from excessive intake of
vitamin A by pregnant women. The evidence that
vitamin A is essential for immunity derives from
epidemiological observation, in vitro studies, clin-
ical trials and experiments in animal models [9].
Results obtained from human studies, supports this
evidence. Acute measles is associated with low
serum levels of vitamin A [13]. Vitamin A supple-
mentation reduces severe morbidity and mortality
from infectious diseases in children [9]. The admin-
istration of a high dose vitamin A supplement to
children with acute complicated measles reduces
mortality by 50% [14]. Previous attempts have been
made to extend these observations to children at
higher risk for respiratory infections.
The action of FRs in the pathogenesis of AOM has
been reported in several studies [2,15]. There are
two suspected mechanisms of FR release into the
middle ear cavity during AOM. Both neutrophils,
responding to infection and S. pneumoniae, the
most common infecting organism in AOM, produce
toxic FRs and their intermediates. Excessive produc-
tion of FRs which is known as oxidative stress,
leading to damage of nuclear and mitochondrial
DNA, cell membrane lipids and intracellular pro-
teins, culminating in cell death [5]. This type of
injury to middle ear mucosa may not only delay
healing but also predispose the ear to reinfection
and subsequent sequelae that include hearing loss
and speech delay.
In the management of AOM, in addition to anti-
biotics, several therapies have been recommended
including topical and systemic decongestants, cor-
ticosteroids, anti-inflammatory agents and muco-
lytics. In spite of all alternative therapies, AOM is
frequently persists or recurs in childhood. It has
been known for more than a century that vitamin A
deficiency is more common during infection than in
its absence [9]. Our hypothesis depends on the fact
that vitamin A is essential for immunity and main-
tenance for respiratory epithelium. In a recent
report by Cemek et al. [16], nonenzymatic antiox-
idants levels such as vitamin A, vitamin C and
vitamin E were determined lower in children with
AOM compared with healthy control subjects. Unal
et al. [17] have studied the therapeutic role of
vitamin A in acute sinusitis based on histopathologic
examinations. However, they could not be able to
find any significant difference between groups. In
our study, although there was no statistically sig-
nificant difference between groups for inflamma-
tion, epithelial integrity was found better in
vitamin A added group. We speculate that this point
is very important for healing process of the middle
ear infection.
Nitric oxide is present in the epithelium of the
healthy airways including middle ear mucosa
[18]. Contrary to reported protective effect of
NO, participations of NO and its metabolites in
the pathogenesis of AOM have also been reported
[2,15]. In a recent study it was declared that NO and

Table 2 Summary of the biochemical results

Group I (n = 15) Group II (n = 15) t p
NO 95.06  8.91 68.61  5.21 9.93 <0.001
MDA 7.15  1.54 5.71  0.78 0.25 0.004
SOD 13.88  1.68 17.72  1.09 7.44 <0.001
GSH 371.34  51.48 724.19  121.41 10.36 <0.001
Efficacy of vitamin A
FRs involves in every steps of AOM pathogenesis and
postulated that noxious effect of these substances
may be abolished by new therapeutic tools [19].
MDA, an end product of lipid peroxidation, is one of
the most widely used marker for free radical
mediated damage. Parks et al. [2] demonstrated
that mucosal MDA level was increased in experi-
mental AOM and FRs in mucosa of infected middle
ear might cause damage by lipid peroxidation.
Döner et al. [20] reported that MDA levels of serum
and infected maxillary sinus mucosa were signifi-
cantly higher than that of controls. Although, our
results are in accordance with literature, NO and
MDA levels in animals receiving both antibiotic and
vitamin A, were significantly lower than the animals
receiving only antibiotic.
FRs have short half-lives and are difficult to
isolate and study. However, indirect measurements
of FRs are available. These include assays of
enzymes that are involved in their biochemical
cycles, such as SOD and GSH-Px, as well as measure-
ments of stable products of FR damage. These
scavenger enzymes, control the effects of FRs as
a part of antioxidant defense system and they are
increase in blood and tissue, in order to save the
balance with FRs during oxidative stress. Several
studies reported increased antioxidant enzyme
activity during infections. Nevertheless in a recent
report, antioxidant enzyme activities were deter-
mined with a diminished level in patient group with
AOM compared with normal population [16]. In
current study, blood SOD and GSH-Px activities were
significantly increased in the group receiving both
antibiotic and vitamin A compared to the group
receiving only antibiotic. Also vitamin A is known
to be a part of nonenzymatic antioxidant defense
systems, it may play another role by increasing
antioxidant enzymes levels.
Although damaging effects of FRs may be demon-
strated by means of biochemical and histologic exam-
ination of the mucosa in animals, in the current study
we could not analyze the activities of SOD and GSH-Px
and the levels of MDA and NO in tissue specimens due
to the trivial amount of middle ear mucosa in rats.
This was the shortcoming of our study.
Whatever the cause is, the inflammation of the
middle ear increases the level of FRs. FR level is
normally maintained at a steady state by antioxi-
dant enzymes. When the antioxidant defense sys-
tem is weakened, the increased FRs may contribute
to otitis formation. Although antibiotics are main-
stays for treatment of otitis media, they do not
specifically treat tissue damage due to FRs. Our
results may explain the rapid improvement in
AOM in the group treated with both antibiotic and
vitamin A.
627
8. Conclusions
In our study, epithelial integrity as well as blood SOD
and GSH-Px activities was better in the group receiv-
ing both antibiotic and vitamin A than the group
receiving only antibiotics. However, blood NO and
MDA levels were decreased in the same group. We
suggest that, antioxidant, antiinfective, immuno-
modulator vitamin A may be added in the manage-
ment of AOM. In conclusion, further clinical studies
are needed to evaluate the therapeutic role of
vitamin A in AOM in humans.
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