Advances in Bioscience and Biotechnology, 2013, 4, 24-29 ABB
http://dx.doi.org/10.4236/abb.2013.411A2004 Published Online November 2013 (http://www.scirp.org/journal/abb/)
Chronic exposure to low doses of ozone produces a state of
oxidative stress and blood-brain barrier damage in the
hippocampus of rat
Selva Rivas-Arancibia1*, Luis Fernando Hernández-Zimbrón1, Erika Rodríguez-Martínez1,
Gabino Borgonio-Pérez1, Varsha Velumani1, Josefina Durán-Bedolla2
1Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México D. F., México
2Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, México
Received 26 September 2013; revised 25 October 2013; accepted 3 November 2013
Copyright © 2013 Selva Rivas-Arancibia et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Chronic exposure to low doses of ozone similar to a
day of high pollution in Mexico City causes a state of
oxidative stress. This produces a progressive neu-
rodegeneration in hippocampus of rats exposed to the
gas. The aim of this study was to analyze the effect of
chronic exposure on the changes in the blood-brain
barrier in rats exposed to low doses of ozone. Method:
each group received one of the following treatments,
control group received air without ozone, and groups
2, 3, 4, 5, and 6 received ozone doses of 0.25 ppm for 4
h daily during 7, 15, 30, 60 and 90 days respectively.
Each group was processed to inmunohistochemical
technique against of the following antibody: blood-
brain barrier, guanylyl cyclase, Iba-1, GFAP, NFκ-B,
TNF-α. The results show that there is a correlation
between the time exposure of ozone and the progres-
sive damage, on the blood-brain barrier rupture, fi-
nally causing edema of endothelial cell, increase in
guanylyl cyclase type 1, thickening of the processes
and astrocytes foot, and an increase in the expression
of factors NFκ-B and TNF-α at 30 and 60 days of ex-
posure to this gas. All the above indicates that the
chronic state of oxidative stress causes a neurodegen-
eration process, accompanied by disruption of the
blood-brain barrier likely to occur in the Alzheimer’s
disease.
Keywords: Ozone; Oxidative Stress; Blood-Brain
Barrier; Neurodegenerative Diseases
1. INTRODUCTION
The environmental contamination is a major health prob-
lem in the densely populated and industrialized cities
[1,2]. The ozone, which is a product of the photochemi-
cal air pollution, is the principal contaminant [3]. High
levels of air contamination have been associated with an
increase and deteriorating of chronic degenerative respi-
ratory illness and also cardiopulmonary and neurodegen-
erative disease [4-6].
It is well established that an acute as well as chronic
inhalation of this gas produces a state of oxidative stress.
When a redox imbalance occurs by an acute exposition
to high doses of ozone (0.8 - 1.0 ppm) for 4 h, it causes cell
damage, accompanied with swelling, inflammation and
changes in mitochondrial as in the endoplasmic reticu-
lum occurs [6,7]. All these changes are reversible, since
the increase in reactive oxygen species (ROS) in the or-
ganisms induces the stimulation of antioxidants systems
which increases their activities [7]. In addition these sys-
tems also have the reparation function that permits to
revert the pathological changes that have been found.
The changes in the redox balance are also produced dur-
ing the respiratory burst by the activation of immune
system as a response of an acute defense of the organism
against the pathogens [8]. Once the infection has been
handed over the balance of the oxide reduction will be
recovered. However, the chronic oxidative stress state is
present in degenerative diseases; this state is character-
ized by a chronic increase of reactive oxygen species
(ROS), reactive nitrogen species (RNS), and reactive
species of transition metals [9]. In this state, the antioxi-
dant systems are unable to revert the oxidative damage
and as they are progressing it also alters the intracellular
signaling. As well as other physiological regulatory sys-
tems such as the loss of regulation of the inflammatory
response, this contributes to increase even more the oxi-
dative stress state. Also there is a progressive mitochon-
*Corresponding author.

S. Rivas-Arancibia et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-29 25
drial damage and alteration which produces a deficit of
ATP [7]. Oxidative stress causes cell damage which will
not permit the cells to maintain homeostasis together
with the alteration in the intracellular signaling. It also
activates phosphorylation pathway and inhibits the de-
phosphorylating pathway. Altogether, these changes fi-
nally lead to neuronal death. Some characteristics of the
chronic neurodegenerative disease are the loss of the re-
gulation of inflammatory response, progression of patho-
physiological complications which takes it to a loss in
the cognitive functions, progressive neuronal death and
an incapability of cerebral reparation mechanisms (since
the process of neurogenesis is blocked by the oxidative
stress) [6] which causes the neurodegeneration process to
progress.
Prolonged exposure to ozone creates a chronic oxida-
tive stress state; in our laboratory we have developed a
progressive neurodegeneration animal model. When the
animals are exposed chronically to low doses of ozone
similar to those of a high contaminated day in Mexico
City (0, 25 ppm for 4 h a day during 7, 15, 30, 60 and 90
days), with an increase of ozone exposure days, we ob-
served a rise in oxidized lipids and proteins, the expres-
sion of Cu/Zn superoxide dismutase increases, however
its activity reduces. It also has been found that the glu-
tathione system is altered (article in preparation); with
this model, we show that a chronic disease caused by an
oxidative stress state after 30 days of ozone exposure
becomes to be irreversible. All these changes go along
with the loss of the regulation of inflammatory response.
During the progressive neurodegeneration, besides the
neuronal changes, it also produces astrocytes alteration and
activated microglia [6]. In this model, we demonstrated
that in healthy animals with no other factor added; only
oxidative stress, caused by low doses of ozone is capable
to trigger a process of progressive neurodegeneration in
the hippocampus of rats. Furthermore, this process be-
comes irreversible after 30 days of exposure and neu-
rodegeneration continues to progress in relation to time.
In addition, the brain antioxidant systems are at a dis-
advantage in comparison with other organs such as the
liver, pancreas and lungs. Brain has very low levels of
catalase, moderated levels of superoxide dismutase de-
spite its high content of phospholipids and high con-
sumption of oxygen. However, the brain has a strict
regulation of the compounds that may be in contact with
neurons through blood-brain barrier (BBB) [10]. The
selectivity of the BBB is vital to brain tissue, since it
prevents toxic substances from getting easily in contact
with the nerve cells [11]. This is because the BBB is
formed by endothelial cells, pericytes and astrocytes that
make it impermeable to water and permeable to very
small lipid molecules, due to the above, the cells that
form the barrier have specific proteins which allow the
passage of small molecules like oxygen, glucose and
micronutrients through it [11,12].
BBB properties change according to the needs of the
brain and exist in a close relationship between neurons,
microglia, astrocytes and BBB for which it is considered
that they form a neurovascular unit which also includes
immune system cells [10]. The barrier has the ability to
respond to changes, both inside and outside the CNS, it
is also capable of secreting or inducing the secretion of
substances, in some cases such as in Alzheimer’s disease
it has been found an elevated activity of nitric oxide
synthase [13]. Our aim is to study the effect of chronic
oxidative stress on the BBB in the hippocampus of rats
exposed to low doses of ozone.
2. MATERIALS AND METHODS
2.1. Materials
Monoclonal mouse antiglial fibrillar acidic protein (GFAP)
and polyclonal mouse anti-ionized calcium-binding ada-
pter molecule 1 (Iba-1) antibodies were obtained from
Biocare, polyclonal mouse anti-guanylyl cyclase and
anti-BBB antibodies were obtained from Abcam. Mouse
polyclonal anti-nuclear factor kappa-B (NFκ-B) was
obtained from Santa Cruz Biotechnology, CA, USA.
2.2. Animals
Animal care and handling were done according to the
Norma Official Mexicana NOM-036-SSA2-2002, the
National Institutes of Health guidelines for Animal Treat-
ment and the Ethics Committee of the Facultad of Medi-
cina at the Universidad Nacional Autónoma de México.
Thirty six male Wistar rats weighing 250 - 300 g were
individually housed in acrylic boxes with free access to
water and food (Purina, Minnetonka, MN) and kept in a
clear air room.
They were randomly divided into six experimental
groups: group 1) exposed to an air stream free of ozone
during 30 days; group 2) exposed for 7 days to ozone;
group 3) exposed for 15 days to ozone; group 4) exposed
for 30 days to ozone; group 5) exposed for 60 days to
ozone and group 6) exposed for 90 days to ozone. Ozone
exposure was done daily for 4 h with a dose of 0.25 ppm.
Immediately after the exposure to ozone, animals were
returned to their home cages.
2.3. Ozone Exposure
Animals were put daily, for 4 h, inside a chamber with a
diffuser connected to a variable-flux ozone generator (5
l/s). The procedure used has been described elsewhere
(Rivas-Arancibia et al., 2010). The air feeding the ozone
converter was filtered purified air. Ozone production
levels were proportional to the current intensity and to
the airflow. A PCI Ozone & Control System Monitor

S. Rivas-Arancibia et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-29 26
(PCI Ozone & Control Systems, West Caldwell, NJ) was
used to measure the ozone concentration inside the
chamber during the experiment and to keep the ozone
concentration constant.
Air exposure: The same chamber was used for treating
the control group where a flow of ozone-free purified air
was used.
2.4. Immunohistochemistry Techniques
After the completions of the treatments, animals from
each group were anesthetized with sodium pentobarbital
(50 mg/kg ip) and intracardially perfused with 4% para-
formaldehyde (Sigma-Aldrich, St Louis, MO). In 0.1M
phosphate buffer (PB) pH 7.4 (Tecsiquim, México), the
brains were postfixed with 10% formaldehyde for 24 h
and embedded in paraffin (Merck, Darmstadt, Germany).
Sagittal brain slices containing the hippocampus of these
animals were cut at 5 μm on a microtome, mounted on
slides, and processed for immunohistochemistry. The
immunohistochemistry for GFAP, NFκ-B, Iba-1, Guany-
lyl cyclase, tumor necrosis factor α (TNF-α) and BBB
was done as follows: Slices of each brain containing the
hippocampus had the paraffin removed, treated with
heat-retrieval solution (Biocare Medical, Concord, CA),
and put into an electric pressure cooker (Decloaking
Chamber; Biocare Medical) for 5 min. After being washed
with distilled water and treated with hydrogen peroxide
(J.T. Baker) (diluted 1:5) for 5 min, the slices were rinsed
again and treated with a blocking reagent (Background
Sniper; Biocare Medical) for 10 min. They were washed
with PBS (Merck), pH 7.4, and incubated overnight at 4
C with anti-GFAP (purified monoclonal mouse antibody,
diluted 1:200; Biocare), anti-NFκ-B (purified polyclonal
mouse antibody, diluted 1:200), anti-Iba-1 (purified poly-
clonal goat antibody, diluted 1:300), anti-guanylyl cy-
clase (purified polyclonal mouse antibody, diluted 1:200),
anti-TNF-α or anti-BBB (purified monoclonal mouse
antibody, diluted 1:200).
Sections were rinsed with PBS and treated with a sec-
ondary antibody using Trekkie Universal Link (Starr
Trek Universal HRP Detection; Biocare Medical) for 1 h.
The sections were later washed with PBS and then
treated with Trekavidin-HRP Label (Starr Trek Universal
HRP Detection; Biocare Medical) for 30 min or the
bound antibody was visualized using 3, 3-diaminoben-
zidine (DAB Substrate Kit; ScyTek, Logan, UT) as the
chromogen. The slices were washed with distilled water
and contrasted with hematoxylin buffer solution (ScyTek,
Concord, CA.). For double immunofluorescence, fol-
lowing primary antibody incubation the sections were
washed with TBS and incubated with the appropriate
Alexa, 488, or 594-conjugated secondary antibody (Invi-
trogen).
Representative brain slices from each group were
processed in parallel. After cover slipping with Entellan
(F/550 ml; Merck), the slices were examined with an
Olympus BX41 Microscope (Olympus, Japan) using a
100× magnification and photographed (Evolution VF-F-
CLR-12, Media Cybernetics camera; Bethesda, MD).
3. RESULTS
3.1. Double Fluorescent Immunohistochemistry
against Blood-Brain Barrier and
Guanylyl Cyclase
The results show that the blood-brain barrier damage
increases with the time of exposure to ozone as shown in
Figure 1. We can also observe alterations in the endothe-
lial cells that are expressing guanylyl cyclase. There is an
increase in the expression of this enzyme after sixty days
of exposure as well as an increase in endothelial cells
volume and at 90 days of exposure to ozone, BBB and
endothelial cells are completely destroyed.
3.2. Double Fluorescent Immunohistochemistry
against Astrocytes and Microglia
We found an increase in the immunoreactivity of Iba-1
and GFAP protein. The results indicate the activation of
both microglia and astrocytes with respect to exposure
time.
3.3. Immunohistochemistry against astrocytes,
NFκ-B and TNF-α
The results show an increase in the thickness of the
Figure 1. Effects of ozone treatment on Blood Brain Barrier
(green) and Guanylyl cyclase (red) double immunofluorescence
in the dentate gyrus of rats. Photomicrographs show not mor-
phological alterations of endothelial cells and blood brain bar-
rier (arrows) in control animals (A) and show s morphological
alterations of endothelial cells and blood brain barrier (arrows)
getting different times of ozone administration in the dentate
gyrus of rats treated with air only (A), 7 days of ozone expo-
sure (B),15 days of ozone exposure (C), 30 days of ozone ex-
posure (D), 60 days of ozone exposure (E), and 90 days of
ozone exposure (F). Magnification 100×, n = 6 per group.

S. Rivas-Arancibia et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-29 27
3(A.B)-(F.B), 3(A.C)-(E.C)) accompanied by thickening
of endothelium wall, product of astrocytes stimulation.
The results retrieved (Figures 3(A.B)-(F.B), and 3(A.C)-
(E.C)) also indicate that these changes are accompanied
by alteration of the inflammatory response, in these re-
sults we can observe changes in the expression of factor
NFκ-B and also the expression of TNF-α. All the above
indicates that in the process of progressive neurodegen-
eration caused by exposure to low doses of ozone within
neuronal death, glial activation presents a loss of selec-
tive permeability of the BBB, this fact is of vital impor-
tance when we consider that BBB has the function of
protecting the brain tissue and for this purpose its opera-
tion is very complex since it must maintain the normal
homeostasis of the central nervous system through selec-
tive transport metabolism of substances present in blood
and brain and moreover has numerous mitochondria and
efficient enzymatic system [12]. Several pathologies are
associated with an increase in the permeability of the
BBB (ischemia and osmotic shock, infections and in-
flammatory processes that cause neurodegenerative dis-
eases) [11,14]. Alterations in the permeability of the
BBB is correlated with an increase of cytokines in blood
and cerebrospinal fluid, with a raised TNF-α TNwhich
also induces the expression of IL-Ib Et-L6 which like-
wise contribute to increase the permeability of the barrier.
The experimental results of this work show that coupled
with the rupture of BBB Figure 1 also increases the ex-
pression of NFκ-B and TNF-α in endothelial cells.
astrocytes processes as well as an increase in the thick-
ness and of the astrocytes foot length, which tend to wrap
around the damaged vascular endothelium as time passes
to ozone exposure. We can observe an increase in the
NFκ-B expression in the nucleus of endothelial cells
from 15 days on of exposition of ozone until 90 days of
exposure to this gas. The results show an increase in
TNF-α expression of endothelial cells from 7 days on of
exposure to ozone and also in capillary endothelium
from 15 days on of treatment, which shows an increase at
15 and 30 days of exposure.
4. DISCUSSION
Previous studies conducted in our laboratory showed that
chronic exposure to low doses of ozone in healthy ani-
mals without other factor added causes a chronic state of
oxidative stress [6] which produces a progressive neu-
rodegeneration process in the hippocampus of rats ex-
posed to this gas. This process is characterized by a neu-
ronal cell damage which provoke cellular death accom-
panied by lack of microglia, astrocytes and mitochon-
drial activation [6,7], loss of the regulation of inflamma-
tory response and increased oxidative stress that causes
to form a vicious cycle which maintains the neurodegen-
erative process.
We can establish that all the changes above mentioned
are accompanied by an alteration of the BBB as shown in
Figures 1(B)-(F) where we can observe that the small
capillaries found in the hippocampal dentate gyrus ex-
hibit continuity loss, edema of the endothelial cell and
alterations in vascular endothelium itself, accompanied by
glial and astrocytosis activation (Figures 2(B) and (C)).
Further studies are needed to enhance and complement
these finding, since it is very important to understand the
involvement of oxidative stress in the blood-brain barrier
disruption, and how these factors are participating in the
development and progression of neurodegenerative dis-
eases.
We can also observe that exposure to low doses of
ozone induces thickening of astrocytes foot and proc-
esses that targets to capillary endothelial cells (Figures
3(A.A)-(F.A)). Therefore we have a rupture of the BBB,
endothelial changes, edema of the endothelial cell and
an increase in nuclear volume (Figures 1(B)-(F) and
5. CONCLUSION
Exposure to low levels of ozone causes a chronic state of
Figure 2. Effects of ozone treatment on GFAP (green) and Iba-1 (red) double immunofluo-
rescence in the hippocampus. Photomicrographs show immunoreactivity in dentate gyrus of
rats treated with air only, arrows indicates an astrocyte surrounding a normal blood vessel (A).
30 days of ozone exposure, morphological alterations of astrocytes surrounding an abnormal
blood vessel (arrows) (B), 60 days of ozone exposure, observe GFAP (right arrow) and Iba-1
(left arrow) hyperactivity and in 60 days treatment in the dentate gyrus of this animals (C).
Magnification 100×, n = 6 per group.

S. Rivas-Arancibia et al. / Advances in Bioscience and Biotechnology 4 (2013) 24-29 28
TNF-α NF
κ
-B GFAP
Figure 3. Effects of ozone treatment on GFAP, NFκ-B and TNF-α immunoreactivity in the rat hip-
pocampus. Light photomicrographs show GFAP immunoreactivity in the dentate gyrus of rats
treated with air only (A.A), 7 days of ozone exposure (B.A), 15 days of ozone exposure (C.A), 30
days of ozone exposure (D.A), 60 days of ozone exposure (E.A) and 90 days of exposure (F.A).
Observe the increased cell immunoreactivity of GFAP in astrocytes surrounding blood vessels in
dentate gyrus starting at 15 days of ozone treatments and ozone long exposures (arrows) (B.A-F.A).
Observe NFκ-B normal expression in endothelial cells (red arrows) on tissue control (A.B.) and in-
creased expression in endothelial cells of 7 days of ozone exposure (B.B,), 15 days of ozone expo-
sure (C.B), 30 days of ozone exposure (D.B), 60 days of ozone exposure (E.B) and 90 days of ex-
posure (F.B) (red arrows). In addition, observe some edematized endothelial cells (C.B, D.B, and
F.B.). TNF-α expression in endothelial cells without ozone treatment (A.C) and increased expres-
sion in endothelial cells of 7 days of ozone exposure (B.C), 15 days of ozone exposure (C.C), 30
days of ozone exposure (D.C), 60 days of ozone exposure (E.C) and 90 days of exposure (F.C).
Also observe some edematized endothelial cells (Arrows) in B.C, E.C. and E.C. Magnification
100×, n = 6 per group.
oxidative stress which produces rupture of BBB with
thickened astrocytes foot accompanied by edema of en-
dothelial cells and an increase in the expression of in-
flammatory cytokines by the endothelium. All the above
forms part of a progressive neurodegeneration process
which forms a vicious circle that leads to a loss of con-
trol of cerebral repair processes and a slow increase in
cell death.
6. ACKNOWLEDGEMENTS
This work was supported by the Dirección General de Apoyo al Per-
sonal Académico [Grant numbers: IN219511 to S. R-A].
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