Neuroscience & Medicine, 2012, 3, 287-293 Published Online September 2012 (
Growth Hormone Prevents the Memory Def ici t C aus ed by
Oxidative Stress in Early Neurodegenerative Stage in Rats
Diana Verónica Castillo-Padilla1, Gabino Borgornio-Pérez1, Alejandro Zentella-Dehesa2,3,
Adrian Sandoval-Montiel3, José Luis Ventura Gallegos3, Selva Rivas-Arancibia1
1Facultad de Medicina, Departamento de Fisiología, Universidad Nacional Autónoma de México, Ciudad Universitaria, México;
2Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, México; 3Unidad de
Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, México.
Received May 3rd, 2012; revised June 5th, 2012; accepted June 12th, 2012
Oxidative stress has been involved in neurodegenerative diseases. The growth hormone (GH) counteracts the levels of
reactive oxygen species. Previously, we showed that the prolonged exposure to ozone causes oxidative stress in the
hippocampus and memory deficits. In this work, we analyzed the effects of the growth hormone on the memory deficit
generated by ozone exposure, growth hormone effects on the Insulin-like growth factor I (IGF-I), and the serine-
threonine protein kinase (Akt) activation in the dentate gyrus. Our results show that GH prevents memory deficits in
early stages of the neurodegenerative process.
Keywords: Oxidative Stress; Growth Hormone; Insulin Growth Factor; Ozone, Serine-Threonine Protein Kinase;
Water Maze; Passive Avoidance
1. Introduction
Oxidative stress in cells is generated because of an im-
balance between the reactive oxygen species (ROS) and
antioxidants. It has been suggested that oxidative stress is
a mechanism for the pathogenesis of neurodegenerative
diseases. Previously, it has been demonstrated that ozone
inhalation generates oxidative damage through the for-
mation of the ROS [1,2]. Oxidative stress produced by
ozone inhalation causes damage in the central nervous
system [3-5], increases lipid peroxidation (LPO) levels in
brain structures [5,6], and causes memory deficits [5-7].
Oxidative stress and the decrease in the growth hor-
mone-insulin-like growth factor I (GH-IGF-I) axis are
well-characterized markers during aging and the cogni-
tive dysfunction that occurs with age [8,9]. It has been
reported that GH attenuates the increase in oxidative-
stress levels [9,10], and improves the cognitive function
deteriorated by aging [11]. The expression of GH recap-
tors has been described in the central nervous system in
rodents [12]. Furthermore, it has been demonstrated that
the mutation of the GH gene causes deficiencies in hip-
pocampus-dependent spatial memory in rats [13]. In ad-
dition, it has been reported that the GH-IGF-I axis is in-
volved in cognitive functions [12,14,15]. Moreover, it
has been demonstrated that IGF-I via Akt is a neuropro-
tective pathway in neurons [16,17]. In previous studies
we demonstrated that oxidative stress generated by ex-
posure to ozone causes memory deficit [5], and neuro-
genesis alterations in rats [6].
In this work we evaluated the effects of the growth-
hormone treatment on the memory deficit generated by
the oxidative stress, produced by low doses of ozone, in
the water maze and passive-avoidance tasks and on the
IGF-I-Akt signaling in the dentate gyrus.
2. Materials and Methods
2.1. Animals
Seventy-nine male Wistar rats weighing 250 to 300 g
were used for this experiment. They were housed indi-
vidually under a 12-h: 12-h light:dark cycle, with food
and water ad libitum. Adequate measures were taken to
minimize pain or discomfort, as outlined in the NIH
Guide for the Care and Use of Laboratory Animals.
Animals were distributed into the groups: 1) CTRL, con-
trol group exposed to air free of ozone during 30 days, 2)
GH groups, which received an injection (sc) of 0.8 mL of
recombinant human growth hormone (r-hGH) (Saizen)
daily, 3) O3 groups, exposed to ozone and received a
injection (sc) of 0.8 mL of saline daily, and 4) O3 + GH
groups, which received GH after ozone exposure.
Groups 2, 3, and 4 were divided into 3 subgroups for 7,
Copyright © 2012 SciRes. NM
Growth Hormone Prevents the Memory Deficit Caused by Oxidative Stress in Early Neurodegenerative Stage in Rats
15, and 30 days of each treatment. The GH was injected
sc at doses of 0.08 UI, alone or after each ozone exposure.
The groups exposed to ozone received 0.25 ppm for 4
hours daily. All groups were evaluated in a water maze
and passive-avoidance tasks. After the last training, three
animals in each group were randomly chosen, killed, and
the dentate gyrus dissected, and the corresponding sam-
ples used for immune-western-blot analyses for IGF-I
and phospho-Akt (a downstream product of phospho-
inositide-3 kinase (PI-3K)).
2.2. Ozone Exposure
Animals were put daily for 4 h into a chamber with a
diffuser connected to a variable flux ozone generator (5
L/s). The procedure has previously been described 4,5.
Ozone was generated from a tube through which a high-
voltage current circulated. Ozone production levels were
proportional to the current intensity and airflow. A PCI
Ozone & Control System Monitor was used to measure
the ozone concentration inside the chamber during the
experiment and to keep the ozone concentration constant.
2.3. Water Maze
Two types of tests were used, training trials and a reten-
tion trial at the end of the experiment. Rats were trained
to find a hidden 10-cm platform in a metallic, dark, and
circular pool (1.5-m diameter and 60-cm height) with
spatial clues surrounding it, and animals were allowed to
swim for 100 seconds to locate the platform in each trial.
Each rat had five trials (100 seconds each) per day, dur-
ing the last three days of each treatment. In the training
sessions all rats were released at different points of the
pool and were taken to a dry cage after finding the plat-
form. In the last trial, the platform was removed, the tank
was divided into four quadrants and the time spent by
rats in the correct quadrant was measured during 60 s.
The time spent by rats in the correct quadrant was used
as a direct measurement of memory retention.
2.4. Passive Avoidance
Two hours after the last treatment, all groups were
trained in a one-trial passive-avoidance conditioning and
tested for short-term and long-term memory (10 min and
24 h after training). Training was done in a conditioning
chamber with two compartments of the same size (30 ×
30 × 30 cm) separated by a guillotine-type door. The
floor of the safe compartment was a grid made of alumi-
num bars (0.5-cm diameter) separated 1.5-cm center to
center. The floor and the walls of the shocking compart-
ment were made of stainless steel. Each wall was con-
tinuous with half the floor, and each half floor was sepa-
rated by a 1-cm slot. The floor was connected to a con-
stant current unit (Mod, PSIU6; Grass) fed by a Grass
stimulator (Mod. S48, Grass technologies), which deliv-
ered 50 square pulses per second at 1.5-mA intensity and
5-ms duration for 5 s. The duration of the stimulus was
automatically monitored, and latencies were manually
measured with chronometers. During training (acquisi-
tion), each animal was placed in the safe compartment
for 10 s. Then, the sliding door was lifted, and the time
required for the animals to cross the threshold of the
shocking compartment was recorded (acquisition la-
tency). If an animal required more than 100 s to cross to
the other side, it was dropped from the experiment and
substituted with another rat exposed to the same condi-
tions. Once the animals crossed with all four paws into
next compartment, the door was closed and a 3-mA foot
shock was delivered for 5 s. Then, the door was opened,
and the time required for the animal to return to the safe
compartment was measured (escape latency). The animal
was again placed in the safety compartment for 10 s, the
door was opened, and the time that the animal remained
was recorded (retention latency). The test session ended
when the animal either entered the shock compartment or
remained in the safe compartment for 600 s.
2.5. Western Blot
The dentate gyrus was microdissected [18]. The test was
done in triplicate on three animals from each group. The
tissue was homogenized and centrifuged. The Bradford
method was used to quantify the protein, and 100 μg of
protein from each sample were used. The protein was
separated with SDS-polyacrylamide gel electrophoresis
(10%) (Sigma-Aldrich) and transferred to nitrocellulose
membranes (Sigma-Aldrich). The membranes containing
the samples of different rat groups were blocked with 5%
skimmed milk in TRIS buffer solution with 0.01%
Tween 20 (TBS-T) (Sigma-Aldrich) for 2 h at 37 ˚C and
incubated overnight individually with IGF-I (1:500 Ab-
cam), phospho-Akt (1:1000 Chemicon), total Akt
(1:1000, Chemicon), and actin (1:10,000) Santa Cruz
Antibodies) antibodies under gentle shaking at 4˚C
(BrinkmannOrbiMix 110, Brinkmann, Germany). The
membranes were rinsed three times with TBS-T, incu-
bated with goat anti-rabbit IgG (Vector, Burlingame, CA)
and anti-mouse conjugated to horseradish peroxidase
(1:10,000) (Biotechnology, Santa Cruz, CA) for 1 h, and
then rinsed three times with TBS-T. The recognized
bands were visualized by chemiluminescence (ECL;
General Electric, Santa Clara, CA). The band densities
were first normalized to the untreated control (100%).
The IGF-I was normalized to actin and the phospho-Akt
was then normalized to total Akt with the values ex-
pressed as a percentage. We did the densitometry using
the software off-line ImageJ (NIH, USA).
Copyright © 2012 SciRes. NM
Growth Hormone Prevents the Memory Deficit Caused by Oxidative Stress in Early Neurodegenerative Stage in Rats 289
2.6. Statistical Analysis
Training trials with the hidden platform in the water-
maze task were analyzed using a repeated-measures
ANOVA. To measure the time spent in the correct quad-
rant with the platform removed, a one-way ANOVA and
Fisher’s post-hoc comparison test were used. In the pas-
sive-avoidance memory task we used the nonparametric
Kruskal-Wallis test and Mann-Whitney U tests to com-
pare the medians in all groups. Western Blots were ana-
lyzed with a Student’s t-test.
3. Results
For the acquisition of spatial memory, the repeated-
measures ANOVA indicated no significant differences
between the groups in the latency to reach the hidden
platform during the training block for 3 days (each block
consisted of 5 trials) (Figure 1(a)). Although no signify-
cant differences were shown by an ANOVA, the ozone
groups showed an apparent longer time to acquire the
learning task during days 1 and 2, as seen in Figure 1(a).
No significant differences in spatial memory retention
(after removing the platform) were found between the
GH (at 7, 15, and 30 days) and the O3 + GH (at 7 and 15
days) groups compared with the control group. However,
the one-way ANOVA (F(9 - 69) = 6.55), P < 0.001, de-
tected significant differences between all groups and the
Fisher’s test comparison showed significant differences
between animals treated with ozone at 7, 15, and 30 days
(P < 0.01), when compared to the control group during
the water-maze retention test (Figure 1(b)).
As shown in Figure 1(b), for the long-term memory in
passive avoidance (24 minutes after training), the
Kruskal-Wallis test showed significant differences be-
tween all groups (P < 0.001). Furthermore, the latencies
in the retention avoidance in animals treated with ozone
plus saline at 7, 15, and 30 days were significantly
shorter than those of the control group (Mann-Whitney U
test (P < 0.05). The GH (at 7, 15, and 30 days) and O3 +
GH (at 7 and 15 days) groups showed no significant dif-
ferences compared to the control group (Figure 2). We
measured significant differences in the O3 + GH group at
30 days compared to the control group in both tasks (P <
0.05) (Figure 2).
The Western Blot analyses showed that GH is able to
raise the expression of IGF-I at 7, 15, and 30 days (P <
0.05). We also found that the O3 treatment is able to in-
crease IGF-I expression at 7 and 15 days of treatment (P
< 0.05). In the 30-day ozone group, the IGF-I expression
is decreased and showed significant differences with the
control group (P < 0.01), whereas in the O3 + GH groups
at 7, 15, and 30 days the expression of IGF-I showed no
significant differences compared to the control group
(Figure 3(a)).
Figure 1. The effect of growth hormone treatment on spa-
tial memory in rats exposed to ozone. (a) The average la-
tency times to reach the hidden platform for all groups;
control group, (CTRL, n = 10), GH = Growth hormone
groups: GH7 = 7 days (n = 8), GH15 = 15 days (n = 7), and
GH30 = 30 days (n = 8). O3 = Ozone treatment: O37 = 7
days (n = 7), O315 = 15 days (n = 7), and O330 = 30 days
(n=7), and O3GH = ozone plus growth hormone groups:
O3GH7 = 7 days (n = 8), O3GH15 = 15 days (n = 10), and
O3GH30 = 30 days (n = 7); (b) Time spent in the correct
quadrant after platform was removed (spatial memory re-
tention) in all groups. Data points are the mean values ± SE
in all groups. *P < 0.01.
The ozone treatment at 7 and 15 days had significant
differences compared to the control group in the phos-
phorylated Akt (P < 0.05). This activation decreases at
30 days of treatment with ozone (P < 0.01). The O3 + GH
groups at 7, 15, and 30 days showed significant differ-
ences with the control group and did show an increase in
Akt activation (P < 0.05). The GH groups showed no
significant differences compared to the control group in
the phosphorylation of Akt (Figure 3(b)).
4. Discussion
Our results showed that ozone exposure at 7, 15, and 30
Copyright © 2012 SciRes. NM
Growth Hormone Prevents the Memory Deficit Caused by Oxidative Stress in Early Neurodegenerative Stage in Rats
Figure 2. The effect of growth hormone treatment on the
passive-avoidance task in rats exposed to ozone. Long-term
memory retention latency, 24 hours after acquisition of
passive-avoidance task. Data points are median values (in
seconds) in all groups. (CTRL, n = 10), GH7 = 7 days (n = 8),
GH15 = 15 days (n = 7), GH30 = 30 days (n = 8), O37 = 7
days (n = 7), O315 = days (n = 7), O330 = 30 days (n = 7),
O3GH7 = 7 days (n = 8), O3GH15 = 15 days (n = 10), and
O3GH30 = 30 days (n = 7). *P < 0.05).
*P < 0.05, **P < 0.01.
Figure 3. The effects of growth hormone on IGF-expression
and Akt activation in rats exposed to ozone. Representative
western blots show normalized immunoreactivity for IGF-I
expression and Akt activation. (a) IGF-I expression in dif-
ferent treatments; (b) Akt phosphorylation in different
treatments. Bars are the mean ± SE in all groups (n = 3).
days generates memory—retention impairments in the
water maze and the passive-avoidance learning (Figures
1(b) and 2(b)). These data indicate that ozone is able to
cause long-term memory deficits. We found that treat-
ment with GH alone shows a tendency to facilitate the
execution of two tasks in 7, 15, and 30 days (Figures 1 (b)
and 2(b)) and activates the expression of IGF-I (Figure
3(a)). This is in agreement with previous studies showing
that GH participates in the improvement of the memory
function [6,19]. In addition, GH has been reported to
reduce oxidative stress in the central nervous system
[10,11]. Our results show that GH treatment at 7 and 15
days is able to prevent the long-term memory deficit
caused by oxidative stress generated by exposure to
ozone (Figures 1(b) and 2(b)). However, at 30 days of
exposure the GH injections do not prevent the long-term
memory deficits, demonstrating that ozone causes irre-
versible oxidative damage at 30 days. Our results suggest
that the GH has a neuroprotective effect on memory al-
terations caused by oxidative stress in an early stage of
the neurodegenerative process. Ramsey [9] reported that
GH treatment attenuated the alterations generated by age
in hippocampal plasticity and spatial memory in rats. We
had also previously reported that the hippocampus is a
region susceptible to oxidative stress caused by ozone
[5,7]. Moreover, the memory deficits caused by ozone
could be caused by morphologic damage in nerve tissue
[3,4] and by neurogenesis alterations in the hippocampus
Much evidence has shown that GH through IGF-I is
involved in structural and cognitive functions [12,14,15].
The GH is directly related to the mRNA IGF-I expres-
sion in the brain [12]. Furthermore, it has been demon-
strated that IGF-I has neuroprotective effects against
oxidative damage through the PI-3K-Akt pathway in
neurons [16,22,23].
In addition, the hippocampal function and adult neu-
rogenesis appears to be related to both the passive-
avoidance and water-maze memory retention, as has
been reported [20,21,24]. We found that GH is able to
increase the expression of IGF-I at 7, 15, and 30 days in
the dentate gyrus. Ozone inhalation itself caused in-
creases in the IGF-I expression and Akt phosphoryla-
tion at 7 and 15 days, probably to prevent cell death
caused by oxidative stress in dentate gyrus. Previous
studies that shown ROS are capable to activate IGF-I and
Akt [22,25,26]. In this sense, the IGF and Akt activation
might modulate the cell survival or death in an oxidative-
stress in a dependent manner [26-29]. Moreover, a pre-
vious study showed that ROS, growt factors, and PI3K-
Akt signaling has been involved as modulators in the
neurogenesis process [30]. In this sense IGF/Akt signal-
ing pathway might promote neurogenesis in early states
Copyright © 2012 SciRes. NM
Growth Hormone Prevents the Memory Deficit Caused by Oxidative Stress in Early Neurodegenerative Stage in Rats 291
of neurodegenerative process caused by ROS increases
produced by ozone chronic inhalation [5]. Previously a
study found in CaMKIIαh KO model that is high
neurogenesis levels and an increase in the expression of
genes involved in oxidative stress [31]. In addition,
CaMKIIαh KO animals show increased neurogenesis,
neuron immaturity and memory deficits [32], as we
shown in our results (in ozone exposure groups), in this
and a previous study [5]. These results suggest that ex-
acerbated oxidative stress could dysregulate the normal
neurogenesis process in rats and can cause memory
deficits. We also found that at 30 days of ozone exposure,
IGF-I expression and Akt activation are completely
inhibited (Figures 3(a) and 3(b)). This is in concordance
with a previous study that showed that the Akt-activity
status is dependent on oxidative-stress levels in neuronal
cells [17] and with the theory that IGF-I supression
induced by DNA damage might be a protective effect of
the cell against oxidative stress [33]. These results
suggest that IGF-I-Akt signaling pathway might parti-
cipate in cell survival during the early processes of
neurodegeneration in an oxidative stress state caused by
ozone exposure; however, after the chronic oxidative
stress, the cell trigger mechanisms to downregulate these
On the other hand, in the O3 + GH groups at 7 and 15
days, the IGF-I expression appears attenuated slightly,
but had no effects on ozone activated Akt (Figure 3(b)).
These results are in accordance with a previous study
where IGF-I expression is caused by oxidative stress, but
is decreased by antioxidant agents [34]. In addittion, GH
might regulate different signalling pathways [35]. More-
over, at 30 days of O3 + GH treatment IGF-I-Akt acti-
vation was maintained, but the GH treatment is unable to
prevent memory deficit, probably because at 30 days the
oxidative stress levels are high and there is a deregulation
in the oxidation-reduction balance and the GH activates
Akt independently of the IGF-I signaling pathway [36].
5. Conclusion
In summary, our results suggest that the GH may prevent
memory deficit caused by ozone in the adult brain only
in early stages of the neurodegenerative process. Also,
GH probably activates different signaling pathways de-
pending on the oxidation-reduction balance.
6. Acknowledgements
This study was supported by DGAPA-UNAM Postdoc-
toral scholarship (2009-2010) and DGAPA IN215408D
and IN219511-3 to S.R-A. Thanks to E. Jalpa-Hernandez
for her review of this manuscript and Dr. Ellis Glazier for
editing this English-language text.
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