AUTHOR QUERY SHEET

Author(s):

Mara Garca-Fernandez, Estela Castilla-Ortega, Carmen Pedraza, Eduardo Blanco,

Isaac Hurtado-Guerrero, Miguel Angel Barbancho, Jerold Chun, Fernando Rodrguez-de-Fonseca,
and Luis Javier Santn Nu nez

Guillermo Estivill-Torrus,

Article title:

Chronic Immobilization in the malpar1 Knockout Mice Increases Oxidative Stress in the
Hippocampus
693998
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Given name(s)

Surname

Mara

Garca-Fernandez

Estela

Castilla-Ortega

Carmen

Pedraza

Eduardo

Blanco

Isaac

Hurtado-Guerrero

Miguel Angel

Barbancho

Jerold

Chun

Fernando

Rodrguez-de-Fonseca

Guillermo

Estivill-Torrus

10

Luis Javier Santn

Nu nez

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International Journal of Neuroscience, 00, 17, 2012

Copyright 2012 Informa Healthcare USA, Inc.
ISSN: 0020-7454 print / 1543-5245 online
DOI: 10.3109/00207454.2012.693998

Chronic Immobilization in the malpar1 Knockout Mice

Increases Oxidative Stress in the Hippocampus

Mara Garca-Fernandez,1 Estela Castilla-Ortega,2 Carmen Pedraza,2 Eduardo Blanco,2

Isaac Hurtado-Guerrero,3 Miguel Angel Barbancho,1 Jerold Chun,4
3 and Luis Javier Santn Nu nez
2,
Fernando Rodrguez-de-Fonseca,3 Guillermo Estivill-Torrus,
1

Departamento de Fisiologa y Medicina Deportiva, Universidad de Malaga, Malaga, Spain

Departamento de Psicobiologa y Metodologa de las CC, Universidad de Malaga, Malaga, Spain

Unidad de Investigacion, Fundacion IMABIS, Hospital Regional Universitario Carlos Haya, Malaga, Spain

3
4

10

15

20

25

Department of Molecular Biology, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, Californea, USA
A B STRA CT
The lysophosphatidic acid LPA1 receptor has recently been involved in the adaptation of the hippocampus to
chronic stress. The absence of LPA1 receptor aggravates the chronic stress-induced impairment of both hippocampal neurogenesis and apoptosis that were accompanied with hippocampus-dependent memory deficits.
Apoptotic death and neurogenesis in the hippocampus are regulated by oxidative stress. In the present work,
we studied the involvement of LPA1 receptor signaling pathway in the regulation of the hippocampal redox after
chronic stress. To this end, we used malpar1 knockout (KO) and wild-type mice assigned to either chronic stress
(21 days of restraint, 3 h/day) or control conditions. Lipid peroxidation, the activity of the antioxidant enzymes
catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), as well as mitochondrial function
stimulation, monitored through the activity of cytochrome c oxidase (COX), were studied in the hippocampus.
Our results showed that chronic immobilization stress enhanced lipid peroxidation as well as the activity of the
antioxidant enzymes studied (CAT, SOD, and GPX). This effect was only observed in absence of LPA1 receptor.
Furthermore, only malpar1 KO mice submitted to chronic stress exhibited a severe downregulation of the COX
activity, suggesting the presence of mitochondrial damage. Altogether, these results suggest that malpar1 KO
mice display enhanced oxidative stress in the hippocampus after chronic stress. This may be involved in the
hippocampal abnormalities observed in this genotype after chronic immobilization, including memory, neurogenesis, and apoptosis.
KEYWORDS: LPA receptors, lysophosphatidic acid, predictable chronic stress

Received 1 March 2012

This work was supported by grants from Spanish Ministries of Health and of
Science (MEC SEJ2007-61187, cofunded by ERDF-, MICINN

PSI2010-16160, to Luis Javier Santn Nu nez;

PI07/0629 and PI10/02514,
Red de Trastornos Adictivos
cofunded by ERDF, to Guillermo Estivill-Torrus;
RD06/001/0000, to Fernando Rodrguez-de-Fonseca), Andalusian Ministry of

Innovation, Science and Enterprise (SEJ-4515, to Luis Javier Santn Nu nez;

and SAF2010-20521, to Fernando
CTS643, to Guillermo Estivill-Torrus;
Rodrguez-de-Fonseca), PCI-A/023328/09 (MAEC-AECID), Pre-competitive
Research Project of the University of Malaga, and first and second University
International Cooperation for Development Project of the University of
Malaga (to Eduardo Blanco). Estela Castilla-Ortega is supported by a FPU
Grant of the Spanish Ministry of Education (AP-2006-02582) and University
of Malaga (Ayuda para la actividad productiva del PIF, III Plan Propio).
Eduardo Blanco is a recipient of postdoctoral fellowship (Juan de la Cierva,
2008) from the Spanish Ministry of Education and Science.

Address correspondence to Luis Javier Santn Nu nez,

Ph.D., Departamento
de Psicobiologa y Metodologa de las CC. Facultad de Psicologa. Universidad
de Malaga. Campus de Teatinos, S/N., 29071 Malaga, Spain. E-mail:
luis@uma.es

INTRODUCTION
Lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3phosphate) is a simple, bioactive phospholipid involved
in several biological activities that can act as an extracellular signal through, at least, 6 G-protein coupled receptors (LPA16 ) [13]. The LPA1 receptor has been linked
to the proliferation, migration, survival, and apoptosis
of the neural progenitor cells during brain development
[48]. This receptor is expressed in hippocampal progenitors and it is involved in their differentiation [9, 10].
Functionally, LPA1 receptor has been demonstrated
to be an important modulator for the normal adult
hippocampal neurogenesis and hippocampal-dependent
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function [1114]. Mice lacking the LPA1 receptor show

defective proliferation and differentiation of the newly
born neurons, in addition to a blunted increase of cell
proliferation and survival in response to environmental enrichment and voluntary exercise [11]. Recently,
we have reported that the absence of LPA1 receptor aggravates the damage induced by chronic stress on hippocampal neurogenesis. In this regard, knockout (KO)
mice for the LPA1 receptor (malpar1 KO mice) showed
reduced cell proliferation together with increased apoptosis in the granular zone of the dentate gyrus after
21 days of restraint (3 h/day). In contrast, chronic restraint in the wild-type (WT) mice was not able to affect significantly these parameters of neurogenesis in the
hippocampus [13]. Concomitantly with the altered hippocampal neurogenesis, severe cognitive deficits in spatial long-term memory were also reported in the stressed
KO mice [13]. Accumulating evidence has implied that
oxidative stress plays a critical role in the detrimental effects of the chronic stress on hippocampal neurogenesis
and apoptosis [1517], having a crucial role in the development of the cognitive deficits usually observed under
chronic stress conditions [16]. The present study was
conducted to study the LPA1 receptor signaling pathway as a modulator of the hippocampal redox in chronic
stress situations. To address this issue, we used malpar1
KO mice and their WT littermates submitted to either
chronic restraint or control conditions. Under these experimental conditions, hippocampus was examined for
various parameters of oxidative stress, namely, lipid hydroperoxides (LOOHs), and the activities of the antioxidant enzymes catalase (CAT) [18], superoxide dismutase (SOD), and glutathione peroxidase (GPX). In addition, activity of cytochrome c oxidase (COX) was also
measured for evaluating mitochondrial function.

METHODS
Animals and Experimental Groups

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The generation and genotyping of malpar1 KO mice

have been previously described [4]. Experiments were
conducted on age-matched male littermates from the
WT and KO genotypes that were approximately 3months old. Mice were single housed on a 12-h
light/dark cycle (lights on at 07:00 h) with water
and food provided ad libitum. Procedures were carried out in accordance with the European animal research laws (European Communities Council Directives
86/609/EU, 98/81/CEE, 2003/65/EC, and Commission
Recommendation 2007/526/EC) and the Spanish National Guidelines for Animal Experimentation and the
Use of Genetically Modified Organisms (Real Decreto
1205/2005 and 178/2004; Ley 32/2007 and 9/2003). All

animal procedures were approved by the Institutional

Animal Care and Use Committee of Malaga University
(approval ID: PSI2010-16160).
A total of 12 animals of both genotypes were randomly submitted either to control or chronic stress treatment (control-WT (n = 6); chronic stress-WT (n =
6); control-KO (n = 6); chronic stress-KO (n = 6)).
Mice from the chronically stressed groups endured 3 h
of daily restraint for 21 consecutive days in a modified 50 ml clear polystyrene conical centrifuge tube with
multiple air holes for ventilation, starting at 10:00 a.m.
[13]. Control mice remained undisturbed in their home
cages.

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Preparation of Hippocampal Homogenates

Twenty-four hours after the last immobilization session
or an equivalent time in the control groups, animals were
sacrificed by decapitation and the hippocampus was dissected out on ice-cooled iron racks and immediately
stored at 80 C until analysis. Hippocampus (a pool of
three animals) were homogenized in digitonin-Tris-HCl
buffer (0.1 mg/ml digitonin, 20 mmol/liter (pH 7.4);
1 g tissue/15 ml) at 0 C and centrifuged at 2,500 g for
30 min at 4 C. All measurements were performed in the
supernatant.

Measurement of Hippocampal Lipid

Peroxidation
LOOHs were evaluated by the FOX2 (ferrous oxidation) automated by Arab and Steghens [19] and adapted
to Cobas Mira (wavelength 600 nm) for studying lipid
peroxidation in serum samples [20, 21]. Xylenol orange (180 l167 M), the first reagent, was added
after the sample (25 l). The first optical reading was
recorded before the addition of 45 l of 833 M iron
II D-gluconate. LOOH was calculated using a standard
curve of tert-butylhydroperoxide and LOOH levels were
expressed in mol/mg of protein. Intra- and interassay
variation coefficients were 3% and 8%, respectively.

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Determination of Antioxidant Enzymes and

COX Activities
CAT (E.C.1.11.1.6) activity was determined by incubating samples for 1 min at 37 C in 66 mM phosphate buffer solution, pH 7.4, and 65 M H2 O2 [18,
22]. The reaction was stopped with 32.4 mM ammonium molybdate and the molybdateH2 O2 complex
was measured at 405 nm. One unit of CAT activity represented the decomposition of 1 mol H2 O2 in
1 min at 37 C. SOD (E.C.1.15.1.1) activity was determined at 37 C using the commercial kit Ransod (Randox, UK). CuZn-SOD activity was differentiated from
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Mn-SOD on the basis of its sensitivity to 3 mM sodium

cyanide. Its activity was expressed in units (1U equals
1 mol substrate turnover per minute) per milligram of
protein [20, 21]. GPX (E.C.1.11.1.9) activity was determined at 37 C using the commercial kit Ransel
(Randox, UK). One activity unit was defined as the
oxidation of 1 mol NADPH to NADP/min at 37 C.
COX activity was assessed using the Cytochrome c Oxidase Assay Kit (Sigma-Aldrich, USA) that uses an optimized colorimetric assay based on observation of the
decrease in absorbance of ferrocytochrome c measured
at 550 nm, which is caused by its oxidation to ferricytochrome c by COX. Unit definition: one unit was defined as the oxidation of 1.0 mol of ferrocytochrome c
per minute at pH 7.0 at 37 C. Protein content was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, USA), based on the method of Bradford and
using bovine serum albumin as standard.

Statistical Analysis
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All data were analyzed by two-way ANOVA test (factors:

genotype (WT vs. KO) and treatment (chronic stress vs.
control)) following by post-hoc comparisons when necessary (Fishers least significant difference (LSD)). p values less than .05 were considered statistically significant.
All data are expressed as mean +/ SEM. The statistical package STATISTICA 7.0 (StatSoft, Inc., USA) was
used for statistical analyses.

RESULTS

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In Figure 1(a), it is showed how lipid peroxidation

level in the hippocampus, estimated from LOOH
oxidative mediators fraction, was markedly increased
after chronic stress only in the malpar1 KO animals
(genotype treatment: F1,20 = 39.15; p < .001; LSD,
p < .05). The analysis of the hippocampal activity of
the antioxidant enzymes showed that the GPX activity
(Figure 1(b)) was enhanced in the malpar1 KO mice
in basal conditions (genotype: F1,20 = 27.75; p < .001)
and after chronic stress in both genotypes (treatment:
F1,20 = 17.26; p < .001). The CAT activity in the hippocampus (Figure 1(c)) was augmented in the malpar1
KO mice but only when exposed to chronic stress
(genotype treatment: F1,20 = 3.88; p < .05 LSD,
p < .05). Similarly, SOD activity in the hippocampus
(Figure 1(d)) was also increased in the malpar1 KO
mice only when they were exposed to chronic stress
(genotype treatment: F1,20 = 23.01; p < .001; LSD, p
< .05). Figure 1(e) shows that the hippocampal activity
of COX was significantly lower in the malpar1 KO mice

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only after chronic stress (genotype treatment: F1,20 =

13.54; p < .001; LSD, p < .05).

DISCUSSION
Oxidative stress is the deleterious effect of the imbalance between free radical production and the available
antioxidant defence capacity. Indeed, when free radicals are not adequately removed or neutralized by antioxidants, they react with lipids, proteins, and nucleic
acids, damaging the cellular functions and causing cell
death [2325]. The brain is particularly susceptible to
oxidative stress due to its high oxygen consumption rate,
high abundance of both redox-capable transition metal
ions and unsaturated lipids, and low availability of antioxidant enzymes compared with other organs [26]. We
have studied the induction of oxidative stress in the hippocampus of WT and malpar1 KO mice after chronic
immobilization and compare it with control conditions.
Our results showed that the chronic stress protocol used
here is not able to induce an overall oxidative stress enhancement in the hippocampus of WT mice, as 21 days
of restraint treatment did not increase the production of
free radicals and/or reduce the antioxidant defence capacity. These results might depend on the habituation
of the WT mice to the chronic stress procedure because
repeated exposure to the same stressor leads to a process of adaptation to the aversive stimulus. The chronic
immobilization protocol used in our study is highly predictable and of mild or moderate intensity compared
to other protocols [27]. According to previous studies, both features of the protocol may facilitate habituation and adaptation of our WT mice to chronic stress
[28, 29]. In this way, some studies have demonstrated
the capacity of the animals to develop adaptive mechanisms to predictable stressors when they are presented
chronically [30, 31]. Indeed, we have previously found
that WT animals submitted to this same stress protocol present changes neither in corticosterone levels nor
in hippocampal neurogenesis [13]. The only change observed in the WT mice under chronic stress was a light
increase of GPX activity. GPX reduces both free hydrogen peroxide and LOOH [32], and the action of GPX
on LOOH can prevent their enhancement in the hippocampus of restrained mice, reducing the cell damage.
In this way, the increased GPX activity in absence of
any oxidative damage is likely an adaptation to repeated
stress allowing the regulation of cellular peroxides. The
increased activity of some antioxidant enzymes results in
the rapid scavenging of ROS and consequently less cell
damage [33].
The lack of LPA1 receptor by itself does not produce either any change in the antioxidant capacity or
in the free radicals production under basal conditions.

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LOOH (nmol/mg)

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COX (Umg/protein)

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Chronic restraint

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Control
Chronic restraint

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KO

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KO

FIGURE 1. Oxidative variables and mitochondrial function in the hippocampus of

malpa1 KO mice submitted to chronic restraint (21 days/3 hours a day). (a) LOOH,
(b) GPX activity, (c) CAT [18] activity, (d) SOD activity and (e) COX activity. Bars represent means SEM of 6 mice. p < .05 according to LSD. KO = malpa1 KO mice; WT
= wild-type mice.

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However, when the predictable chronic stress is endured

by the malpar1 KO mice, several changes are revealed in
both oxidant variables and antioxidant enzymes activity
(i.e., enhanced lipid peroxidation, SOD, CAT, and GPX
activities and COX activity downregulation). Increased
SOD, CAT, and GPX activity is a normal response to
the increased production of free radicals. SOD converts
superoxide radical into H2 O2 and both CAT and GPX
are responsible for detoxification of H2 O2 [32]. Nevertheless, the enhancement of the hippocampal activity of
these enzymes is not efficient to protect the cells against
free radicals toxicity, because the increased LOOH pro-

duction is still very high in the malpar1 KO mice after chronic restraint. Therefore, this LOOH overproduction indicates the inability of the GPX enhancement
for their detoxification, leading to increase the peroxidative stress that will contribute to the overproduction
of free radicals and to the oxidative damage [21, 34].
The peroxidation of membrane lipids as the observed
in our study is one of the most common consequences
of oxidative stress. Since the hippocampus (and brain
in general) includes high content of polyunsaturated
fatty acids, this reaction produces marked damage to
the structure and function of cell membranes [35, 36].

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Therefore, enhanced lipid peroxidation observed in the

malpar1 KO mice after chronic stress might be one of
the major biochemical alterations underlying oxidantinduced cell injury in this genotype under chronic stress
situations.
Interestingly, COX activity downregulation in the
hippocampus of the malpar1 KO submitted to chronic
stress clearly indicates that the mitochondria have been
seriously damaged. COX is the last enzyme in the respiratory electron transport chain of mitochondria, and
its activity is close related to the oxidative state of the
membrane phospholipid cardiolipin [37]. The observed
enhancement of the lipidic peroxidation may be account
for the COX downregulation activity and mitochondrial
damage. This mitochondrial damage with the consequent dramatic decrease in cellular energy supply will
become an important source of free radicals and contribute to apoptosis [15].
Oxidative stress has been involved not only in the
enhancement of apoptosis but also in the reduced
hippocampal neurogenesis [16, 3840]. Conversely,
administration of antioxidants reduces oxidative stress
and prevents the reduction of proliferation and the
enhancement of apoptosis in the hippocampus [16,
17, 41]. Furthermore, increased oxidative stress is
also thought to have an important role in cognitive
impairment, and administration of antioxidant agents
has been shown to improve such deficits [16, 17, 41,
42]. Increased apoptosis and reduced formation of new
neurons in the hippocampus accompanied by spatial
long-term memory deficits have recently been reported
in malpar1 KO mice undergoing chronic stress [13].
Considering all these data, one might speculate that the
apoptosis, neurogenesis, and cognitive deficits exhibited
by malpar1 KO mice after chronic stress are, at least in
part, consequence of the increased oxidative damage of
the hippocampus. However, further studies need to be
carried out to explore this hypothesis.
The enhanced hippocampal oxidative stress linked to
the absence of LPA1 receptor after chronic stress suggests a crucial role of LPA1 receptor pathway in the
adaptative response to stress. To date, there is not available information to support the mechanism through the
LPA1 receptor may be involved in this adaptation. Nevertheless, we have recently reported that chronic stress
in the malpar1 KO mice induces hypocortisolism [13], a
phenomenon usually attributed to an initial hyperreactivity of the hypothalamic-pituitary-adrenal (HPA) axis,
and where the deregulation of the glucocorticoid receptor (GR) [42] is crucially involved [43, 44]. Both highlevel corticosterone in the serum and GR-deregulated
function have been linked to increased oxidative stress,
leading to oxidative damage in the hippocampus [45,
46]. Therefore, an anomalous regulation of the HPA
axis function in response to chronic stress in the ab
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sence of LPA1 receptor may be, at least in part, responsible for the increased oxidative stress observed. Although
this study was not designed to test this hypothesis, future works will elucidate more clearly the relationship
between these factors.

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CONCLUSIONS
Taken together, our results showed upregulation of antioxidant enzyme activity and lipid peroxidation in combination with a downregulation of COX activity in the
hippocampus of malpar1 KO mice when submitted to
chronic stress. Increases in antioxidant enzyme activities and lipid peroxidation are consistent with enhanced
oxidative stress. This increased oxidative stress may contribute to explain the enhanced vulnerability of this
genotype to chronic stress. Furthermore, these results
highlight the relevance of the LPA1 pathway signaling
in the hippocampus to regulate the response to chronic
stressors.

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Declaration of interest: The authors report no conflicts of interest.

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