Int. J.

Devl Neuroscience 26 (2008) 487–495

www.elsevier.com/locate/ijdevneu

Role of p38 MAPK and pro-inflammatory cytokines expression

in glutamate-induced neuronal death of neonatal rats
V. Chaparro-Huerta a, M.E. Flores-Soto a, G. Gudiño-Cabrera d,
M.C. Rivera-Cervantes c, O.K. Bitzer-Quintero b, C. Beas-Zárate a,d,*
a
Laboratorio de Neurobiologı́a Molecular, División de Neurociencias, Centro de Investigación Biomédica de Occidente (CIBO),
Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Mexico
b
Laboratorio de Neuroinmunomodulación, División de Neurociencias, Centro de Investigación Biomédica de Occidente (CIBO),
Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Mexico
c
Laboratorio de Neurobiologı́a Celular, Instituto de Neurobiologı́a, Departamento de Biologı́a Celular y Molecular,
Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA), Universidad de Guadalajara, Guadalajara, Mexico
d
Laboratorio de Desarrollo y Regeneración, Instituto de Neurobiologı́a, Departamento de Biologı́a Celular y Molecular,
Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA), Universidad de Guadalajara, Guadalajara, Mexico
Received 24 January 2008; received in revised form 18 February 2008; accepted 18 February 2008

Abstract
Pro-inflammatory cytokines TNF-a, IL-1b and IL-6 rises significantly during neuronal damage and activate the signaling p38 MAPK pathway,
which is involved in the apoptotic (AP) neuronal death. Systemic administration of glutamate as monosodium salt (MSG) to newborn animals
induces neuronal death, however whether neurons die by AP or necrosis through MAPK p38 pathway activation it is unknown. In this study, TNF-
a, IL-1b and IL-6 expression levels, AP neuronal death and cellular type that produces TNF-a was also identified in the cerebral cortex (CC) and
striatum (St) of rats at 8, 10, and 14 days of age after neonatal exposure to MSG. TNF-a production and AP neuronal death was significantly
increased in the CC at PD8–10, and in the St in all ages studied by excitotoxicity effect induced with MSG. This effect was completely inhibited by
SB203580 (p38 inhibitor) in both regions studied. TNF-a, IL-1b and IL-6 RNAm increased after MSG administration, whereas SB203580 did not
modify their expression. These data indicates that neuronal death induced by excitotoxicity appears to be mediated through p38 signaling pathway
activated by TNF-a and their inhibition may have an important neuroprotective role as part of anti-inflammatory therapeutic strategy.
# 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Keywords: Apoptosis; Cytokines; Glutamate neurotoxicity; p38 MAPK; Cerebral cortex; Striatum; Rat

1. Introduction induces oxidative stress a commun feature present in different

Activation of glutamate receptors may play important roles
in the neuronal death that occur naturally during development
of the nervous system and in various neurodegenerative
disorders. Overactivation of the glutamate ionotropic receptors
lead to excitotoxic cell death and such excitotoxic neuronal
death can induce either apoptosis or necrosis depending on the
intensity of receptor activation (Johnston, 2005). Also, that
neuronal death by excitotoxic phenomenon may eventually
* Corresponding author at: Laboratorio de Neurobiologı́a Celular y Mole-
cular, División de Neurociencias, CIBO, Sierra Mojada #800, col. Independ.,
Guadalajara, Jalisco 44340, Mexico. Fax: +52 33 3618 1756.
E-mail address: CarlosBeas@cucba.udg.mx (C. Beas-Zárate).
0736-5748/$34.00 # 2008 ISDN. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijdevneu.2008.02.008
neurodegenerative diseases, such as Parkinson, Alzheimer, etc.
and neurological diseases with expression of protein involved
in cell-cycle control (Zhu et al., 2003; Camins et al., 2007).
The signals generated by glutamate receptors activate the
stress-sensitive MAP kinase, JNK and p38, which are implicated
in neuronal apoptosis (Cao et al., 2004). JNK appears to be
strongly activated by neuronal stress in cerebellar neurons
(Coffey et al., 2002). Whereas, there are even reports concerning
proapoptotic functions of p38, in spontaneous apoptosis of
neutrophils, and apoptosis induced by withdrawal of trophic
factors, glutamate and sodium salicylate (Cao et al., 2004).
On the other hand, cytokine overexpression in the brain is
an important factor in the pathogenesis of neurotoxic and
neurodegenerative disorders. In the CNS after injury expressed
various genes of pro-inflammatory cytokines. In rats, IL-1b
488 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495
protein and its mRNA are detected in neurons from several
brain structures as cerebral cortex, hippocampus, striatum,
thalamus, amygdala, and cerebellum (Wang and Shuaib, 2002).
IL-1b protein and its mRNA increase following focal ischemic
injury, and also TNF-a protein and mRNA elevated have been
shown in various experimetal models of traumatic brain injury
in rats (Wang and Shuaib, 2002). Several evidences suggest a
relationship between p38 activation, the production of pro-
inflammatory cytokines and apoptosis (Kummer et al., 1997;
Juo et al., 1997). The p38 active several substrates such as ATF-
2, CREB, CHOP, MEFC2, Elk-1 and Mnk-1. Once activated,
Mnk-1 phosphorylates the eukaryotic initiation factor 4E (eIF-
4E), recruiting protein-synthesizing ribosome’s and additional
protein synthesis initiation factors to the mRNA. Mnk-1 can be
blocked by PD98059 MEK1 and SB203580 p38 inhibitors,
respectively (Waskiewicz et al., 1997), supporting the idea that
Mnk-1 integrates signals from these two kinases pathway.
Recently our work group showed an important activation of p38
pathway under neuroexcitotoxicity conditions, which it was
associated with neuronal death in cerebral cortex of neonatal
rats (Rivera-Cervantes et al., 2004). However, the pro-
inflammatory cytokines levels and their cellular co-expression
associated with p38 activation may play a key factor in that
cellular death control, which it is unknown. Therefore, the aim
of this work was evaluate the TNF-a, IL-1b and IL-6
expression levels, as well as the cellular localization of
TNF-a associated with apoptosis and p38 pathway activation at
the cerebral cortex and striatum rats at 8, 10 and 14 days of age
with neuronal damage induced by neonatal exposure to
glutamate as monosodium salt (MSG).
2. Materials and methods
2.1. Animal preparation
Pregnant Wistar rats were used and kept under optimal environmental
conditions, i.e., free access to water and food, with 12–12 h light–dark cycles, at
temperatures ranging between 23 and 25 8C, and in separate cages. On the day
of birth, all litters were adjusted to eight offspring per female. Offspring were
given MSG (4 mg/g body weight) subcutaneously (s.c.) on postnatal days (PD)
1, 3, 5, and 7, according to Beas-Zárate et al. (2001). A group of untreated
animals was used as external control. A third group was treated with SB203580
(0.42 mg/g body weight), a p38 inhibitor, administered s.c. 30 min before MSG,
and a fourth group was treated with SB203580 alone at the same doses as
previously determined (Rivera-Cervantes et al., 2004).
All animals were used on PD8, PD10, and PD14, some treated and control
animals were fixed by perfusion and used for histological analysis and other
were killed by decapitation and the cerebral cortex and striatum were dissected
out for further molecular analysis.
All animals in the various groups were from the same litters or from parallel
litters. Animal care and handling were in accordance with Mexican General
Health Laws and their corresponding chapters (1987) and the National Institutes
of Health Guidelines for the Care and Use of Laboratory Animals (NIH
publication no. 80.23, revised 1996). All efforts were made to minimize the
number of animals used and their suffering.
2.2. Semi-quantitative reverse transcriptase-polymerase chain
reaction (RT-PCR)
Unfixed cerebral frontal cortices and striatum collected from each group
were dissected out at 4 8C until used for molecular biology studies.
Table 1
PCR primers used for amplification of target genes
Gen Sequences Cycles
TNF-a 0
Sense 5 -CGA GTG ACA AGC CCG TAG CC-3 0
38
Antisense 30 -CC GCT GAC CGC ACA AGT AGG-50
IL-1b Sense 50 -CCA GGA TGA GGA CCC AAG CA-30 30
Antisense 30 -CC TTT GTC GTT ACC AGC CCT-50
IL-6 Sense 50 -CTT CCA GCC AGT TGC CTT CT-30 40
Antisense 30 -GG GGT TGA AGG TTA CGA GAG-50
b-Actin Sense 50 -CACCACAGCTGAGAGGGAAATCGTG 22
CGTGA-30
Antisense 30 -ATTTGCGGTGCACGATGGAGGGGC
CGGACT-50
Total RNAwas isolated according the method described by Chomczynski and
Sacchi (1987). Briefly, brain tissue was homogenized using a polytron system in
the presence of TRIzol; chloroform was added, the aqueous phase was collected,
and the RNA precipitated with isopropanol at 4 8C overnight. The quantity and
RNA integrity were routinely assessed by absorbance (A260/280) and the ethidium-
bromide fluorescence of RNA separated electrophoretically on 1% formaldehyde-
containing agarose gels (Armendáriz-Borunda et al., 1997).
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed
according to a previously described methodology (Delgado-Rizo et al., 1998;
Beas-Zárate et al., 2001). A standardized semi-quantitative PCR method based
on the amplification of the target gene and a constitutively expressed gene, b-
actin, was used. Target genes were for TNF-a, IL-1b and IL-6 pro-inflammatory
cytokines and the primers used are depicted in Table 1.
Total RNA (2 mg) was reverse transcribed in 0.05 M Tris–HCl (pH 8.3),
40 mM KCl, 7 mM MgCl2 buffer containing 0.05 U/ml RNase inhibitor and
200 U/ml Maloney’s murine leukemia virus (M-MLV) reverse transcriptase.
Samples were incubated for 10 min at 70 8C and then 60 min at 37.5 8C.
Reverse transcriptase was inactivated by heating the sample tubes at 95 8C for
10 min. cDNAs thus obtained were used immediately in PCR or were stored at
20 8C until use. The optimal PCR conditions were determined to detect the
expression of each particular cytokine. Amplification was performed in a PCR
buffer of 50 mM Tris–HCl (pH 9.0) and 50 mM NaCl containing a mix of
100 mM dNTPs and 1 U Taq DNA polymerase. Amplification reactions were
overlaid with light mineral oil and held at 95 8C for ‘hot-start’ PCR for 5 min,
then run in an automated thermal cycler for the number of cycles specified in
Table 1. Each cycle consisted of 95 8C for 1 min, 60 8C for 1 min, and 72 8C for
1.5 min, with a final cycle of extension for 5 min at 72 8C.
2.3. Photographic and densitometric scanning
Densitometric analysis of PCR products was performed with a Kodak
computational system. Sample electrophoreses were photographed with Kodak
Digital Science Scanner and analysis was performed using the same system.
After the scanning was complete, the area corresponding to each band, which
represented the amplified PCR product, was automatically calculated and
normalized against the area representing the expression of the constitutive
gene. Results were expressed as relative intensity in arbitrary units compared to
the control value.
Each parameter was determined in duplicate for each animal, using at least six
animals, and then averaged for each group before comparison between groups.
2.4. Immunohistochemical study
Animals from each group were anesthetized at PD8 for intraperitoneal
injection of pentobarbital (50 mg/kg) and intracardially perfused with Ringer
phosphate solution (pH 7.2) for 5 min at a pressure of 130 cm H2O, then with
fixing solution (0.1 M phosphate-buffered 4% paraformaldehyde, pH 7.2) for
15 min. Brains were extracted and fixed for 48 h in the same fixing solution at
room temperature.
 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495 489

Coronal sections of the brains, including the cerebral cortex and striatum,
were washed in 0.1 M phosphate buffer and sections (40-mm thick) cut with a
vibratome were collected.
For dual-label with rabbit anti-TNF-a (Biosurce) and rabbit anti-GFAP
(Chemicon) (1:500) all steps were performed at room temperature and sepa-
rated by a minimum of three 15 min PBS washes. Free-floating sections were
initially treated with 0.5% Triton X-100 for 1 h and normal goat serum in PBS-
sodium azide (1:100) to reduce nonspecific staining, then incubated 72 h with
rabbit anti-GFAP. Sections were then incubated sequentially with biotinylated
goat anti-rabbit (overnigth 1:500 Vector Laboratories, CA) and incubated for
2 h with Vectastain ABC solution. Finally were incubated with diaminobenzi-
dine (DAB kit, Vector) for a maximum of 5 min, until staining was observed.
For staining with rabbit anti-TNF-a were similarity and color developed using
NovaRED kit (Vector) for approximately 10 min.
Preparations were examined under a light microscope (40) connected to a
Leica Image Analyser and equipped with an analog photomicroscopy system.
Cells with GFAP-TNF-a positive were observed on the Fr1 region from cerebral
cortex and complete striatum. Data obtained corresponded to six animals for
each group studied and four sections were examined for each animal, analyzing
a total of eight fields per section.

2.5. Evaluation for apoptosis

For immunohistochemical detection and quantification of apoptosis at

single cell level it was based on labeling of DNA strand breaks following
the procedure of TUNEL technique according to procedure described into the
manual of in situ cell death detection kit, POD (Roche cat. no. 1 684 817) using
paraffin-embedded tissue sections. Briefly, sections were dewaxed, rehydrated
and treated with K proteinase. Permeabilization and TUNEL reaction were
further performed. Incorporated fluorescein was detected by anti-fluorescein
antibody Fab fragments from sheep, conjugated with horseradish peroxidase
(POD) and finally stained cells with diaminobenzidine were analyzed under a
light microscope. The apoptotic cells from four experiments were counted of
four sections and were examined for each animal, analyzing a total of eight
fields per section.
2.6. Statistical analysis
Fig. 1. Upper panel depicts the RT-PCR expression products of TNF-a mRNA
at cerebral cortex from different groups: control (CTL), MSG, SB-203580
(0.42 mg/g body weight [30 mM]) MSG plus SB-203580: (A) TNF-a mRNA
expression at postnatal day 8; (B) 10th postnatal age; (C) 14th postnatal day of
age. Lower panel shows the densitometry analysis of gene expression under
different conditions described in the upper panel. Values are means  S.E.M.
from six experiments by duplicate. Statistically different respect to control
group at *p < 0.001, ^p < 0.05. Comparison between MSG and SB203580 at
ä
p < 0.05.

All morphological data were analyzed by double blind. All data collected administered during neonatal period and compared to control
from control rats were compared among different groups of the study. Statistical group (Fig. 2). Also, TNF-a mRNA levels were reduced after
comparisons were made with ANOVA and a p < 0.05 was considered sig-
the p38 signaling pathway was inhibited by SB203580 in either
nificant. Data were represented as mean  standard error of the mean (S.E.M.).
MSG or alone groups compared with what MSG treated group
(Fig. 2).
3. Results
3.3. IL-1b and IL-6 mRNA levels
3.1. TNF-a mRNA levels in cerebral cortex
It is widely documented that the high level of TNF-a is
High levels in TNF-a mRNA were observed by the effect of
accompanied by the synthesis and release of others cytokines
MSG on PD8 and remained along all ages studied (PD10 and
such as IL-1b and IL-6 in different cellular responses. Thus,
PD14), when it was administered during neonatal period and
high levels of IL-1b in cerebral cortex and striatum were
compared to control group (Fig. 1). However, the presence of
induced at PD8 by an early exposure to MSG at 41 and 48%
SB203580 previous to MSG administration was able to avoid
respectively compared to control group (Fig. 3) and the levels of
that increase in the TNF-a mRNA levels in all ages studied by
IL-6 showed 60 and 47%, respectively (Fig. 4). Whereas,
MSG effect, including at PD10 where TNF-a mRNA levels
SB203580 alone induced an important reduction in IL-1b and
were less than control group (Fig. 1). Whereas, the SB203580
IL-6 mRNA compared to both control and MSG groups (Figs. 3
alone reduced significantly the TNF-a mRNA levels on PD10
and 4). Likewise, IL-6 mRNA levels in MSG plus SB203580
in comparison with the control group (Fig. 1).
group was also reduced respect to control group, while IL-1b
was not changed compared to the control group (Fig. 4).
3.2. TNF-a mRNA levels in striatum
3.4. Apoptotic cells and co-localization of TNF-a
High levels in TNF-a mRNA but in minor intensity were
also observed in the striatum by effect of MSG on PD8 and With the purpose to determine the cellular death suscept-
remained along all ages studied (PD10 and PD14), when it was ibility at cerebral cortex and striatum, a semi-quantitative study
490 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495
Fig. 2. Upper panel shows the RT-PCR expression products of TNF-a mRNA at
striatum from different groups: control (CTL), MSG, SB-203580 (0.42 mg/g
body weight [30 mM]) MSG plus SB-203580: (A) TNF-a mRNA expression at
postnatal day 8; (B) 10th postnatal age; (C) 14th postnatal day of age. Lower
panel shows the densitometry analysis of gene expression under different
conditions described in the upper panel. Values are means  S.E.M. from
six experiments by duplicate. Statistically different respect to control group at
*
p < 0.001, äp < 0.01, ^p < 0.005, &p < 0.05.
Fig. 3. Upper panel depicts the RT-PCR expression products of IL-1b mRNA at
cerebral cortex (A) and striatum (B) from different groups: control, MSG and
SB203580 (0.42 mg/g body weight [30 mM]) alone or combined with MSG at
postnatal day 8. Lower panel shows the densitometry analysis of gene expres-
sion. Values are means  S.E.M. obtained from six experiments by duplicate.
Statistically different respect to control group at *p < 0.001. MSG compared
with SB203580 alone and MSG plus SB203580 groups were at ^p < 0.001.
SB203580 group compared to MSG plus SB203580 at äp < 0.001, p < 0.05.
Fig. 4. Upper panel shows the RT-PCR expression products of IL-6 mRNA at
cerebral cortex (A) and striatum (B) from different groups: control, MSG and
SB203580 (0.42 mg/g body weight [30 mM]) alone or combined with MSG at
postnatal day 8. Lower panel depicts the densitometry analysis of gene
expression Values are means  S.E.M. obtained from six experiments by
duplicate. Statistically different respect to control group at *p < 0.001. MSG
compared with SB203580 alone and MSG plus SB203580 groups were at
^
p < 0.001, &p < 0.05.
of cells with positive characteristics to TUNEL was performed
in all groups and ages studied. Results shown an important
increase in the apoptotic cells from PD8 and PD10 induced by
MSG at cerebral cortex region compared to control group
(Fig. 5A). While the striatum region showed an increase, but
less than that observed in the cerebral cortex in all ages studied,
however, a major number of apoptotic cells were observed at
PD14 in this region (Fig. 6A).
The increase in cellular death induced by MSG effect was
reduced by SB203580 inhibitor in both groups with MSG
treatment or alone at PD8–PD14 in both regions studied (Figs. 5
and 6). In the Fig. 5(B) and 6(B) we can observe representative
microphotographs of the neuronal death by apoptosis in a
TUNEL reaction. In the controls groups does not exist positive
stained to this reaction, whereas the MSG group is with the
nuclear material condensed and SB203580 alone and plus with
MSG are similar to control groups (Fig. 5(B) and 6(B)).
It is known that one of the main functions of TNF-a is
associated with cellular death signals; therefore it was interest to
known the main cellular source of production and release of
TNF-a at cerebral cortex and striatum. Thus, a differential
immunohistochemistry to neuronal and glial cells in both regions
was performed. TNF-a content in the cerebral cortex was
majorly visualized in neurons in the MSG treated group than
GFAP immunopositive glial cells (Fig. 7). In contrast, in the
striatum region TNF-a was mainly visualized in GFAP
immunopositive glial cells (Fig. 8), indicating different response
in the source of TNF-a synthesis to be both neuronal and/or glial
in function of stimuli and cerebral region under study.
 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495 491

Fig. 5. (A) Number of apoptotic neurons at cerebral cortex at 8, 10 and 14 postnatal days in rats from control, MSG, SB203580 (0.42 mg/g body weight [30 mM])
alone or combined with MSG groups. Cerebral cortex slices were visualized using TUNEL technique. (B) Representative microphotograph of neurons from the
cerebral cortex under different conditions studied. Values are means  S.E.M. of four experiments according described in Section 2. Statistically different respect to
control, MSG vs. SB, vs. MSG + SB, SB vs. MSG + SB group at *p < 0.001, &p < 0.005, |p < 0.01, ^p < 0.05.

4. Discussion ways [p38, Jun N-terminal kinase (JNK)1/2 and extracellular-

Cytokines, as major effectors of the inflammatory cascade,
play a key role in the nerve cell response to brain injury and
may be beneficial or, when secreted in an imbalanced fashion,
detrimental to neurons (Kriz, 2006). TNF-a and IL-1b are
usually the first cytokines to be up-regulated after challenge
(Morganti-Kossmann et al., 2002; Correale and Villa, 2004)
and normally induce the synthesis of anti-inflammatory
cytokines, such as IL-6, creating a feedback loop to re-
establish the resting immunological status (Xing et al., 1998;
Correale and Villa, 2004).
Production and release of cytokines depend on inducible gene
expression mediated by activation of cell signaling. The primary
inflammatory stimulus may act through membrane receptors
such as TNF-a receptor (TNFR1 and TNFR2), causing the
activation of three major intracellular signaling cascades: the
three distinct mitogen-activated protein kinase (MAPK) path-
regulated kinase (ERK)1/2] (Saklatvala et al., 2003). Here, we
investigated the participation of signaling pathways by cytokines
associated with their mRNA expression, and examined the
relevance of these cytokines into the apoptosis observed under
neuroexcitotoxicity conditions induce by early treatment with
MSG.
Our results indicate that excitotoxicity by MSG induces a
rapid production of cytokines and activation of p38 MAPK
(Chaparro-Huerta et al., 2005). In a fashion similar to that found
in the presence of classical inflammatory agents like lipopoly-
saccharide (LPS) (Bhat et al., 1998; Hua et al., 2002). In vivo
studies have shown that increased expression of TNF-a and its
membrane receptors, mainly TNFR1, are detected as early at
15 min after traumatic injury in neurons, astrocytes and
oligodendrocytes (Yan et al., 2003), transient focal ischemia
and peripheral nerve injury in neurons and astrocytes (Yin et al.,
2004; Ohtori et al., 2004).
492 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495

Fig. 6. (A) Number of apoptotic neurons at striatum at 8, 10 and 14 postnatal days in rats from control, MSG, SB203580 (0.42 mg/g body weight [30 mM]) alone or
combined with MSG groups. (B) Representative microphotograph of neurons from the cerebral cortex under different conditions studied. Striatum slices were
visualized using TUNEL technique. Values are means  S.E.M. of four experiments according described in Section 2. Statistically different respect to control, MSG
vs. SB, vs. MSG + SB, SB vs. MSG + SB group at *p < 0.001, &p < 0.005, äp < 0.01.

The astrocytes activation levels, as a potent source of
immunologically relevant cytokines, play a pivotal role in the
type and extent of CNS inflammatory response (Dong and
Benveniste, 2001). In general, astrocytes induce a peak of
cytokine mRNA levels during the first 3–6 h after an
inflammatory stimulus (Bhat et al., 1998; Harry et al.,
2002), whereas cytokine protein levels become maximal
during the first 24 h and are maintained for longer time periods
(Lieberman et al., 1989). However, each cytokine usually
shows a differential patron of expression. Our results also
shown that pro-inflammatory cytokines TNF-a, IL-1b and IL-
6 were inhibited by SB203580, a specific inhibitor of p38
MAPK, under excitotoxicity conditions, indicating a specific
response to that stimulus mediated through p38 pathway
activation. It is known that p38 MAPK plays a central role in
the regulation of a wide range of immunological responses,
thus an extracellular stimuli including a variety of cytokines
(i.e., IL-1, IL-2, IL-7, IL-17, IL-18, TGF-b and TNF-a) and
several pathogens as staphylococcal peptidoglycan, staphy-
lococcal enterotoxin-B, echovirus-1 and herpex simplex-1 and
(LPS), among others activates p38 through different TOLL
receptors (Hommes et al., 2003; Arbabi and Maier, 2002; Qi
and Elion, 2005).
In this work, also it was investigated the cytokins pathway
and its by cytokines and its association with their mRNA
expression, and we examined the relevance of those cytokines
with the apoptosis observed in the cerebral cortex and in the
striatum regions after treatment with MSG. The results
indicated that neuronal death appears to be mediated through
p38 pathway activation, since its inhibition avoided that
neuronal death induced by an early glutamate excitotoxicity.
Recent studies have shown that TNF-a through its signaling
TNFR1 receptors type, mediate astrocytosis, as well as
apoptotic cellular death, which it could be prevented with
caspase inhibitors (Scheller et al., 2006; Alexander et al., 2007).
Furthermore, previous results from our work group showed a
persistent high glial and microglial reactivity under this model
of study, it appears to suggest a possible chronic over-expressed
TNFR1 receptors in the brain regions studied.
However, it was evident that TNF-a was localized in both
type of cells, neurons and non-neurons with some dominance
according the regions studied, thus the relationship between
 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495 493

Fig. 7. Immunohistochemistry for TNF-a in the cerebral cortex of rats at eighth of postnatal day. (A and B) Immunopositive astrocytes expressing glial fibrillary
acidic protein (GFAP)-positive (arrow). (C and D) rat neurons expressing TNF-a protein-positive (asterisk). (E and F) Double-label with TNF-a-GFAP expression
(arrow for glia and asterisk for neurons). (A, C and E) Control groups and (B, D and F) MSG groups. Magnification 40.

both type of cells on the final decision on neuronal survival or
death may be determinant overall when the apoptosis plays a
key role in normal aging and neurodegeneration implying
some-grade of inflammation. Thus, taken together these results
and in agreement with the results obtained on neuronal death
induced by glutamate-neurotoxicity observed from PD8 to
PD10, but not on PD14 in particular at the cerebral cortex, it
appears suggest that the mechanism of apoptotic neuronal death
also involve mainly the anti-apoptotic protein Bcl2. Previous
results from our work group shown an important increase in the
Bcl2 expression at PD8, but not at PD14 (Segura Torres et al.,
2006), although the TNF-a levels remain increased at this age,
as well as, the interesting, cytokine (TNF-a)-elicited Bcl-2 up-
regulation age-dependent showed by Xu et al. (2007),
indicating that, at least, on PD14, the p38 pathway could not
be completely activated by a reduction in the pro-inflammatory
cytokines release, instead of, other signaling pathways may be
activated in a cytokine independent form. Nevertheless, in the
striatum region, high neuronal death was observed to continue
at PD14 (Fig. 6). It could be explained, at least, on the role of
pro-inflammatory cytokines, which remain increased in this
region at this age studied, it may due mainly to TNF-a, which
appears to leading to a cycle of neuronal damage or death by
microglial activation extending the inflammatory process as
proposed recently by Wilms et al. (2007) and by Sawada et al.
(2006). This idea is supported by previous results from our
work group, where high and chronically glial and microglial
reactivity was observed in the several brain regions including
striatum region (in preparation). Therefore, whether this early
changes may have behaviour and learn implications at the
adulthood phase, it should be investigated. Although, a deficit
in both place learning acquisition and retrieval abilities
impaired were observed in mature rats treated neonatally with
MSG suggesting that possibility (Olvera-Cortes et al., 2005).
494 V. Chaparro-Huerta et al. / Int. J. Devl Neuroscience 26 (2008) 487–495

Fig. 8. Immunohistochemistry for TNF-a at striatum in rats at eighth of postnatal day. (A and B) Immunopositive astrocytes expressing glial fibrillary acidic protein
(GFAP)-positive (arrow). (C and D) Neurons expressing TNF-a protein-positive (asterisk). (E and F) double-label with TNF-a-GFAP expression (arrow labels glia
and asterisk for neurons). (A, C and E) Control groups and (B, D and F) MSG groups. Magnifications 40.

Acknowledgements expression are associated with neurotoxicity induced neonatally by gluta-

This work was partially supported by CONACyT grant
30901-M to CBZ and scholarship no. 158761 to VChH.
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