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Neurochemistry International 51 (2007) 459–466
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Cell type-specific activation of p38 MAPK in the brain
regions of hypoxic preconditioned mice
Xiangning Bu a, Ping Huang a, Zhifeng Qi a, Nan Zhang a, Song Han a, Li Fang b,*, Junfa Li a,**
a
Institute for Biomedical Science of Pain, Beijing Key Laboratory for Neural Regeneration and Repairing, Department of Neurobiology,
Capital Medical University, #10 You An Men Wai Xi Tou Tiao, Beijing 100069, China
b
Division of Neurosurgery, Department of Surgery, Neuroscience and Cell Biology, The University of Texas Medical Branch,
301 University Boulevard, Galveston, TX 77555-0517, USA
Received 23 December 2006; received in revised form 20 April 2007; accepted 25 April 2007
Available online 17 May 2007
Abstract
Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral
injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by
observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution
of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly ( p < 0.05) in the
regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when
compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an
immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To
further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38
MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific
maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of
hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute
to the development of cerebral HPC mechanism in mice.
# 2007 Elsevier Ltd. All rights reserved.
Keywords: P38 mitogen-activated protein kinases (p38 MAPK); Hypoxic preconditioning (HPC); Protein expression; Phosphorylation; Brain
1. Introduction
It is well accepted that a sublethal ischemic/hypoxic exposure
can improve the tissue or organ’s tolerance to a subsequent lethal
ischemic/hypoxic insult; this protects cells against injuries and
death, known as ischemic/hypoxic preconditioning (I/HPC)
(Jones and Bergeron, 2004; Vannucci and Hagberg, 2004).
Information regarding the signal transduction mediators of this
phenomenon is complex and uncertain (Tsai et al., 2004). To
investigate the molecular mechanism of cerebral I/HPC, we have
developed a unique ‘‘auto-hypoxia’’-induced HPC mouse model
that mimics clinical asphyxia (Li et al., 2005; Lu and Liu, 2001).
* Corresponding author. Tel.: +1 409 772 2944; fax: +1 409 772 4687.
** Corresponding author. Tel.: +86 10 8391 1475; fax: +86 10 8391 1491.
E-mail addresses: [email protected] (L. Fang), [email protected] (J. Li).
0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2007.04.028
Using this model, we have demonstrated a series of protein
kinases mediated the signalling cascades of cerebral HPC as
follows: (1) increased membrane translocation of conventional
protein kinase C bII and g (cPKCbII, g), novel protein kinase C e
(nPKCe), and phosphorylation of its substrate, neurogranin (Li
et al., 2005, 2006; Niu et al., 2005); (2) decreased phosphorylation levels and protein expression of extracellular signalregulated kinases 1/2 (ERK1/2) (Long et al., 2006); (3) enhanced
phosphorylation of a nuclear transcriptional factor, cyclic AMP
response element binding protein (CREB) (Gao et al., 2006).
The p38 kinase is one of the mitogen-activated protein
kinase (MAPK) family members that include extracellular
signal-regulated kinases (ERK), c-Jun N-terminal kinases
(JNK), and p38 MAPK. They function as critical mediators of
signal transduction pathways (Johnson and Lapadat, 2002). The
activation of p38 MAPK requires dual phosphorylations of
threonine 180 (Thr180) and tyrosine 182 (Tyr182) residues
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within the conserved TGY motif; this is reported to be involved
in conveying extracellular stress to cellular response such as
inflammation and the processes of cell differentiation, growth,
and death (Widmann et al., 1999). Studies demonstrated that
both global and focal cerebral ischemia caused neuronal death
and activation of p38 MAPK in the brain of rat or gerbil (Irving
et al., 2000; Piao et al., 2002; Sugino et al., 2000; Walton et al.,
1998). Hypoxia increased cell death via a nPKCe-p38 MAPK
pathway (Jung et al., 2004) and inhibition of p38 MAPK
phosphorylation reduced cytotoxicity after hypoxia-reoxygenation procedures (Lee and Lo, 2003). In addition, inhibition
of p38 MAPK activation during ischemia reduces injury
and contributes to ischemia/reoxygenation preconditioninginduced cardioprotection in rat neonatal ventricular cardiocytes
and isolated rat hearts, respectively (Marais et al., 2001; Saurin
et al., 2000). However, little is known about the role of p38
MAPK in the development of cerebral HPC of mice in vivo.
Both ERK1/2 and p38 MAPK pathways activate common
transcription factors such as Ets-like transcription factor-1
(Elk-1) and CREB (Tan et al., 1996; Whitmarsh et al., 1997).
However, we found that enhanced phosphorylation of CREB
accompanied the decrease of ERK1/2 phosphorylation in the
brain of HPC mice (Gao et al., 2006; Long et al., 2006). In this
study, our goal was to investigate whether p38 MAPK was
activated by observing the changes in phosphorylation and
protein expression levels of p38 MAPK in the frontal cortex,
hippocampus, and hypothalamus of mice following repetitive
hypoxic exposure.
2. Experimental procedures
2.1. Materials
Phosphatase inhibitors (okadaic acid, sodium pyrophosphate, and potassium
fluoride), proteinase inhibitors (leupeptin, aprotinin, pepstatin A, and chymostatin), mouse monoclonal anti-b-actin antibody, and other reagents, such as
EDTA, EGTA, SDS, dithiothreitol (DTT), and Nonidet P-40 were purchased
from Sigma Company (St. Louis, MO, USA). Protein assay reagents, horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG,
were purchased from Bio-Rad Company (Hercules, CA, USA).
2.2. ‘‘Auto-hypoxia’’-induced HPC mouse model
Experiments were conducted on male BALB/c (Bagg albinos inbred ‘‘c’’
strain) mice at the age of 8–10 weeks (weighing 18–22 g) at room temperature
(20–22 8C). The animal protocol was approved by the University Institutional
Animal Care and Use Committee of Capital Medical University and is consistent with the NIH Guide for the Care and Use of Laboratory Animals (NIH
Publication No. 80-23). ‘‘Auto-hypoxia’’-induced HPC mouse models that
could mimic the clinical aspect of asphyxia were prepared as in our previous
reports (Li et al., 2005; Lu and Liu, 2001). Briefly, mice were placed individually in a 125-ml airtight jar with fresh air and sealed with a rubber plug to
mimic an environment of progressive auto-hypoxia. The mice were removed
from the sealed jars as soon as the first gasping appeared, and tolerant time was
recorded. A minimum of 30 min was allowed for recovery under normoxic
conditions. Next, the mice were switched to another hermetically sealed jar with
the same volume of fresh air after recovery from the previous hypoxic exposure.
This procedure was repeated one to four times. It was subsequently designated
as the normoxic control group (H0), which was kept in an open jar; hypoxic
group (H1) was exposed to hypoxia once; and the repetitive hypoxic exposure
became groups H2, H3, and H4. After four hypoxic exposures (H4), some mice
were kept in the normoxic condition for 24 h for recovery before the fifth and
sixth hypoxic exposures on the following day. Then, we made the experimental
group of delayed HPC mice as group H5 and H6, respectively. At the end of the
experiments, the mice were sacrificed. The frontal cortex, hippocampus, and
hypothalamus regions of the brain were collected, frozen in liquid nitrogen, and
kept frozen at 70 8C for later analysis.
2.3. Whole tissue homogenate preparation
To determine the phosphorylation and protein expression levels of p38
MAPK, the frontal cortex, hippocampus, and hypothalamus were extracted as
in our previous reports (Li et al., 2005, 2006). The whole tissues were
homogenized at 4 8C in homogenizing buffer (50 mM Tris–Cl, pH 7.5,
containing 2 mM DTT, 2 mM EDTA, 2 mM EGTA, 5 mg/ml each of leupeptin,
aprotinin, pepstatin A, and chymostatin, 50 mM KF, 50 nM okadaic acid,
5 mM sodium pyrophosphate, and 2% SDS) and sonicated to dissolve the
tissue completely. The protein amounts of samples were determined by BCA
kit (Pierce Company, USA).
2.4. SDS–PAGE and Western blot analysis
The phosphorylation and protein expression levels of p38 MAPK were
analyzed by Western blot as reported previously (Long et al., 2006; Niu et al.,
2005). Briefly, 50 mg of protein from the whole tissue homogenate of each
sample was loaded in 10% SDS–PAGE gel. Then, the proteins were transferred
onto the nitrocellulose membrane (NC membrane, Bio-Rad, USA) at 4 8C,
400 mA for 4 h. The transferred NC membrane was washed for 10 min with
TTBS (20 mM Tris–Cl, pH 7.5, containing 0.15 M NaCl and 0.05% Tween 20)
followed by the blocking solution with 10% nonfat milk in TTBS for 1 h.
First, the blocked membrane was incubated with primary rabbit polyclonal
antibodies against Thr180/Tyr182-phosphorylated p38 MAPK (p-p38 MAPK,
Cell Signal Technology, USA) and total p38 MAPK (T-p38 MAPK), including
phospho- and nonphospho-p38 MAPK (Santa Cruz Biotechnology Inc., USA)
at a 1:1000 dilution for 4 h at room temperature, respectively. For the second
antibodies, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Company, USA) at a 1:5000 dilution for
2 h. The membranes were washed three times (each for 10 min) in TTBS after
the incubation with the primary or secondary antibodies. Finally, immunoblotting signals were visualized using ECL-plus Kit (Chemicon International,
USA). The ratio of p-p38 MAPK to T-p38 MAPK in H0 was recognized as
100%, and the values of groups H1–H6 were expressed as a percentage of the
value of group H0.
To analyze the protein expression levels of p38 MAPK, the expression of bactin was determined at the same time with the same protocol, and individual
bands were quantified. Those membranes were blotted with antibodies against
the T-p38 MAPK stripped using buffer containing 62.5 mM Tris–Cl, pH 6.7,
100 mM 2-mercaptoethanol, and 2% SDS, for 50 min at 55 8C following a
period of constant shaking. Next, they were reprobed with primary mouse
monoclonal antibody against b-actin (Sigma Company, USA) at a 1:1000
dilution for 3 h at room temperature. The value of the relative optical density of
each band corresponding to T-p38 MAPK was normalized to the value of bactin to demonstrate protein expression level. The ratio of the values of relative
units in group H0 was regarded as 100%, and data from groups H1–H6 were
expressed as a percentage of the value of group H0.
2.5. Immunochemistry study
Mice were anesthetized with 6% chloral hydrate (240 mg/kg, i.p.) and
perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde.
Brains were quickly removed and fixed in 4% paraformaldehyde for 24 h and
cryoprotected with 20% sucrose solution. A 30-mm section of frozen mouse
brain was placed in ice-cold 10 mM phosphate buffered saline (PBS). The slices
were incubated with 3% H2O2 for 10 min to exhaust the endogenous peroxidase
and washed in PBST (10 mM PBS, pH 7.4, containing 0.5% Triton X-100).
Next, they were blocked with 10% goat serum for 1 h. The blocked slices were
incubated with primary polyclonal antibodies against p-p38 MAPK at a 1:400
dilution for 12 h at 4 8C. The specimens were incubated in secondary antibodies
X. Bu et al. / Neurochemistry International 51 (2007) 459–466
461
of biotinylated goat anti-rabbit IgG (ZYMED company, USA) at a 1:200
dilution for 2 h, and then were incubated with third antibodies of streptavidin-conjugated horseradish peroxidase (Sigma–Aldrich Company, USA) at a
1:200 for 2 h at room temperature. Finally, the slices were washed extensively in
PBS, and a solution containing H2O2 (0.03%) and 3,30 -diaminobenzidine
(DAB, 60%) was added to visualize the slices. The images were captured
by a Leica microscope imaging system (Leica Company, Germany).
For double-labeled immunofluorescent staining with p-p38 MAPK and
neurofilaments heavy chain (NF-H, a neuron-specific maker), S100 (astrocytespecific marker), and CD11b (microglia-specific maker), sections were incubated with a mixture of primary rabbit polyclonal anti-p-p38 MAPK antibody
with mouse monoclonal anti-NF-H antibody (Cell Sciences Company, USA),
mouse monoclonal anti-S100 antibody (Chemicon International, USA), and
mouse monoclonal anti-CD11b antibody (Chemicon International, USA) all at
a 1:400 dilution overnight at 4 8C, respectively. Then, a mixture containing
rhodamine-labeled anti-rabbit IgG at a 1:200 dilution (red color, Sigma
Company, USA) and fluorescein isothiocyanate-labeled anti-mouse IgG at a
1:200 dilution (green color, Sigma Company, USA) was incubated for 30 min at
37 8C. The images were captured by fluorescent microscope (Leica Company,
Germany). For negative control of the immunohistochemical staining (assessment of nonspecific immunostaining), the alternating sections from each
experimental group were incubated with PBS without primary antibody.
2.6. Statistics analysis
Images of immunostained sections captured by a Leica microscope imaging
system were used for quantitative analysis. The number of immunoreactive cells
was counted in a blinded fashion. Every second section was picked from a series of
consecutive sections (30 mm), and six sections were counted for each region of the
brain. We also performed a quantitative analysis for the immunoblot bands by
using GelDoc-2000 Imaging System (Bio-Rad Company, USA). Presented values
are expressed as means S.E. Statistical analysis was conducted by one-way
analysis of variance followed by all pairwise multiple comparison procedures
using the Bonferroni test. Significance was regarded as p < 0.05.
3. Results
3.1. Changes in phosphorylation and protein expression
levels of p38 MAPK in the brain of HPC mice
To determine the phosphorylation levels of p38 MAPK, the
ratio of p-p38 MAPK to T-p38 MAPK in the frontal cortex,
hippocampus, and hypothalamus of mice was quantified by
using Western blot with primary antibodies against p-p38
MAPK and T-p38 MAPK, respectively. A typical result of
Western blot in Fig. 1A and the quantitative analysis in Fig. 1B
indicated that p38 MAPK phosphorylation levels increased
significantly ( p < 0.05) in the frontal cortex, hippocampus, and
hypothalamus regions of mice from the H2–H6 groups (n = 6
for each group) in comparison to the control group
(H0 = 100%, n = 6, Fig. 1B).
In contrast to p-p38 MAPK, we did not find significant
changes in T-p38 MAPK protein expression levels in the frontal
cortex, hippocampus, and hypothalamus of mice following
repetitive hypoxic exposure (H1–H6 versus H0: 100%, n = 6
for each group) by using b-actin as internal reference (Fig. 1A,
the negative data of quantitative analysis does not show).
3.2. Distribution of p-p38 MAPK in the brain of HPC mice
To investigate the regional and cellular distribution of p-p38
MAPK in brain coronal slices of mice from H0, H3, and H6
Fig. 1. Increased phosphorylation of p38 MAPK in the brain of HPC mice. (A)
Representative results of Western blot showed that the levels of p-p38 MAPK,
not T-p38 MAPK, gradually increased in the frontal cortex, hippocampus, and
hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6). (B)
Quantitative analysis indicated a significant increase of p-p38 MAPK in the
frontal cortex and hippocampus of mice from H2–H6 groups, and in hypothalamus from H3–H6 groups (vs. H0: *p < 0.05, n = 6 for each group). H0:
normoxic control; H1–H6: hypoxic exposure times; p-p38 MAPK: Thr180/
Tyr182-phosphorylated p38 MAPK; T-p38 MAPK: total protein of p38 MAPK
including both phospho- and nonphospho-p38 MAPK.
groups, we also performed immunostaining with primary rabbit
polyclonal antibody against p-p38 MAPK. This method
allowed us to visualize the phosphorylation levels as well as
the localization of p-p38 MAPK inmmunoreactive cells in the
brain regions affected by repetitive hypoxic exposure.
Fig. 2 shows the immunostaining of p-p38 MAPK in cortex
(Fig. 2A, B, and C) and hippocampus (Fig. 2D, E, and F) of
mice from normoxic control H0 (Fig. 2A and D), three hypoxic
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Fig. 2. Immunostaining of p-p38 MAPK in the cortex and hippocampus of HPC mice. Brain slices (30 mm thickness) of frozen mouse brain from H0, H3, and H6
groups were cut in the coronal section and stained with primary rabbit polyclonal antibody against Thr180/Tyr182-phosphorylated p38 MAPK (p-p38 MAPK).
Sparsely p-p38MAPK-immunostained cells were detected in both cortex (A) and hippocampus (D) of mice from the H0 group. Following three (H3) and six (H6)
hypoxic exposures, the number of p-p38 MAPK immunostained glia-like cells increased significantly both in cortex (B and C) and hippocampus (E and F) of mice.
Scale bars represent 50 mm in Fig. 3A, B, and C, and 100 mm in Fig. 3D, E, and F.
exposures H3 (Fig. 2B and E) and six hypoxic exposures H6
(Fig. 2C and F) groups. After three or six hypoxic exposures,
the number of p-p38 MAPK positive cells in the frontal cortex
(H3: 43.0 3.6, H6: 35.3 0.9 versus H0: 18.1 2.0,
p < 0.05, n = 3) and the CA1 region of hippocampus (H3:
32.3 2.1, H6: 34.1 9.4 versus H0: 9.3 4.2, p < 0.05,
n = 3) markedly increased, which was consistent with the
results of Western blots. Such p-p38 MAPK immunostained
cells exhibited a glia-like phenotype with small, round-shaped,
and intensely stained cell bodies.
Unlike the changes of p-p38 MAPK in cortex and
hippocampus, Fig. 3 indicated that repetitive hypoxic exposures
(H3 in Fig. 3B, B1, and B2; H6 in Fig. 3C, C1, and C2)
increased the numbers of p-p38 MAPK positive neuron-like
cells in the dorsomedial hypothalamic nucleus (DM, Fig. 3A1,
B1, and C1) and the zona incerta (ZI, Fig. 3A2, B2, and C2) of
the hypothalamic area of mice. The results of quantitative
analysis showed as follows: H3 (69.9 5.1), H6 (56.9 13.2)
versus H0 (29.1 4.5) in DM ( p < 0.05, n = 6), and H3
(19.4 4.8), H6 (21.5 0.7) versus H0 (5.3 1.1) in ZI
( p < 0.05, n = 6). Fig. 3A, A1, and A2 showed the distribution
of p-p38 MAPK positive neuron-like cells in the hypothalamic
area—DM and ZI of mice from the normoxic control H0 group.
3.3. Determination of p-p38 MAPK positive cell type in the
brain of HPC mice
To further define the cell type of p-p38 MAPK positive cells
in the brain of HPC mice, double-labeled immunofluorescent
staining was performed on brain slices of mice after three
hypoxic exposures (H3). We used primary rabbit polyclonal
antibody against p-p38 MAPK (red color) and primary mouse
monoclonal antibody against NF-H (green color, neuronspecific markers), S100 (green color, astrocyte-specific
marker), and CD11b (green color, microglia-specific maker)
to visualize the cell-specific localization of p-p38 MAPK in the
X. Bu et al. / Neurochemistry International 51 (2007) 459–466
463
Fig. 3. Immunostaining of p-p38 MAPK in the hypothalamus regions of HPC mice. Brain slices (30 mm thickness) of frozen mouse brain from H0, H3, and H6
groups were cut in the coronal section and stained with primary rabbit polyclonal antibody against Thr180/Tyr182-phosphorylated p38 MAPK (p-p38 MAPK). The pp38 MAPK positive neuron-like cells increased in the dorsomedial hypothalamic nucleus (DM, 4A1, B1, and C1) and zona incerta (ZI, 4A2, B2, and C2) of mice
following three (H3: 4B, B1, and B2) and six (H6: 4C, C1, and C2) hypoxic exposures, when compared to the normoxic control group (H0: 4A, A1, and A2). Scale
bars represent 300 mm for A, B, and C, and 10 mm for A1–A2, B1–B2, and C1–C2.
cortex (Fig. 4A, B, and C), hippocampus (Fig. 4D, E, and F) and
dorsomedial hypothalamic nucleus (DM, Fig. 4G, H, and I) of
H3 mice, respectively. From the merged images of Fig. 4C, F,
and I (yellow color), we found that the p-p38 MAPK positive
cells (Fig. 4A, D, and G, red) co-localized with the CD11b
(Fig. 4B and E, green) not S100 (data not show) both in cortex
and hippocampus, and the NF-H immunoreactive cells
(Fig. 4H, green) in the DM area of mice from H3 group.
This suggests that the increased p-p38 MAPK occurred in
microglia of cortex and hippocampus as well as in neurons of
hypothalamus of HPC mice.
4. Discussion
Preconditioning with ischemia/hypoxia induces tolerance in
the brain to protect against a subsequent lethal insult (Jones and
Bergeron, 2004; Vannucci and Hagberg, 2004). For example,
pre-treatment with hypoxia protects against injury caused by
oxygen–glucose deprivation (OGD) in neuronal cell culture
(Bruer et al., 1997). In adult rodent brain, a sublethal global
ischemia insult protects against neuronal damage induced by a
lethal insult and stimulates the activation of MAPK/ERK
kinase 1/2 (MEK1/2) and its downstream target ERK1/2
(Shamloo et al., 1999). In addition, ischemic preconditionioning reduces the magnitude of MEK1/2, ERK1/2 and JNK
activation that is normally observed after a lethal ischemic
insult (Gu et al., 2001). Although the cellular mechanisms
underlying cerebral HPC are still unclear, recent evidence
suggests a role for the MAPK signaling pathways in the
development of tolerance to brain injury.
We used an in vivo HPC mouse model and found that the
phosphorylation of p38 MAPK increased significantly in the
frontal cortex, hippocampus, and hypothalamus regions of mice
during the development of cerebral HPC both at the early and
delayed phases. We also observed the activation of two
substrates, CREB and mitogen- and stress-activated protein
kinase 1 (MSK1) of p38 MAPK was involved in cerebral HPC
(Gao et al., 2006). The activation of p38 MAPK may
subsequently activate a group of substrates, such as MSK1,
and then MSK1 phosphorylate the transcription factor CREB.
CREB activation participated in the transcriptional activation of
several immediate early genes, such as c-fos, junB, and egr1,
and some antiapoptotic proteins expression requires the
phosphorylation of CREB (Deak et al., 1998; Lonze and
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X. Bu et al. / Neurochemistry International 51 (2007) 459–466
Fig. 4. Double-labeled immunofluorescent staining of p-p38 MAPK and CD11b or NF-H in different cerebral regions of HPC mice. Brain slices (30 mm thickness) of
frozen H3 mouse brain were cut in the coronal section. We used primary rabbit polyclonal antibody against p-p38 MAPK (red color, A, D, and G), primary mouse
monoclonal antibody against CD11b (green color, B and E) and neurofilament heavy chain (NF-H, green color, H) to visualize the cell-specific localization of p-p38
MAPK in the cortex (A and B), hippocampus (D and E), and dorsomedial hypothalamic nucleus (DM, G, and H) of H3 mice. From the merged images of C, F, and I
(yellow color), we found that the p-p38 MAPK positive cells (red color, A, D, and G) co-localized with the CD11b and NF-H immunoreactive cells in the cortex (B,
green color), hippocampus (E, green color) and DM area (H, green color) of mice from H3 group, respectively. Scale bars represent 20 mm. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of the article.)
Ginty, 2002; Schiller et al., 2006). Therefore, our present study
suggests that p38 MAPK signaling is involved in hypoxiainduced tolerance in brain of mice.
The role of p38 MAPK in cardio- and neuroprotection
induced by ischemia/hypoxia has been surrounded by
controversy (Hausenloy and Yellon, 2006). Studies have been
designed to determine the role of p38 MAPK in ischemic/
hypoxic preconditioning or injury using SB203580, a specific
inhibitor of the p38 MAPK. The inhibition of p38 MAPK
suppressed the hypoxia-induced apoptosis of HL-60 cells
(Ikeda et al., 2006). p38 MAPK inhibition significantly reduced
ischemia/reperfusion-induced elevation of left ventricular enddiastolic pressure accompanying decreased myocardial tumor
necrosis factor, interleukin-1beta, and interleukin-6 protein
levels, and reduced active myocardial caspase-1, caspase-3, and
caspase-11 after myocardial ischemia (Wang et al., 2005).
Cerebral endothelial cell death after hypoxia/reoxygenation
was mediated by interactions between caspase-3 and p38
MAPK, and SB203580 significantly reduced cytotoxicity (Lee
and Lo, 2003). Moreover, myocytes expressing a dominant
negative p38a MAPK, which prevented ischemic p38 MAPK
activation, were resistant to lethal simulated ischemia (Saurin
et al., 2000). However, the activation of p38 MAPK may also be
implicated in playing a protective role in neurons that
underwent ischemic stimulation. For example, pretreatment
with SB203580 could aggravate the infarction size of brain and
cerebral vascular leakage induced by focal cerebral ischemia
(Lennmyr et al., 2003). Limb ischemic preconditioning induced
brain ischemic tolerance via p38 MAPK activation, and
SB203580 blocked the protection to CA1 hippocampal
pyramidal neurons against delayed neuronal death (Sun
et al., 2006). However, these studies were lack of morphological evidences of p-p38MAPK and used different kind of
models. Therefore, the opposing roles of p38 MAPK during
ischemic/hypoxic stimulation might be a result of the difference
in tissue-specific, ischemic/hypoxic procedures or stimulation
intensity.
The activation and expression of p38 MAPK are closely
related to both the glia and neurons in brain regions. Previous
studies showed that transient focal cerebral ischemia caused
X. Bu et al. / Neurochemistry International 51 (2007) 459–466
intense immunoreactivity of p-p38 MAPK in astrocyte-like cells
of the cortex, while increased p-p38 MAPK immunoreactivity
was detected in neurons of Caudate nucleus (Irving et al., 2000).
Using double-labeled immunofluorescent staining, activated p38
MAPK induced by permanent focal cerebral ischemia was
identified both in neurons and in astrocytes of mice (Wu et al.,
2000). In this study, we found that p-p38 MAPK localized
differently with NF-H in both cortex and hippocampus, but colocalized with NF-H in DM area and CD11b (microglia-specific
marker) in the cortex and hippocampus of HPC mice,
respectively. This suggests that increased phosphorylation of
p38 MAPK occurred in microglia of cortex and hippocampus as
well as in neurons of hypothalamus of mice in response to
repetitive hypoxia. Some studies showed that ischemia causes
severe damages to the pyramidal neurons especially in the CA1
region of hippocampus. This kind of pyramidal cell death takes a
delayed time-course, which called delayed neuronal death. This
delayed neuronal death in the hippocampus appears to be
accompanied by glial cell activation involving both astrocytes
and microglia (Kirino, 1982). Activated microglia and astrocytes
have been shown to release a variety of cytotoxic agents that lead
to neuronal injury (Boje and Arora, 1992). Since the activation of
p38 MAPK has been implicated in transcription of numerous
genes involved in inflammatory processes, including proinflammatory cytokines (Beyaert et al., 1996; Matsumoto et al.,
1998) or inducible nitric oxide synthase (Bhat et al., 1998; Da
Silva et al., 1997), the induction of p-p38 MAPK in glial cells in
the brain might be responsible for the synthesis of inflammatory
mediators. In contrast, activation of p38 MAPK in neurons may
be involvled in neuroprotection against ischemia. Isoflurane
preconditioning induces the phosphorylation of p38 MAPK in
neurons of different brain regions including cortex, hippocampus
and subventricular zone (Zheng and Zuo, 2004). Our results
showed that the activation of p38 MAPK was found in neurons in
hypothalamus but not in cortex and hippocampus. The
hypothalamus is considered a critical region of the brain for
regulation of homeostatic process such as feeding and
thermoregulation in the central nervous system (Schwartz
et al., 2000). Studies have suggested that p38 MAPK was
differentally activated in distinct regions of the hypothalamus
depending on the condition of energy balance (Morikawa et al.,
2004; Ueyama et al., 2004). However, the precise mechanisms of
p38 MAPK phosphorylation in cortex, hippocampus, and
hypothalamus regions remain unclear. We speculated that cell
type-specific activation of p38 MAPK might attribute to the
different cerebral regions, which have different sensitivity to
hypoxia.
In summary, our study demonstrated that the development of
cerebral HPC is accompanied by increased phosphorylation of
p38 MAPK without affecting its total protein expression levels
in the frontal cortex, hippocampus, and hypothalamus of mice.
These results are further supported by histological studies in
which enhanced p38 MAPK phosphorylation in microglia and
neurons occurred in the cortex, hippocampus, and hypothalamus of HPC mice, respectively. Further in vivo studies may be
necessary to understand the role of p38 MAPK in neural
protection during the development of cerebral HPC.
465
Acknowledgments
This work was supported by the following grants: National
Natural Science Foundation of China (30470650 and
30670782), Beijing Natural Science Foundation (5072008),
China 973 program (2006CB504100), and NIH DE 15814. The
authors thank Steve Schuenke for editorial assistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.neuint.2007.04.028.
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