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Introduction
Cerebral ischemia evokes secondary inflammation in the
brain that contributes to ischemic
insults[1]. In the delayed progression of ischemic stroke, postischemic inflammation
may play an important role in brain
damage[2]. As one of the pro-inflammatory molecules, 5-lipoxygenase (5-LOX) is the
rate-limiting enzyme in the metabolism of arachidonic acid to
produce leukotrienes, including leukotriene
B4 (LTB4) and cysteinyl leukotrienes [CysLTs, namely leukotriene
C4 (LTC4), LTD4 and
LTE4]. The importance of 5-LOX in stroke has
been proven in reports where the gene encoding the 5-LOX
activating protein confers the risk of
stroke[3,4]. Experimental studies have shown that the expression and the metabolite
production of 5-LOX in the brain are increased after cerebral
ischemia, and 5-LOX inhibitors exert neuroprotective effects
on cerebral ischemic injury[5_10].
On the other hand, minocycline, a semisynthetic
tetracycline antibiotic, has been reported to possess neuroprotective
effects on cerebral ischemic
injury[11_15] and other brain
injuries[16_18]. The neuroprotective effect of minocycline relates
to its anti-inflammatory and anti-apoptotic activities, such
as inhibiting the activation and proliferation of microglia, the
expressions of inducible nitric oxide synthase (iNOS),
interleukin-1β converting enzyme and
cyclooxygenase-2[11,12], and the caspase-dependent and independent cell apoptotic
pathways[16_18]. However, the anti-inflammatory mechanisms
of minocycline are not fully understood.
Recently, we reported that minocycline protected PC12
cells against in vitro ischemic-like injury or
N-methyl-D-
aspartate (NMDA)-induced excitotoxicity, and it could
inhibit 5-LOX translocation to the nuclear membranes (a
phenomenon of 5-LOX activation)[19,20]. These findings
indicate that the in vitro protective effects of minocycline
on ischemic or excitotoxic injuries may be partly mediated by
inhibiting 5-LOX activation. In the present study, we
determined whether minocycline exerts an in
vivo anti-inflammatory effect that is mediated by inhibiting 5-LOX activation
after focal cerebral ischemia in rats.
Materials and methods
Measurements of physiological variables Male
Sprague-Dawley rats weighing 250_300 g (Experimental Animal Center,
Zhejiang Academy of Medical Sciences, Hangzhou, China)
were used in this study. The animals were housed under a
controlled temperature (22±2 °C), 12 h light/dark cycle, and
allowed free access to food and water. All experiments were
carried out in accordance with the National Institute of Health
Guide for the Care and Use of Laboratory Animals.
The rats were anesthetized with chloral hydrate (400
mg/kg, ip). A polyethylene tube was inserted into the right femoral
artery for continuously monitoring the blood pressure using
a computer-assisted system (MedLab-U/4cs, Nanjing MedEase, Nanjing, China), and for measuring
PaO2, PaCO2, and
arterial blood pH (Blood Gas Analyzer ABL 330, Leidu,
Copenhagen, Denmark). Blood glucose was monitored by
the One Touch Basic Blood Glucose Monitoring System
(Lifescan, Los Angeles, CA, USA). The rectal (core)
temperature was measured and maintained at 37.0±0.5 °C with a
heating pad and a heating lamp during the surgery. Percent
changes in the regional cerebral blood flow (rCBF) over the
middle cerebral arterial (MCA) territory (2 mm in diameter;
6 mm lateral and 2 mm caudal to bregma) were recorded as
described[21] using a laser Doppler flowmeter (ML191, AD
Instruments, Bella Vista, New South Wales, Australia), and
the steady baseline value of rCBF before ischemia was 100%.
Middle cerebral artery occlusion Transient focal
cerebral ischemia was induced by the suture occlusion method
as previously described[22]. Briefly, after anesthesia, a
midline incision was made in the neck, the right external carotid
artery (ECA) and the right internal carotid artery (ICA) were
carefully exposed and dissected, and a 3_0 (0.26 mm diameter)
monofilament nylon suture was inserted from the ECA into
the ICA to occlude the origin of the right MCA. After
occlusion for 30 min, the suture was withdrawn to allow reperfusion,
the ECA was ligated, and the incision was closed. The
sham-operated rats underwent identical surgery, except that the
intraluminal filament was not inserted. The achievement of
the middle cerebral artery occlusion (MCAO) was confirmed
by a reduction of 50% or more in the rCBF from the baseline
value[23]. After surgery, the rats were kept for about 2 h in a
warm box heated by lamps to maintain the body temperature.
Minocycline (Syowa Hakko, Tokyo, Japan) dissolved in
sterile saline (22.5 and 45 mg/kg) was ip injected at 0.5 and 2
h after reperfusion on the first day, and twice daily on the
second and third days. This dosage regimen was based on
previous reports in studies of rat global and focal cerebral
ischemia[11,12]. Equal volume (1 mL/kg) of saline was ip
injected as the control.
Behavioral assessments Neurological deficit scores were
evaluated 72 h after reperfusion according to the
described method[24]; 0, no deficit; 1, flexion of contralateral forelimb
upon lifting of the whole animal by the tail; 2, decrease of
thrust toward contralateral plane; and 3, circling to the
contralateral side. An inclined board test was performed to
assess balance and coordination [25] based on the method
developed by Yonemori et al[26]. The rats were placed on a
board (50 cm×30 cm). Once they were stable, the board was
inclined horizontally to vertically. The degree at which the
animal fell from the board (holding angle) was recorded. The
test was repeated 3 times and the average degree was used.
All the behavioral and morphological changes were observed
by the investigators who were blind to the treatments.
Histological examination After the behavioral
assess-ments, the rats were anesthetized 72 h after reperfusion, and
perfused transcardially with 4% paraformaldehyde after a
saline prewash. The brains were removed and postfixed in
4% paraformaldehyde overnight, and transferred to 30%
sucrose for 3 d. Six serial coronal slices were cut at 2 mm
intervals from the frontal pole. Then, 2 sets of coronal
sections (10 and 20 µm) were cut by cryomicrotomy (CM1900,
Leica, Wiesbaden, Germany) from the slices. After being
stained with 1% toluidine blue, the 20 µm thick sections were
used for gross photographic examination, while the 10 µm
sections were used for microphotographic or
immunohistochemical examination.
In the gross photographs, the lesion area of the brain
tissue was defined as an area with reduced Nissl staining,
and confirmed by light microscopy to have dark
pyknotic-necrotic cell bodies. The lesion areas were determined using
an image analysis program (AnalyPower1.0, Zhejiang
University, Hangzhou, China). The lesion volume of each
section was calculated as: lesion area×slice thickness (2 mm),
and the total lesion volume was the summation of the lesion
volumes of all sections. In the microphotographs, the
neurons in the temporoparietal cortex III and IV layers adjacent
to infarcted area (0.2_0.4 mm caudal to bregma) were
immunostained with a mouse monoclonal antibody against
neuronal nuclei (NeuN) as described later, and neuron
density was counted. The neurons or immunostained cells were
randomly counted in three 200 µm2 squares at the upper,
middle, and lower sites of the boundary zone adjacent to the
ischemic core, and then were averaged.
Immunohistochemical analyses In the 10 µm sections,
endogenous peroxidase activity was eliminated by reaction
with 3% hydrogen peroxide for 30 min, and non-specific
binding of IgG was blocked by incubation with 5% normal goat
serum for 2 h at room temperature. The brain sections
reacted overnight at 4 °C with a rabbit polyclonal
anti-myeloperoxidase (MPO, a marker of neutrophils) antibody
(1:200, Neomarkers, Fremont, CA,
USA)[27], a mouse monoclonal anti-CD11b (a marker of macrophage/microglia)
antibody (1:200, Serotec, Oxford,
UK)[12], or a rabbit polyclonal antibody against 5-LOX (1:200, Cayman Chemical, Ann Arbor,
MI, USA)[28] overnight at 4 °C, then incubated with
biotinyl-ated goat anti-rabbit or goat anti-mouse IgG (1:200,
Zhong-shan Biotechnology, Beijing, China) for 2 h at room
tempera-ture, and horseradish peroxidase-streptavidin (1:200,
Zhong-shan Biotechnology, China) for 60 min. Finally, the sections
were exposed for 5_20 min to 0.05% 3, 3'-diamino-benzidine
and 0.03% H2O2.
To visualize the localization of 5-LOX in different cell
types, double immunofluorescence was employed. Briefly,
after blocking non-specific binding of IgG with 5% normal
goat serum for 2 h at room temperature, each section was
incubated overnight at 4 °C with a mixture of a rabbit
poly-clonal antibody against 5-LOX and a mouse monoclonal
antibody against neuronal nuclear antigen (NeuN, 1:100, a
specific marker of neurons, Chemicon, Temecula, CA,
USA)[29], glial fibrillary acidic protein (GFAP, 1:800, a specific marker
of astrocytes, Chemicon, USA)[29] or CD11b (1:200). Then,
the sections were incubated with the mixture of
fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG and
Cy3-conjugated goat anti-mouse IgG (Chemicon, USA). The
anti-NeuN antibody alone was also used for detecting the
neurons.
Endogenous IgG immunostaining was performed to
detect blood-brain barrier (BBB)
disruption[30]. Cortical sections (3.8_4.0 mm caudal to bregma) reacted respectively
and successively with a biotinylated anti-rat IgG antibody
(1:200, Sigma Chemical Co, St Louis, MO, USA), horseradish
peroxidase-streptavidin (1:200) and 0.05%
3,3'-diamino-benzidine. The stained sections were photographed using a
digital camera (FinePix S602 148 Zoom, Fuji, Tokyo, Japan),
and analyzed with a computer using image analysis software
(Imagetool 2.0, University of Texas Health Science Center,
San Antonio, TX, USA). The percentage of
immunoreactivity in the sections was determined as follows: [IgG-positive
area in ischemic hemisphere/(contralateral hemisphere
area)]×100%[30].
RT-PCR In another experiment, the cortical tissues were
dissected on ice from the ischemic core and the boundary
zone adjacent to the ischemic core 72 h after reperfusion,
and stored at -70 °C until use. The control tissues were
removed from the cortex of the sham-operated rats
corresponding to the ischemic core. The total RNA was extracted
using Trizol reagents (Invitrogen, Carlbad, CA, USA)
according to the manufacture's protocol. For the cDNA
synthesis, 2 µg total RNA was mixed with 1 mmol/L dNTP,
0.2 µg random primer, 20 U RNasin, and 200 U M-MuLV
reverse transcriptase in 20 µL reverse reaction buffer. The
mixture was incubated at 42 °C for 60 min and then heated at
72 °C for 10 min to inactivate the reverse transcriptase.
PCR reactions were performed on an Eppendorf Master
Cycler (Eppendorf, Hamburg, Germany). The reaction
conditions were as follows: 1 µL cDNA mixture reacted in 20 µL
reaction buffer containing 1.5 mmol/L
MgCl2, 0.2 mmol/L dNTP, 20 pmol/L primer, and 1 U
Taq DNA polymerase. The reactions were initially heated at 94 °C for 1 min, then at 94 °C
for 30 s, 60 °C for 60 s, and 72 °C for 30 s, repeated for 35
cycles, and finally elongating at 72 °C for 10 min. Ten
μL of the PCR products were separated by 2% agarose gel
electrophoresis and visualized by ethidium bromide staining. The
density of each band was measured by the UVP gel analysis
system (Bio-Rad, Richimond, CA, USA). The mRNA
expression of 5-LOX was reported as ratios of
5-LOX/β-actin. The primer sequences were as follows: 5-LOX, forward: 5'-AAA
GAA CTG GAA ACA CGT CAG AAA-3', and reverse: 5'-AAC TGG TGT GTA CAG GGG TCA GTT-3';
β-actin, forward 5'-TCA TGA AGT GTG ACG TTG AC-3', and reverse 5'-CCT
AGA AGC ATT TGC GGT GC-3'. The product sizes were 514 bp and 285 bp, respectively. The specificity of the
primers was verified using the BLASTN program
(http://www.ncbi.nlm.nih.gov/blast/).
Leukotriene measurement In a separate experiment, the
ischemic cortexes were quickly isolated on ice 3 h after
reperfusion, which was confirmed as the peak time of
leukotriene production in our laboratory. The cortical tissues
were weighed and homogenized in ice-cold absolute ethanol.
After centrifugation of the homogenates at 15
000×g at 4 °C for 30 min, the supernatant was collected and filtrated through
a 0.2 µm filtrator. The filtration was dried under nitrogen and
resuspended in an ELISA buffer. The tissue contents of
LTB4 and CysLT (the 5-LOX metabolites) were measured
according to the protocol of the enzyme immunoassay kit
(Cayman Chemical, USA). All measurements were carried
out in duplicate.
Statistical analysis Data are expressed as the mean±SD.
The differences between the groups were analyzed by
one-way ANOVA followed by Student-Newman-Keuls
t-test; the neurological deficit score and the percent area of IgG
immunoreactivity were analyzed by non-parametric Mann-Whitney
U-test (SPSS 10.0 for Windows, 1999, SPSS, Chicago, IL,
USA). A value of P<0.05 was considered statistically
significant.
Results
Physiological parameters There were no
significant differences in the mean arterial blood pressure, arterial blood
PaO2, PaCO2, and blood glucose between 30 min before and 30
min after MCAO among the groups treated with saline and
minocycline (22.5 and 45 mg/kg) as well as the
sham-operation group. Treatment with minocycline did not affect these
physiological parameters. In the ischemic rats, rCBF was
reduced by approximately 50%_60% during 30 min MCAO
and recovered to nearly baseline levels 15 min after
reperfu-sion; treatment with minocycline did not alter rCBF
reduction and recovery (Table 1).
Ischemic injuries The neurological deficit score
increased (Figure 1A) and the inclined degrees decreased
significantly (Figure 1B) 72 h after reperfusion in the MCAO
control rats. Minocycline (22.5 and 45 mg/kg) significantly
reduced the neurological deficit scores (P<0.05 or 0.01
vs MCAO control, Figure 1A), and significantly increased the
inclined degrees only at the concentration of 45 mg/kg
(P<0.01 vs MCAO control, Figure 1B).
The infarct volume in the ischemic hemispheres was
significantly reduced by minocycline (22.5 and 45 mg/kg) 72
h after reperfusion (P<0.01 vs MCAO control, Figure 1C).
The reduced NeuN-positive neuron density in the boundary
zone was significantly attenuated by minocycline (22.5 and
45 mg/kg) 72 h after reperfusion
(P<0.01 vs MCAO control, Figure 1D).
Inflammatory reactions The endogenous IgG
immunoreactivity was intensively increased 72 h after reperfusion;
minocycline (22.5 and 45 mg/kg) remarkably reduced IgG
exudation (P<0.05 or 0.01 vs MCAO control, Figure 2).
The amount of MPO-positive neutrophils and
CD11b-positive macrophage/microglia increased in the ischemic
hemispheres 72 h after reperfusion (Figure 3A, 3B).
Mino-cycline (22.5 and 45 mg/kg) significantly inhibited
neutrophil and macrophage/microglial accumulation
(P<0.01 vs MCAO control, Figure 3C, 3D), as evidenced by MPO and
CD11b immunoreactivity, respectively.
Changes in 5-LOX expression and enzymatic activity
5-LOX-positive cells were markedly increased in the ischemic
core and boundary zone 72 h after reperfusion (Figure 4A),
which was inhibited by minocycline (45 mg/kg,
P<0.01,
Figure 4B). Double immunofluorescence showed that 5-LOX
was primarily localized in NeuN-positive neurons in the
ischemic core, and in GFAP-positive astrocytes and
CD11b-positive macrophage/microglia in the boundary zone (Figure
5); while 5-LOX expression was much weaker in astrocytes and
macrophage/microglia in the ischemic core as well as in
neurons in the boundary zone (data not shown). The
5-LOX-positive and CD11b-positive cells were reduced by
mino-cycline (right panels in Figure 5).
5-LOX mRNA expression was significantly increased in
both the ischemic core and the boundary zone 72 h after
reperfusion (Figure 6A), which was significantly inhibited
by minocycline (45 mg/kg, P<0.01
vs MCAO control, Figure 6B). The contents of
LTB4 and CysLT in the ischemic cortex increased 3 h after reperfusion, which was significantly
reduced by minocycline (45 mg/kg, P<0.01
vs MCAO control, Figure 6C, 6D).
Discussion
In the present study, we found that minocycline exerted
the anti-inflammatory effects after ischemia/reperfusion
injury in rats as previously
reported[5_10]. Minocycline inhibits 5-LOX expression and activation in ischemic brain tissue
after focal cerebral ischemia, confirming its ability in PC12
cells[19,20]. These findings indicate that minocycline might
inhibit postischemic inflammation via modulating 5-LOX
expression and activation.
In postischemic brain inflammation in the subacute phase
(days after ischemia), the brain-blood barrier(BBB) disruption,
as well as the accumulation of neutrophils and
macrophage/microglia in the brain, are the determinants in pathogene-
sis[31,32]. The present study indicates that minocycline
inhibits endogenous IgG exudation, an indicator of BBB
disruption, as previously reported[30]. This effect of
minocycline might result from the inhibition of neutrophil
accumulation because the disrupted BBB has been reported
to promote blood leukocyte
recruitment[33]. However, resident microglia and hematogenous macrophages play similar
roles in the pathogenetic cascade after cerebral ischemia;
both are CD11b-positive, but the distinction between these
cells has not been possible due to a lack of discriminating
cellular markers[34]. Since minocycline inhibits microglial
activation[11,12] and BBB disruption, it might directly inhibit
resident microglia activation and/or indirectly result from the
inhibition of BBB disruption.
The most important finding in the present study is that
minocycline may inhibit postischemic inflammation by
inhibiting 5-LOX expression and enzymatic activation.
Similarly, we also found that minocycline inhibited 5-LOX
activation and exerted a protective effect on in
vitro ischemic-like and excitotoxic injury in PC12
cells[19,20]. 5-LOX activation has been reported in transient global
ischemia[5,6] and focal cerebral
ischemia[7,9], and in the human brain tissue
from stroke patients[8]. Recently, we found that 5-LOX
expression and enzymatic activity increased after focal
cerebral ischemia in rats, and were spatio-temporally involved in
neuron injury in the acute phase and astrocyte proliferation
in the late phase in vivo[10]. These
findings indicate that
5-LOX may be a target of minocycline neuroprotection.
Mino-cycline inhibits the activation of p38 mitogen-activated
protein kinase (MAPK)[35,36], which may explain its effect on
5-LOX translocation or activation. As shown in neutrophils,
activated p38 MAPK phosphorylates MAPK-activated
protein kinase 2 and 3, which then directly phosphorylates
5-LOX; the phosphorylated 5-LOX translocates to the nuclear
membrane and is activated to produce
leukotrienes[37]. There-fore, minocycline possibly inhibits 5-LOX activation by
modulating p38 MAPK activity. However, we could not explain
why minocycline reduces the expression of 5-LOX, which
may result from secondary responses due to attenuation of
inflammation or from a special ability of minocycline.
The inhibition of 5-LOX activation may explain the
anti-inflammatory effects of minocycline. As supporting
evidence, microglia, neutrophils, and macrophages possess
the leukotriene-producing capacity of
5-LOX[38_40]. The
5-LOX metabolites, leukotrienes (LTB4 and CysLT), are
potent inflammatory mediators that can induce a variety of
res-ponses, such as chemotaxis of
leukocytes[41] and BBB
disruption[25,42]. Therefore, reduction of leukotriene production
by minocycline might attenuate postischemic inflammatory
reactions. Additionally, minocycline possesses many other
abilities that also relate to its anti-inflammatory effects, such
as the inhibition of cytokine
production[43], iNOS[43],
metallo-protease activity[13], and antioxidant
ability[14].
However, as discordant evidence, no difference in
ischemic infarcts has been found between 5-LOX-deficient
and wild-type mice with focal cerebral
ischemia[44]. We explained the discrepancy by 2 possibilities: one
possibi-lity is that 5-LOX deficiency may cause compensations as
reported otherwise[45,46], which should be considered as an
explanation for the results from wild-type and
5-LOX-defici-ent animals. Another possibility is that 5-LOX may act only
as one factor in a complex system on ischemic brain injury;
even if in arachidonic acid-metabolizing pathway, 5-LOX
deficiency may enhance cyclooxygenase
activity[47]. Recent-ly, 5-LOX was confirmed as a modulator in rat focal cerebral
ischemia in another laboratory[48]. Therefore, 5-LOX
activation may be reasonably considered as one of the targets of
minocycline.
In conclusion, we found that minocycline inhibited
postischemic inflammation, partly mediated by the inhibition of
5-LOX expression and enzymatic activation.
References
1 Barone FC, Feuerstein GZ. Inflammatory mediators and stroke:
new opportunities for novel therapeutics. J Cereb Blood Flow
Metab 1999; 19: 819_34.
2 Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic
stroke: an integrated view. Trends Neurosci 1999; 22: 391_7.
3 Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S,
Jonsdottir H, Thorsteinsdottir U, et al. The gene encoding
5-lipoxygenase activating protein confers risk of myocardial
infarction and stroke. Nat Genet 2004; 36: 233_9.
4 Lohmussaar E, Gschwendtner A, Mueller JC, Org T, Wichmann
E, Hamann G, et al. ALOX5AP gene and the PDE4D gene in a
central European population of stroke patients. Stroke 2005;
36: 731_6.
5 Ohtsuki T, Matsumoto M, Hayashi Y, Yamamoto K, Kitagawa
K, Ogawa S, et al. Reperfusion induces 5-lipoxygenase
translocation and leukotriene C4 production in ischemic brain. Am J
Physiol 1995; 268: H1249_57.
6 Baskaya MK, Hu Y, Donaldson D, Maley M, Rao AM, Prasad
MR, et al. Protective effect of the 5-lipoxygenase inhibitor
AA-861 on cerebral edema after transient ischemia. J Neurosurg
1996; 85: 112_6.
7 Ciceri P, Rabuffetti M, Monopoli A, Nicosia S. Production of
leukotrienes in a model of focal cerebral ischaemia in the rat. Br
J Pharmacol 2001; 133: 1323_9.
8 Tomimoto H, Shibata M, Ihara M, Akiguchi I, Ohtani R, Budka
H. A comparative study on the expression of cyclooxygenase
and 5-lipoxygenase during cerebral ischemia in humans. Acta
Neuropathol (Berl) 2002; 104: 601_7.
9 Zhang RL, Lu CZ, Ren HM, Xiao BG. Metabolic changes of
arachidonic acid after cerebral ischemia-reperfusion in diabetic
rats. Exp Neurol 2003; 184: 746_52.
10 Zhou Y, Wei EQ, Fang SH, Chu LS, Wang ML, Zhang WP,
et al. Spatio-temporal properties of 5-lipoxygenase expression and
activation in the brain after focal cerebral ischemia in rats. Life
Sci 2006; 79: 1645_56.
11 Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J.
Tetracyclines inhibit microglial activation and are
neuroprotec-tive in global brain ischemia. Proc Natl Acad Sci USA 1998; 95:
15769_74.
12 Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH,
Koistinaho J. A tetracycline derivative, minocycline, reduces
inflammation and protects against focal cerebral ischemia with
a wide therapeutic window. Proc Natl Acad Sci USA 1999; 96:
13496_500.
13 Koistinaho M, Malm TM, Kettunen MI, Goldsteins G, Starckx S,
Kauppinen RA, et al. Minocycline protects against permanent
cerebral ischemia in wild type but not in matrix
metalloprotease-9-deficient mice. J Cereb Blood Flow Metab 2005; 25: 460_7.
14 Morimoto N, Shimazawa M, Yamashima T, Nagai H, Hara H.
Minocycline inhibits oxidative stress and decreases
in vitro and in vivo ischemic neuronal damage. Brain Res 2005; 1044: 8_15.
15 Hewlett KA, Corbett D. Delayed minocycline treatment reduces
long-term functional deficits and histological injury in a rodent
model of focal ischemia. Neuroscience 2006; 141: 27_33.
16 Sanchez Mejia RO, Ona VO, Li M, Friedlander RM. Minocycline
reduces traumatic brain injury-mediated caspase-1 activation,
tissue damage, and neurological dysfunction. Neurosurgery 2001;
48: 1393_9.
17 Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M,
et al. Minocycline inhibits cytochrome c release and delays
progression of amyotrophic lateral sclerosis in mice. Nature 2002; 417:
74_8.
18 Wang X, Zhu S, Drozda M, Zhang W, Stavrovskaya IG, Cattaneo
E, et al. Minocycline inhibits caspase-independent and
-dependent mitochondrial cell death pathways in models of Huntington's
disease. Proc Natl Acad Sci USA 2003; 100: 10 483_7.
19 Song Y, Wei EQ, Zhang WP, Zhang L, Liu JR, Chen Z.
Mino-cycline protects PC12 cells from ischemic-like injury and
inhibits 5-lipoxygenase activation. Neuroreport 2004; 15: 2181_4.
20 Song Y, Wei EQ, Zhang WP, Ge QF, Liu JR, Wang ML,
et al. Minocycline protects PC12 cells against NMDA-induced injury
via inhibiting 5-lipoxygenase activation. Brain Res 2006; 1085:
57_67.
21 Yano T, Anraku S, Nakayama R, Ushijima K. Neuroprotective
effect of urinary trypsin inhibitor against focal cerebral
ischemia-reperfusion injury in rats. Anesthesiology 2003; 98: 465_73.
22 Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible
middle cerebral artery occlusion without craniectomy in rats.
Stroke 1989; 20: 84_91.
23 Prestigiacomo CJ, Kim SC, Connolly ES Jr, Liao H, Yan SF,
Pinsky DJ. CD18-mediated neutrophil recruitment contributes
to the pathogenesis of reperfused but not nonreperfused stroke.
Stroke 1999; 30: 1110_7.
24 Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL,
Bartkowski H. Rat middle cerebral artery occlusion: Evaluation
of the model and development of a neurologic examination.
Stroke 1986; 17: 472_6.
25 Yu GL, Wei EQ, Zhang SH, Xu HM, Chu LS, Zhang WP,
et al. Montelukast, a cysteinyl leukotriene receptor-1 antagonist, dose-
and time-dependently protects against focal cerebral ischemia in
mice. Pharmacology 2005; 73: 31_40.
26 Yonemori F, Yamaguchi T, Yamada H, Tamura A. Evaluation
of a motor deficit after chronic focal cerebral ischemia in rats.
J Cereb Blood Flow Metab 1998; 18: 1099_106.
27 Hua R, Walz W. Minocycline treatment prevents cavitation in
rats after a cortical devascularizing lesion. Brain Res 2006; 1090:
172_81.
28 Zuo L, Christofi FL, Wright VP, Bao S, Clanton TL.
Lipoxygen-ase-dependent superoxide release in skeletal muscle. J Appl Physiol
2004; 97: 661_8.
29 Schmidt W, Reymann KG. Proliferating cells differentiate into
neurons in the hippocampal CA1 region of gerbils after global
cerebral ischemia. Neurosci Lett 2002; 334: 153_6.
30 Muramatsu K, Fukuda A, Togari H, Wada Y, Nishino H.
Vulnerability to cerebral hypoxic-ischemic insult in neonatal but not in
adult rats is in parallel with disruption of the blood_brain barrier.
Stroke 1997; 28: 2281_8.
31 Danton GH, Dietrich WD. Inflammatory mechanisms after
ischemia and stroke. J Neuropathol Exp Neurol 2003; 62: 127_36.
32 Simundic AM, Basic V, Topic E, Demarin V, Vrkic N, Kunovic B,
et al. Soluble adhesion molecules in acute ischemic stroke. Clin
Invest Med 2004; 27: 86_92.
33 Gidday JM, Gasche YG, Copin JC, Shah AR, Perez RS, Shapiro
SD, et al. Leukocyte-derived matrix metalloproteinase-9
mediates blood-brain barrier breakdown and is proinflammatory after
transient focal cerebral ischemia. Am J Physiol Heart Circ Physiol
2005; 289: H558_68.
34 Schilling M, Besselmann M, Leonhard C, Mueller M, Ringelstein
EB, Kiefer R. Microglial activation precedes and predominates
over macrophage infiltration in transient focal cerebral ischemia:
A study in green fluorescent protein transgenic bone marrow
chimeric mice. Exp Neurol 2003; 183: 25_33.
35 Tikka TM, Koistinaho JE. Minocycline provides neuroprotection
against N-methyl-D-aspartate neurotoxicity by inhibiting
microglia. J Immunol 2001; 166: 7527_33.
36 Lin S, Zhang Y, Dodel R, Farlow MR, Paul SM, Du Y. Minocycline
blocks nitric oxide-induced neurotoxicity by inhibition p38 MAP
kinase in rat cerebellar granule neurons. Neurosci Lett 2001;
315: 61_4.
37 Werz O, Klemm J, Samuelsson B, Radmark O. 5-Lipoxygenase is
phosphorylated by p38 kinase-dependent MAPKAP kinases. Proc
Natl Acad Sci USA 2000; 97: 5261_6.
38 Brock TG, McNish RW, Peters-Golden M. Translocation and
leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat
basophilic leukemia cells and alveolar macrophages. J Biol Chem
1995; 270: 21 652_8.
39 Brock TG, McNish RW, Bailie MB, Peters_Golden M. Rapid
import of cytosolic 5-lipoxygenase into the nucleus of
neutrophils after in vivo recruitment and
in vitro adherence. J Biol Chem 1997; 272: 8276_80.
40 Ballerini P, Di Iorio P, Ciccarelli R, Caciagli F, Poli A, Beraudi A,
et al. P2Y1 and cysteinyl leukotriene receptors mediate purine
and cysteinyl leukotriene co-release in primary cultures of rat
microglia. Int J Immunopathol Pharmacol 2005; 18: 255_68.
41 Barone FC, Hillegass LM, Tzimas MN, Schmidt DB, Foley JJ,
White RF, et al. Time-related changes in myeloperoxidase
activity and leukotriene B4 receptor binding reflect leukocyte
influx in cerebral focal stroke. Mol Chem Neuropathol 1995;
24: 13_30.
42 Baba T, Black KL, Ikezaki K, Chen KN, Becker DP. Intracarotid
infusion of leukotriene C4 selectively increases blood-brain
barrier permeability after focal ischemia in rats. J Cereb Blood Flow
Metab 1991; 11: 638_43.
43 Lai AY, Todd KG. Hypoxia-activated microglial mediators of
neuronal survival are differentially regulated by tetracyclines.
Glia 2006; 53: 809_16.
44 Kitagawa K, Matsumoto M, Hori M. Cerebral ischemia in
5-lipoxygenase knockout mice. Brain Res 2004; 1004: 198_202.
45 Takahashi E, Ino M, Miyamoto N, Nagasu T. Increased
expression of P/Q-type Ca2+ channel alpha1A subunit mRNA in
cerebellum of N-type Ca2+ channel alpha1B subunit gene-deficient mice.
Brain Res Mol Brain Res 2004; 124: 79_87.
46 Snell BJ, Day A, Ledent C, Lawrence AJ.
[3H]Adenosine uptake in brainstem membranes of CD-1 mice lacking the adenosine
A(2a) receptor. Life Sci 2004; 75: 225_35.
47 Goulet JL, Snouwaert JN, Latour AM, Coffman TM, Koller BH.
Altered inflammatory responses in leukotriene-deficient mice.
Proc Natl Acad Sci USA 1994; 91: 12852_6.
48 Jatana M, Giri S, Ansari MA, Elango C, Singh AK, Singh I,
et al. Inhibition of NF-kappaB activation by 5-lipoxygenase
inhibitors protects brain against injury in a rat model of focal cerebral
ischemia. J Neuroinflammation 2006; 3: 12. |
