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After cerebral ischemia, the highly complex pathophysiological process that follows can be separated into 3 successive
phases: metabolic stress and excitotoxicity (acute, within hours), inflammation and apoptosis (subacute, hours to days), and
repair and regeneration (chronic, days to
months)[1,2]. Post-ischemic inflammation in the subacute phase is an important
event in which a large number of cells and molecules/mediators are involved. Among the inflammatory cells, the
accumulation of neutrophils and macrophage/microglia in the brain is a determinant in
pathogenesis[3_5]. Cysteinyl leukotrienes (CysLT,
including LTC4, LTD4 and
LTE4), the 5-lipoxygenase metabolites of arachidonic acid, represent one type of pro-inflammatory
mediator involved in cerebral
ischemia[6_9]. CysLT can increase blood_brain barrier (BBB) permeability and induce brain
edema after cerebral ischemia and neutrophil
perfusion[6,9]. However, their actions on inflammatory cells in the ischemic brain
are still unknown, although neutrophil-endothelial cell cooperation has been suggested to induce brain edema via
production of CysLT in guinea pig brains perfused with human
neutrophils[9]. In peripheral tissues, CysLT can increase eosinophil
adhesion[10] and transendothelial
migration[11], which are involved in airway eosinophilic inflammation in
asthma[12].
The pro-inflammatory actions of CysLT are mediated by activating cysteinyl leukotriene receptors
(CysLT1 and
CysLT2)[13].
CysLT1 receptor mRNA has been detected by Northern blotting in the
brain[14], and its protein is primarily expressed in
microvascular endothelium in human brain
tissues[15]. We previously found that the
CysLT1 receptor antagonists pranlukast
(ONO-1078) and montelukast protected against acute and chronic ischemic brain injury in rats and
mice[16_21]. This effect may partly result from inhibiting BBB permeability, brain edema, and glial scar
formation[17,20,21]. However, whether pranlukast
inhibits inflammatory cells in ischemic brain tissue is not yet clear.
Therefore, to further determine whether the protective effect of pranlukast is associated with anti-inflammatory activity,
in the present study we observed its effect on cerebral ischemia-induced neutrophil and macrophage/microglial accumulation
as well as BBB disruption and neuronal injury in mice with focal cerebral ischemia.
Materials and methods Materials Pranlukast (ONO-1078) was kindly provided by Dr Masami TSUBOSHIMA (Ono Pharmaceutical Co, Osaka,
Japan). Chloral hydrate, 2,3,5-triphenyltetrazolium chloride (TTC) and biotinylated anti-mouse IgG antibody were purchased
from Sigma (St Louis, USA). Fluoro-Jade B was purchased from Chemicon International (Temecula,
CA, USA). Biotinylated anti-rabbit IgG, biotinylated anti-rat IgG, horseradish peroxidase streptavidin and 3,3¡¯-diaminobenzi-dine were purchased
from Zhongshan Biotechnology (Beijing, China). Polyclonal rabbit anti-myeloperoxidase (MPO) and monoclonal rat
anti-CD11b antibodies were purchased from Serotec (Oxford, UK).
Physiological parameter monitoring Male Kunming mice weighing 25_30 g (Shanghai Experimental Animal Center,
China; Certificate No 22-001004) were used in this study. Mice were housed in a controlled temperature environment, with
a 12 h light/12 h dark cycle, and allowed free access to food and water. All experiments were carried out in accordance with
the National Institute of Health¡¯s Guide for the Care and Use of Laboratory Animals.
Mice were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg). A polyethylene tube was inserted into
the right femoral artery for continuously monitoring blood pressure, by using a computer-assisted system (MedLab-U/4cs;
Nanjing MedEase, Nanjing, China), and for measuring
PaO2, PaCO2, and arterial blood pH (ABL 330 blood gas analyzer; Leidu,
Denmark). Blood glucose was monitored by using a one touch basic blood glucose monitoring system (Lifescan, USA).
Rectal (core) temperature was measured and maintained at 37.0±0.5 ºC with a heating pad and a heating lamp during the
surgery.
Focal cerebral ischemia Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) as
previously described[20]. Briefly, a 6_0 nylon monofilament suture, blunted at the tip and coated with 1%
poly-L-lysine, was inserted into the right internal carotid artery, and advanced approximately 10 mm distal to the carotid bifurcation to occlude
the origin of the middle cerebral artery. In sham-operated animals, the same procedure was carried out, except that an
intraluminal filament was inserted. Pranlukast (0.01 and 0.1 mg/kg) was ip injected 30 min before and 30 min after MCAO.
Neurological deficits and histopathological assessment
Neurological deficit scores were evaluated 72 h after MCAO as
described by Bederson et al[22]: 0, no deficit; 1, failure to extend left forepaw fully; 2, circling to the left; 3, failing to the left;
4, no spontaneous walking with a depressed level of consciousness.
Mice were re-anesthetized 72 h after MCAO, and brains were quickly removed and dissected into 1.5 mm-thick coronal
slices. The slices were stained with 0.5% TTC at 37
oC for 30 min, and then fixed in a 10% buffered formalin solution. The
stained slices with the caudal face upward were photographed by a charge coupling device CCD camera (CP 230; Panasonic,
Japan) and images were recorded on a computer. Adjusted infarct area and both hemisphere areas of each slice were
determined by using an image analysis program (AnalyPower 1.0; Zhejiang University, Hangzhou, China) as reported
elsewhere[20]. Infarct volume was calculated as lesion area¡Áthickness (1.5 mm). The summation of the infarct volumes of all brain
slices was the total infarct volume.
In another series, mice were deeply anesthetized 72 h after MCAO, and perfused transcardially with saline followed by 4%
paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). The brains were removed, and fixed overnight in the same fixative
as described earlier, and then immersed in 30% sucrose solution in phosphate buffer. The brains were frozen and 20
µm- or 10 µm-thick coronal sections were cut by cryomicrotomy (CM1900; Leica, Germany). The 10
µm-thick sections were used for immunohistochemical analysis, and the 20
µm-thick sections were stained with 0.0004% Fluoro-Jade B to detect degenerated
neurons by fluorescent
microscopy[23].
Immunohistochemical analyses To determine neutrophil and macrophage/microglial accumulation, the brain sections
were immunostained with anti-MPO (marker of
neutrophils)[24] or anti-CD11b (marker of macrophage/microglia)
antibodies[25]. The 10 mm-thick sections were sequentially incubated with 5% goat serum for 2 h, rabbit polyclonal anti-MPO (1:200) or rat
monoclonal anti-CD11b (1:200) antibodies overnight, biotinylated anti-rabbit and anti-rat IgG (1:200) for 2 h, and streptavidin
avidin-biotin-horseradish peroxidase complex (1:200) for 2 h. Finally, the sections were exposed for 5_20 min to 0.01%
3,3¡¯-diaminobenzi-dine.
Endogenous IgG immunostaining was performed to detect BBB
disruption[26]. Brain sections were reacted respectively
and successively with biotinylated anti-mouse IgG antibody (1:500), horseradish peroxidase streptavidin (1:200) and
3,3¡¯-diaminobenzidine. The gray scales in the immuno-stained sections were detected with an image analyzer (Imagetool 2.0;
University of Texas Health Science Center, San Antonio, TX, USA). IgG exudation was evaluated as the percentage increase
of the gray scale in the ischemic hemisphere:
(Gi_G0)/G0×100%, where Gi is the gray scale of the ischemic hemisphere and
G0 is the gray scale of the contralateral non-ischemic hemisphere.
Statistical analysis Values are presented as mean±SD. One-way ANOVA (Student-Newman-Keuls) or the nonparametric
Mann-Whitney U-test was used for statistical analysis using the SPSS software package (version 10.0 for Windows; SPSS,
USA). P<0.05 was considered statistically significant.
Results
There were no significant differences in mean arterial blood pressure, or arterial blood
PaO2, PaCO2, or blood glucose
between 30 min before and 30 min after MCAO among the groups treated with saline and pranlukast (0.01 and 0.1 mg/kg) as
well as those animals that received a sham operation (Table 1).
Focal cerebral ischemia induced neurological deficits (2.20±0.71,
n=16) 72 h after MCAO. Pranlukast reduced the
neurological deficit score by 20% at a dose of 0.01 mg/kg
(1.76±0.75; n=16, P>0.05 vs ischemic control; Mann-Whitney
U-test) and by 37% at 0.1 mg/kg (1.38±0.57;
n=16, P<0.05 vs ischemic control) 72 h after MCAO. Pranlukast at doses of
0.01 and 0.1 mg/kg significantly decreased the infarct volume by 32.2% and 42.9%, respectively, 72 h after MCAO
(P<0.05 vs ischemic control; Figure 1). The number of Fluoro-Jade B-positive cells (degenerated neurons) in the
temporoparietal cortex III and IV layers was increased 72 h after MCAO, and significantly reduced by pranlukast (0.01 and
0.1 mg/kg; Figure 2). In addition, endogenous IgG immunoreactivity was increased by 22.2%±4.2% in the ischemic
hemisphere as compared with the contralateral non-ischemic hemisphere, and pranlukast (0.01 and 0.1 mg/kg) markedly reduced
IgG exudation (Figure 3).
The numbers of MPO-positive neutrophils and CD11b-positive macrophages/microglia were increased in the ischemic
boundary region 72 h after MCAO (Figures 4 and 5). Pranlukast (0.01 and 0.1 mg/kg) significantly inhibited neutrophil
accumulation (Figure 4C, 4D), but did not inhibit macrophage/microglial accumulation (Figure 5C, 5D).
Discussion
In the present study, the most important finding is that pranlukast inhibited neutrophil but not macrophage/microglial
accumulation in ischemic brain tissue 72 h after MCAO in mice, in addition to ameliorating neurological deficits and neuron
degeneration and reducing infarct volume and IgG exudation. These results not only further confirm the neuroprotective
effect of pranlukast on focal cerebral
ischemia as reported elsewhere[16_21], but also demonstrate its anti-inflammatory properties.
The effect of pranlukast on post-ischemic neutrophil
accumulation might result from the inhibition of BBB disruption. The present results indicate that pranlukast inhibits
endogenous IgG exudation, an indicator of BBB disruption, as found in previous studies through endogenous plasma
albumin or Evans blue
staining[16,17,20].
Because neutrophils are hematogenous inflammatory cells, their accumulation in ischemic brain tissues may reflect BBB
disruption because the disrupted BBB has been reported to promote blood leukocyte
recruitment[2,5].
The lack of effect of pranlukast on post-ischemic
macrophage/microglial accumulation might indicate that
CysLT1 receptor is not involved in this response in the subacute
phase (72 h after MCAO). Resident microglia and hematogenous macrophages play similar roles in the pathogenetic cascade
following cerebral ischemia, and both of them are CD11b-positive, but distinction between these cells has not been possible
due to a lack of discriminating cellular
markers[25,27]. However, in mice transplanted with green fluorescent protein
(GFP)-transgenic bone marrow, hematogenous
GFP+ macrophages were rarely observed on d 2, reached peak numbers on d 7, and
decreased thereafter; in contrast, resident
GFP_ microglia rapidly became activated on d 1 after
MCAO[27]. Therefore, most CD11b-positive cells 72 h after MCAO in the present study might be activated resident microglia. Although the activation of
microglia in the subacute phase might not be regulated by
CysLT1 receptor, further observations are needed, because this
receptor may be expressed in later phases, and many other factors may influence the effect of pranlukast.
In summary, pranlukast inhibits post-ischemic inflammation in the brain, mainly acting on the recruitment of
hematogenous inflammatory cells (such as neutrophils) via inhibiting BBB disruption. This finding is consistent with the localization
of CysLT1 receptor in microvascular endothelium in the human
brain[15]. The anti-inflammatory effects of pranlukast have
been reported in peripheral tissues, for example allergen-induced interleukin-5 production in human lung
tissue[28], transendothelial migration of eosinophils across human umbilical vein endothelial cells in response to
LTD4[11], and eosinophil
adhesion[10]. In the present paper, we demonstrated the anti-inflammatory effect of pranlukast in the focal ischemic brain,
but this effect might be limited to BBB disruption-associated neutrophil recruitment. Thus, the present study indicates that
the anti-inflammatory effect of pranlukast may be partly involved in its neuroprotective effect against cerebral ischemic
injury.
Acknowledgement
We thank Dr Masami TSUBOSHIMA, Ono Pharmaceutical Co, Osaka, Japan, for supplying the pranlukast.
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